Improved methods of targeted protein degradation

Abstract

The present invention relates to a modified secreted AY-WB protein 5 (SAP05) protein, and in particular the use of the modified SAP05 protein as part of a fusion compound for use in ubiquitin-independent protein degradation. The invention also relates to methods for the use of the fusion compound in targeted protein degradation, modulating physiological responses and therapy.

Description

[0001] Improved methods of targeted protein degradation

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a modified secreted AY-WB protein 5 (SAP05) protein, and in particular the use of the modified SAP05 protein as part of a fusion compound for use in ubiquitin-independent protein degradation. The invention also relates to methods for the use of the fusion compound in targeted protein degradation, modulating physiological responses and therapy.

[0004] BACKGROUND OF THE INVENTION

[0005] It is possible to reduce protein levels in a targeted manner by disruption at the transcription level through nucleic acid-based tools such as RNA interference (RNAi) and more recently, CRISPR / Cas9 gene editing technology. However, these technologies have specific disadvantages, such as (embryonic) lethality, irreversibility, lack of tissue specificity and off-target effects. In addition, safe and efficient cell delivery is an issue, as well as metabolic stability and problematic off-target effects.

[0006] In view of these issues, targeted protein degradation (TPD) has been explored to reduce protein in cells, which work at the post-translational level to cause degradation. An advantage of this approach is that target proteins can be accurately and rapidly degraded in a tissue / cell specific manner, even if the target protein has a slow turnover rate. This is favourable over degradation at the transcriptional level as experiments using these tools can be difficult to interpret due to the delay between loss of gene expression and resulting protein depletion. Specifically targeting at the protein level also avoids adaption events at the genomic level, such as gene amplification and also offers isoform specificity.

[0007] Targeting at the protein level can also be very advantageous where a target has a function in more than one stage of development of the organism. By targeting degradation at the cell or tissue level it is possible to selectively degrade proteins in many useful contexts. For example, in plants there are a number of transcription factors that have functions in both shoot / root meristem and embryo development and it is difficult to study the function of these proteins in meristems if the plant dies at the embryo stage. Therefore, by specifically targeting proteins at a tissue level, it would be possible to deplete the protein only in the meristem, and not in the embryo. In a similar way, Toll-like receptors are important during embryo development in Drosophila but also have a key role in immunity, therefore conducting protein degradation after embryo development allows for the investigation of these proteins specifically in the context of immunity without causing harmful effects on development.

[0008] Clearly, there are numerous applications for a selective protein degradation technology in both plants and animals, as well as in yeasts and fungi, where it is desirable and / or useful to reduce or eliminate the presence of a particular protein of interest.

[0009] In animal systems, recent examples include Specific and Nongenetic Inhibitor of Apoptosis Protein (lAP)-dependent Protein Erasers (SNIPERs), proteolysis-targeting chimeras (PROTACs), and Degronimids, which have offered the ability to target previously “undruggable” targets that were not capable of pharmacological targeting. Of note, all of these examples induce selective degradation of target proteins through ubiquitin-mediated pathways. Ubiquitination is an enzymatic process involving three steps: activation, conjugation, and ligation; and requiring three enzymes: ubiquitin- activating enzymes (E1s), ubiquitin-conjugated enzymes (E2) and ubiquitin ligases (E3s) (6). Firstly, ubiquitin, a small (8.6kA) regulatory protein is activated by E1s through an ATP dependent reaction. Secondly, the transfer of ubiquitin from E1 to E2 occurs through catalyzation by E2. Lastly, E3s catalyse the production of an isopeptide bond between a lysine of the target protein and the C-terminal glycine of ubiquitin. Further ubiquitin molecules can then be added to the first, creating a polyubiquitin chain which marks the protein for targeted protein destruction by the proteasome. Ubiquitin mediated degradation therefore requires this energy intensive three step enzymatic process to be repeated many times over to effectively degrade the protein. Harnessing this process for targeted protein degradation has also been technically challenging, due to the highly regulated nature of ubiquitination, which depends on cell or tissue specific enzymes. These enzymes need to be identified and characterized in the tissues / cells, and it can be highly challenging to identify the right combination of enzymes to be effective for a given substrate. Even when these enzymes are identified, it is difficult to exclude that these enzymes are active elsewhere in an organism, which increases the risk of off-target effects. It is also possible that ubiquitin chains can be of different compositions, with some ubiquitin compositions not being degraded but instead being transported to another cell location, such as the nucleus.

[0010] There is therefore a need to develop alternative selective protein degradation technologies that bypasses the need for ubiquitination. The present invention addresses this need.

[0011] SUMMARY OF THE INVENTION

[0012] Here, we further investigated how SAP05 mediates ubiquitin-independent degradation by generating the crystal structures of SAP05 complexes with ZnF domain of SPL5 and vWA domain of RPN10 and conducting follow-up mutagenesis and degradation experiments. We found that SAP05 acts like a molecular glue (also referred to herein as a scaffold) by binding ZnF and vWA on oppositely positioned surfaces at 1:1 stoichiometries. SAP05 interaction with vWA doesn’t appear to cause steric hindrance with other 26S proteasome components and their interactors. Our data show that the bacterial SAP05 effector has evolved as a functional adapter of the 26S proteasome to enable ubiquitin-independent degradation of structured eukaryotic proteins.

[0013] Eukaryotic proteins targeted for degradation by the cellular proteolysis pathway are usually 'tagged' by multimers of a protein known as ubiquitin to enable later degradation by the 26S proteasome. However, some proteins mediate targeted protein degradation (TPD) without involving ubiquitin. How these proteins mediate TPD is largely unknown. Here we defined the biochemical short-circuit used by a small bacterial effector protein, the effector protein SAP05, to enable ubiquitin-independent TPD. SAP05 forms a ternary complex by simultaneously interacting with the von Willebrand Factor Type A (vWA) domain of the proteasome receptor RPN10 and (ZnF) domains of SPL and GATA transcription factors (TFs), leading to TPD of the TFs. We obtained crystal structures of the SAP05-vWA complex at 1.7 Angstrom resolution and of the SAP05-ZnF complex at 2.2 Angstrom resolution. These revealed that SAP05 has a unique bimodular architecture with vWA and ZnF binding to opposite sides. Whereas the vWA domain is required for RPN10 docking to the proteasome, SAP05 binds vWA at a location that retains RPN10 interactions with proteasome components. We have further identified that a single amino acid change prevents SAP05 binding to SPL and GATA TFs, whereas binding to vWA was fully retained. Similarly, we have identified the amino acids required for SAP05 binding to vWA while retaining binding to the TFs. Taken together, our data demonstrate that a small protein with a unique bimodular architecture can biochemical short-circuit cellular proteolysis to enable ubiquitin- independent TPD.

[0014] Based on the above data, we have developed a protein knockdown technology that can induce selective proteasomal protein degradation independent of ubiquitin. Specifically, we have identified an effector that directly binds a 26S proteasome component leading to the degradation of target substrates, without the requirement for ubiquitination.

[0015] To date, effectors that directly link host targets to the proteasome system in a ubiquitin independent manner are not known. Thus, the mode of action we have identified involving Von Willebrand factor type A domain (vWA)-binding proteins, such as SAP05, is significant. As discussed above, whereas cellular protein levels may be altered by gene knockout and RNA silencing, some systems require the direct targeting of proteins for degradation. In fact, targeted protein degradation has become one of the most promising approaches for drug discovery in targeted therapies. Current approaches to changing protein abundance in cells rely on substrate ubiquitination: for example, the proteolysis-targeting chimera (PROTAC) technique uses small-molecule ligands that create complexes between E3 ligases and targets, a process that can be challenging. Our study of phytoplasma effectors has revealed an alternative approach whereby bridging targets directly onto proteasome subunits, such as RPN10 - and specifically the vWA domain of RPN10, results in efficient protein degradation. Through further analyses we have identified that the region of vWA that is on the opposite site of the area of vWA that binds the 26S proteasome is responsible for binding SAP05. Specific mutations of two amino acids within this SAP05-binding region of vWA prevents binding of SAP05 to vWA, but do not prevent binding of vWA to the proteasome. On this basis, we have established that any agent that binds the region of vWA that is on the opposite site of the area of vWA that binds the 26S proteasome can be used to mediate degradation of the target protein, through the proteasome, but independently of ubiquitin.

[0016] In one aspect of the invention there is provided a modified secreted AY-WB protein 5 (SAP05) comprising at least one mutation, wherein SAP05 comprises a plurality of loop sequences, and wherein the at least one mutation is within at least one loop sequence and wherein the at least one mutation reduces or abolishes binding of SAP05 to at least one SQUAMOSA promoter binding protein-like protein (SPL) and / or at least one GATA transcription factor.

[0017] The loop sequence may comprise an amino acid sequence selected from SEQ ID NO: 29 to 36 or a variant or fragment thereof. Preferably, the at least one mutation is within at least one of SEQ ID NO: 29 to 36 or a variant thereof.

[0018] The least one mutation may be selected from a mutation at positions 66, 76, 77, 80, 104 and 106 of SEQ ID NO: 1 or a corresponding position in a homologous sequence.

[0019] The homologous sequence may be selected from SEQ ID NO: 7, 9, 11 , 12, 14 and 16 and functional variants thereof.

[0020] The least one mutation may be at position 66 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. Preferably, the mutation is a substitution. More preferably, the mutation is a substitution of D66 to a positively charged amino acid, more preferably a substitution to D66A.

[0021] The SAP05 protein may comprise at least one further mutation, wherein the at least one mutation is at positions 40 to 70 or 120 to 135 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. Preferably the at least one mutation allows binding of SAP05 to a non-plant vWA domain.

[0022] The at least one mutation may be selected from positions 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 124, 125, 126, 127, 128, 129, 130, 131 and 132 or a corresponding position on a homologous sequence.

[0023] In another aspect of the invention, there is provided a modified secreted AY-WB protein 5 (SAP05) comprising at least one mutation, wherein the at least one mutation is at a position selected from positions 40 to 70 or 120 to 135 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. Preferably, the at least one mutation allows binding of SAP05 to a non-plant vWA domain. The at least one mutation is selected from positions 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 124, 125, 126, 127, 128, 129, 130, 131 and 132 a corresponding position in a homologous sequence.

[0024] The homologous sequence may be selected from SEQ ID NO: 7, 9, 11 , 12, 14 and 16 and functional variants thereof.

[0025] In another aspect of the invention, there is provided a fusion compound comprising an Rpn10 binding moiety and a target-binding moiety, wherein the Rpn10 binding moiety binds one or more positions selected from positions 24, 27, 31, 34, 42, 68, 70, 71 and 77 on SEQ ID NO: 5 or a corresponding position on a homologous sequence.

[0026] The Rpn10 binding moiety may be a SAP05 peptide, wherein the SAP05 peptide is the modified SAP05 protein described above.

[0027] The Rpn10 binding moiety may be selected from peptides, cyclic peptides, binding domains, small molecules / chemicals, t-cell receptors, antibodies, functional fragment of an antibody, nanobodies, monobodies, (peptide) macrocycles, DARPins, affibodies, adnectins, affimers, anticalins and aptamers.

[0028] The fusion compound may further comprise a RPN10 protein or a vWA domain, wherein preferably the RPN10 protein comprises an amino acid sequence as defined in SEQ ID NO: 5, 21 or 23 or a functional variant thereof and wherein the vWA domain comprises an amino acid sequence as defined in SEQ ID NO: 18 or a functional variant thereof.

[0029] The target-binding moiety may be selected from peptides, cyclic peptides, binding domains, small molecules / chemicals, t-cell receptors, antibodies, functional fragment of an antibody, nanobodies, monobodies, (peptide) macrocycles, DARPins, affibodies, adnectins, affimers, anticalins and aptamers.

[0030] The fusion compound may be a bi-specific antibody, wherein the bi-specific antibody is capable of binding Rpn10 and a target. In another aspect of the invention, there is provided an expression vector comprising a nucleic acid sequence encoding the modified SAP05 of the invention or the fusion compound of the invention.

[0031] In another aspect of the invention, there is provided a cell comprising the fusion compound of the invention or the expression vector of the invention. Preferably the cell is a eukaryotic cell.

[0032] In another aspect of the invention, there is provided a transgenic organism expressing the expression vector of the invention or comprising the cell of the invention, wherein the organism is not a human.

[0033] In some embodiments, the fusion compound or the expression vector is for use as a medicament. In another embodiment, the fusion compound or expression vector can be used as a biological agrochemical. For example, the fusion compound or expression vector can be used to control plant pests, weeds and other plant pathogens.

[0034] In another aspect of the invention, there is provided the use of the fusion compound of the invention or an expression vector of the invention in targeted protein degradation. Alternatively, there is provided the use of a modified SAP05 protein alone or in combination with a vWA domain in targeted protein degradation, for example as defined in SEQ ID NO: 5 or 18 or a variant thereof. Preferably, the targeted protein degradation is ubiquitin-independent protein degradation.

[0035] In another aspect of the invention there is provided a method of targeted protein degradation, the method comprising applying the fusion compound of the invention or the expression vector of the invention to a sample. Alternatively, there is provided a method of protein degradation, the method comprising applying the modified SAP05 protein of the invention alone or in combination with a vWA domain, as described above. Preferably, the targeted protein degradation is ubiquitin-independent protein degradation.

[0036] In another aspect of the invention, there is provided a method of controlling the level of a target protein, the method comprising applying the fusion compound of the invention, the expression vector of the invention or a modified SAP05 protein of the invention (and optionally additionally a vWA domain herein) or to a sample.

[0037] In one embodiment, a fluorescently tagged antibody is applied to the sample in order to label a protein of interest prior to applying the fusion compound, wherein the targetbinding moiety of the fusion compound is specific for the fluorescent tag, and wherein degradation of the labelled protein of interest in the sample can be detected by a decrease in fluorescent signal.

[0038] In another embodiment, the target protein is a cytosolic protein or a cell surface protein. In another embodiment, the method may further comprise the step of administering a vWA targeting moiety. The purpose of administering a vWA targeting moiety alone is to prevent prolonged degradation of the target protein by the fusion compound, which could lead to toxicity. Hence, a vWA binding moiety may be added to control or limit the amount of fusion compound-mediated degradation by competing for binding to the region at which the fusion compound binds vWA (or RPN10), thereby preventing degradation. As such, in this embodiment, the vWA targeting binding moiety binds the same region on vWA as the fusion compound. More preferably, the vWA targeting moiety may be added in an amount that is higher than the fusion compound - i.e. in excess - to outcompete binding of the fusion compound to vWA.

[0039] In another aspect of the invention, there is provided a method of gene editing, the method comprising: a) introducing a CRISPR-Cas (or Cpf) system comprising a CRISPR enzyme to a cell or organism, b) allowing the CRISPR-Cas (or Cpf) system to edit one or more target nucleic acid sequence, c) delivering a fusion compound or expression vector according to the invention comprising a target moiety that is specific for said CRISPR enzyme; and d) allowing the fusion compound to degrade the CRISPR enzyme so as to inhibit any further activity of the CRISPR-Cas system.

[0040] In one embodiment, the target protein can cause pathology in a target organism or the target protein may be a drug target. In another aspect of the invention, there is provided a method of modulating a physiological response in an organism, the method comprising administering to said organism a fusion compound of the invention, an expression vector of the invention (encoding the fusion compound) or a modified SAP05 protein of the invention (and optionally additionally a vWA domain). The physiological response may be selected from a stress response, an immune response, a hormone response or a light response. For example, the stress response may be an abiotic or biotic stress response in plants; an example of the latter is a response to a plant pathogen.

[0041] In another aspect of the invention there is provided a method of treating a condition in a patient in need thereof, the method comprising administering to said patient a fusion compound of the invention or an expression vector of the invention or a modified SAP05 protein (and optionally additionally a vWA domain). In one embodiment, the condition is characterised by increased expression or activity of a target protein, and the target-binding moiety is specific for said target protein.

[0042] In another aspect of the invention, there is provided a method of treating an infection caused by a microorganism, the method comprising administering to a subject a fusion compound of the invention or an expression vector of the invention, or a modified SAP05 protein of the invention (and optionally additionally a vWA domain), wherein the target binding moiety is specific for a protein expressed by a microorganism.

[0043] In another aspect of the invention there is provided a method of increasing the immunogenicity of a protein, the method comprising administering to a subject a fusion compound of the invention or an expression vector of the invention, or a modified SAP05 protein of the invention (and optionally additionally a vWA domain), wherein the target binding moiety is specific for the protein, and wherein proteasome degradation of the protein results in increased antigen presentation of peptides degraded from the protein. In one embodiment, the fusion compound is administered to a subject suffering from an infection or cancer.

[0044] In another aspect, there is provided a method for creating a protein knockout model, the method comprising administering a fusion compound of the invention or an expression vector of the invention, or a modified SAP05 protein of the invention (and optionally additionally a vWA domain) to a cell or an organism. In one embodiment, the model is a disease model where the disease is caused or characterised by a dysfunction or absence of a target protein, and the target moiety is specific for the target protein.

[0045] In another aspect, there is provided a method of identifying a degradation effect of a target protein in a biological system, the method comprising applying a fusion compound of the invention, an expression vector of the invention (encoding the fusion compound) or a modified SAP05 protein of the invention (and optionally additionally a vWA domain) to the biological system in order to degrade said target protein. Preferably, the target protein is a regulatory protein, and the method further comprises performing RNA sequencing of the RNA of the biological system to determine the transcription status of one or more genes, preferably the targeted gene, possibly a downstream member of a pathway involving said targeted gene, and / or to assess the transcriptome of the system. Preferably, this will further comprise performing RNA sequencing of the RNA of a biological system wherein the method comprising applying the fusion compound of the invention, a modified SAP05 protein of the invention (and optionally additionally a vWA domain), or an expression vector of the invention to the biological system has not been performed (i.e. a control biological system) and comparing the two systems. In some embodiments, the method may be applied as part of a biological or pharmaceutical screening method; for example, by identifying the degradation effect in the presence or absence of a candidate drug, and comparing with a control. In some embodiments the screen may be carried out in a cell line, for example a human cell line, or may be carried out in a model organism, for example, a mouse, or may be carried out in a fungus, for example, a yeast.

[0046] In another aspect of the invention there is provided a method of modulating a characteristic of a fungus, the method comprising introducing the fusion compound of the invention, an expression vector of the invention (encoding the fusion compound), or a modified SAP05 protein of the invention (and optionally additionally a vWA domain) to the fungus, wherein the target-binding moiety is specific for a target protein the expression of which is associated with the characteristic. In some embodiments, the fungus is a yeast (eg, Saccharomyces, Candida, Pichia, etc). In some embodiments, the fungus is detrimental to food production; for example, can cause spoilage of food, or may cause infections or ill-health in an organism that may consume the food. In some embodiments, the characteristic to be modulated is growth and / or reproduction of the fungus. In other embodiments, the characteristic to be modulated is production and expression of a toxic compound; or is production and expression of a compound which alters the taste of a food product.

[0047] In another aspect of the invention there is provided a method of increasing production in a farm animal, the method comprising introducing the fusion compound of the invention, an expression vector of the invention (encoding the fusion compound) or a modified SAP05 protein of the invention (and optionally additionally a vWA domain), to the farm animal, wherein the target-binding moiety is specific for a target protein the expression or activity of which is negatively correlated with production. In some embodiments the farm animal may be a bovid, caprid, ovid, porcid. In other embodiments the farm animal may be avian; for example, a galloanserine such as chicken, duck.

[0048] In another aspect of the invention there is provided a method of increasing yield in a plant, the method comprising introducing the fusion compound of the invention, an expression vector of the invention (encoding the fusion compound), or a modified SAP05 protein of the invention (and optionally additionally a vWA domain), to the plant, wherein the target-binding moiety is specific for a target protein the expression or activity of which is negatively correlated with yield.

[0049] In another aspect of the invention there is provided a method of reducing or removing glutens in a plant, the method comprising introducing the fusion compound of the invention, an expression vector of the invention (encoding the fusion compound), or a modified SAP05 protein of the invention (and optionally additionally a vWA domain), to the plant, wherein the target-binding moiety is specific for gliadin or glutenin.

[0050] In a preferred embodiment, targeted protein degradation is ubiquitin-independent protein degradation.

[0051] In another aspect, there is provided a pharmaceutical composition comprising the fusion compound of the invention, an expression vector of the invention (encoding the fusion compound), or a modified SAP05 protein of the invention (and optionally additionally a vWA domain), and a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, the pharmaceutical composition is for use in treatment of cancer, infection, a neurodegenerative disorder or a proteopathy.

[0052] In the above embodiments where a SAP05 and a vWA domain are used in combination, SAP05 and vWA may be applied or administered separately, sequentially or concurrently. Where used herein, the vWA domain may be a plant vWA domain. The plant vWA domain may comprise or consist of SEQ ID NO: 18 or a functional variant or fragment thereof.

[0053] In another aspect, there is provided a screening library comprising a plurality of fusion compounds according to the invention, wherein each fusion compound comprises a different target-binding moiety.

[0054] In another aspect, there is provided a method for screening target-binding moieties using the screening library of the invention. Said methods may be useful in the pharmaceutical industry, for example to screen candidate target-binding moieties as potential therapeutics.

[0055] In a final aspect, there is provided a kit comprising a fluorescently tagged antibody and a fusion compound of the invention, wherein the target-binding moiety of the fusion compound is specific for the fluorescent tag.

[0056] DESCRIPTION OF THE FIGURES

[0057] The invention is further described in the following non-limiting figures:

[0058] Figure 1 shows the crystal structures of SAP05 - ZnF_SPL5 and SAP05 - vWA_RPN10 complexes reveal the bimodular property of SAP05. Secondary structures are highlighted. (B) Amino acid sequence of SAP05 highlighting the locations of secondary structures and ZnF_SPL5 interacting and vWA_Rpn10 interacting residues. (C) Crystal structure of SAP05 - ZnF_SPL5 complex. (D) Crystal structure of SAP05 - vWA_Rpn10 complex. (E) Bimodular interfaces of SAP05 showing the loop surface residues and sheet surface residues that bind to ZnF_SPL5 and vWA_Rpn10. (F) Ternary structure of ZnF_SPL5 - SAP05 - vWA_Rpn10 obtained by superimposing the crystal structures of ZnF_SPL5 - SAP05 and SAP05 - vWA_Rpn10 complexes. Figure 2 shows the SAP05 loop surface interacts with ZnF_SPL5. (A) Different stereo view of SAP05 - ZnF_SPL5 interaction interface. Pink spheres, Zn2+ions. (B) The electrostatic potential surface view of SAP05 and ZnF_SPL5 during complex formation. (C) Isothermal titration calorimetry of SAP05 and ZnF_SPL5. (D, F) Western blot analysis of proteasomal degradation of SPL5 (D) and GATA19 (F) in presence of wildtype or mutant SAP05 in N. benthamiana leaves. E) Yeast two-hybrid (Y2H) assay to test interactions of SAP05 and its mutant versions with A. thaliana Rpn10, GATAs and SPLs. EV, empty vector control. AD, GAL4-activation domain. BD, GAL4-DNA binding domain.

[0059] Figure 3 shows the SAP05 p-sheet surface binds to vWA_Rpn10. (A) Different stereo views of SAP05 - vWA_Rpn10 interaction interface showing the residues involved in complex formation. (B) Western blot analysis for SPL5 degradation with SAP05 wildtype and mutants in A. thaliana protoplasts. (C) Y2H assay to test interactions of SAP05 and its mutant versions with A. thaliana Rpn10, GATAs and SPLs. EV, empty vector control. AD, GAL4-activation domain. BD, GAL4-DNA binding domain.

[0060] Figure 4 shows the SAP05 loop 3 region is essential for interaction with vWA domains. Structure of SAP05 - vWA_AtRpn10 complex is superimposed on the structure of vWA domains from the yeast Saccharomyces cerevisiae Rpn10 (PDB 5LN1) or human Rpn10 (PSMD4, PDB 6MSD). Arabidopsis thaliana Rpn10 (Uniprot ID: P55034); Homo sapiens Rpn10 (Uniprot ID: Q5VWC4); ScRpnIO, Saccharomyces cerevisiae Rpn10 (Uniprot ID: P38886).

[0061] Figure 5 shows the SAP05 interaction with vWA domain has no hinderance on plant 26S proteasome.

[0062] Figure 6 shows the expression and purification of SAP05 - ZnF_SPL5 and SAP05 - vWA_RPN10 complexes for crystallization. (A) Amino acid sequences of full-length SAP05 (excluding signal peptide), ZnF_SPL5 and vWA_Rpn10 proteins. The highlighted sequence (yellow) represents the region used for protein expression and purification. (B) Gel filtration chromatogram and SDS-PAGE analyses of peak fractions of purified SAP05, ZnF_SPL5, SAP05 - ZnF_SPL5 and SAP05 - vWA_Rpn10 complexes. Figure 7 shows the residues on SAP05 - ZnF_SPL5 and SAP05 - vWA_Rpn10 complex interface and introduced mutations to validate their function. (A) Residues on SAP05 - ZnF_SPL5 complex interface. (B) SAP05 mutants generated to test their contribution during interaction with ZnF_SPL5. (C) Residues on SAP05 - vWA_Rpn10 complex interface. (D) SAP05 mutants generated to test their effect on interaction with vWA_Rpn10.

[0063] Figure 8 shows the structural prediction of SAP05 - ZnF_SPL5 and SAP05 - ZnF_GATA19 complexes. (A) Left, AlphaFold-Multimer model for SAP05 - ZnF_SPL5 complex superimposed on the crystal structure of SAP05 - ZnF_SPL5 complex. Middle, The Local Distance Difference Test (LDDT) value of predicted SAP05 - ZnF_SPL5 structure, showing the quality of predicted models by evaluating local distance differences to the reference. Right, predicted aligned error (PAE) of the rank_1 model, showing the estimate of position error between predicted and true structures. Blue means lower and red higher error scores. (B) Left, AlphaFold-Multimer model of SAP05 - ZnF_GATA19 complex. Middle, LDDT value of predicted SAP05 - ZnF_GATA19 complex. Rank_1 model was used. Right, predicted aligned error (PAE) of the rank_1 model. (C) Left, predicted SAP05 - ZnF_GATA19 interaction interface. Right, interacting residues between SAP05 and ZnF_GATA19 of predicted structure.

[0064] Figure 9 shows a western blot analysis of proteasomal degradation of SPL5 in presence of wild-type or SAP05 single mutants on SAP05 - vWA_Rpn10 interacting interface in N. benthamiana leaves

[0065] Figure 10 shows conservation of SPL, GATA and Rpn10-interacting residues in SAP05 homologs (A). (B) Y2H assay showing SAP05 homologs that bind only SPLs or only GATAs. (C) Y2H assay showing that swapping the loop 4 sequences from GATA binding SAP05 homologs contribute to SPL binding, and swapping loop 4 sequences from SPL binding SAP05 homologs result in GATA binding.

[0066] Figure 11 shows that the SAP05 residue D66 is crucial to maintain the electronegative surface on SAP05 - ZnF_SPL5 complex interface. (A) Wild-type SAP05 D66 interacts with Q77 on ZnF_SPL5 (left) and present negative surface on the interacting interface (right). (B) Mutagenesis of D66 make SAP05 lost the interaction with SPL5 Q77 (left), and the electronegativity on the interaction surface is reduced (right). (C) Wild-type SAP05 D66 interacts with R105 on ZnF_GATA19 from Alphafold-Multimer predicted structure (left) and present negative surface on the interacting interface (right). (D) Mutagenesis of D66 make SAP05 lost the interaction with GATA19 R105 (left), and the electronegativity on the interaction surface is positive (right).

[0067] Figure 12 shows that SAP05 can mediate protein degradation in human 26S proteasome. Purified human 26S proteasomes degrade His-SPL5 in the presence of SAP05 and A. thaliana vWA, and MG132 inhibits this degradation. Western blots shown are from protein extracts of recombinant human 26S proteasome preparations (BostonBiochem) in the presence of purified His-SPL5 and SAP05 with or without A. thaliana RPN10 vWA (AtvWA) or proteasome inhibitor MG132 probed with antibodies to HA, GFP and SAP05 as shown at left. Red dots at left of the blots indicate the expected sizes of TFs. Protein loading was visualized using Amido black staining.

[0068] DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0070] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. Such techniques are explained fully in the literature.

[0071] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences.

[0072] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

[0073] We previously identified the small bacterial effector protein SAP05 (secreted AY-WB protein 5) that mediates ubiquitin-independent targeted protein degradation (TPD). SAP05 forms a ternary complex via interacting with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and Zinc-finger (ZnF) domains of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs), leading to direct TF TPD by the 26S proteasome. However, the structural basis of this type of TPD is unknown.

[0074] As described herein we obtained crystal structures of the SAP05-WVA complex at 2.17 angstrom (A) resolution and of the SAP05-ZnF(SPL5) complex at 2.20 A. These structures along with mutagenesis and in vivo degradation analyses revealed that SAP05 has a surprisingly unique bimodular architecture with two distinct surfaces, including a ‘loop surface’ with protruding loops that form electrostatic interactions with ZnF and a ‘sheet surface’ with -sheets and a-helices that form polar interactions with vWA. SAP05 interactions with members of both TFs involved several single amino acids responsible for multiple interactions, whereas the firmer vWA interaction required multiple separate connections. Notably, the binding of SAP05 to Rpn10 does not cause obvious steric hindrance within the proteasome. Taken together, the present invention demonstrates that a small bacterial bimodular protein can biochemically short-circuit cellular proteolysis enabling ubiquitin-independent TPD in eukaryotic cells, information that is very valuable for the development of novel TPD technologies.

[0075] Further, our demonstration that SAP05 has a bimodal structure with a ‘loop surface’ that interacts with TFs and a ‘sheet surface’ that interacts with vWA will facilitate more efficient engineering of SAP05 as the ideal chassis. That is, by modifying or mutating the SAP05 loop structure, sheet surface and / or key residues, it is possible to engineer a highly effective vehicle for targeted delivery. Furthermore, we have found that by mutating the specific residue(s) in SAP05 responsible for binding SPL and GATA transcription factors and / or vWA, we can selectively degrade target proteins via the proteasome in a ubiquitin-independent manner, and critically without any off-target effects.

[0076] Two advantages are afforded by such a modified SAP05 protein. Firstly, the use of a modified SAP05 protein can be significantly improved if SAP05 fails to additionally bind and degrade GATA and SPL transcription factors, since the un-wanted degradation of GATAs and SPLs would likely interfere with flowering and associated production of fruit. As described herein, we have identified a mutation that maintains SAP05 interactions with vWA, but prevents binding to SPLs and GATAs. Secondly, a modified SAP05 protein that does not bind to GATA and SPL transcription factors is highly desirable for use in animals, and in particular humans, since unmodified SAP05 may also be able to bind non-plant (e.g. animal or human) GATAs. Advantageously therefore, use of the modified SAP05 of the invention in methods of human protein degradation also prevents any adverse target effects on GATA controlled genes.

[0077] The present invention can also be used to selectively degrade target proteins in host cells, tissues or whole organisms. Give, as shown in Figure 5, that SAP05 does not affect the ability of the 26S proteasome to degrade substrates, this effector appears to be an ideal chassis for engineering a new type of degraders that do not depend on E3 ligases. This is important because, while PROTACs, which are promising small molecules that recruit E3 ligases for ubiquitination of targets for degradation, their dependence on recruiting E3 ligases have causes several clinical issues with side effects, resistance and so on. Alternative systems are therefore needed.

[0078] By way of example, we have demonstrated that SAP05 may be engineered to degrade GFP by conjugation of an anti-GFP antibody (Figure 12). By conjugating a targetspecific binding moiety, SAP05 was able to mediate targeted degradation of GFP independent of ubiquitination.

[0079] Accordingly, in a first aspect of the invention, there is provided a modified secreted AY- WB protein 5 (SAP05) comprising at least one mutation, wherein SAP05 comprises a plurality of loop sequences, and wherein the at least one mutation is within at least one loop sequence and wherein the at least one mutation reduces or abolishes binding of SAP05 to at least one SQUAMOSA promoter binding protein-like protein (SPL) and / or at least one GATA transcription factor.

[0080] In one embodiment, the at least one mutation does not affect binding of SAP05 to a vWa domain.

[0081] Binding of the modified SAP05 protein to at least one SQUAMOSA promoter binding protein-like protein (SPL) and / or at least one GATA transcription factor and / or to a vWa domain can be determined using routine binding assays in the art, such as the yeast two hybrid assay, which is described in detail below.

[0082] In one embodiment, binding of the modified SAP05 protein to at least one SQUAMOSA promoter binding protein-like protein (SPL) and / or at least one GATA transcription factor is reduced by at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,

[0083] 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

[0084] 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,

[0085] 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

[0086] 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

[0087] 95%, 96%, 97%, 98%, or at least 99% or more compared to the level of binding of nonmodified SAP05 protein to at least one SQUAMOSA promoter binding protein-like protein (SPL) and / or at least one GATA transcription factor.

[0088] In one embodiment, the at least one mutation prevents or abolishes binding of SAP05 to the zinc-finger domain of SPL or GATA transcription factors.

[0089] In one embodiment, the GATA transcription factor is a plant GATA transcription factor.

[0090] In another embodiment, the GATA transcription factor is a non-plant GATA transcription factor, for example an animal GATA transcription factor, preferably a human GATA transcription factor, or an insect GATA transcription factor.

[0091] By “loop sequence” is meant a generally pattern-less region of amino acid sequence that connects two secondary structures, such as beta-strands and / or alpha-helices. As shown in Figure 1 , the 12.3 kDa SAP05 protein comprises a globular compact structure with five anti-parallel p-strands that form an internal p-sheet core (Fig. 1A, B). p-strand 1 (pi) locates on one of the surfaces and is connected via a loop-helix-loop-helix-loop structure that runs to the opposite surface to p2. This p-strand is connected to p3 and P4 via loop structures that remain on the same side of the protein. p4 then connects via a loop-helix-loop structure that runs to the opposite surface to p5, which runs antiparallel to pi (Fig. 1A). Both the SAP05 - ZnF_SPL5 and SAP05 - vWA_Rpn10 structures comprise 1:1 complexes (Fig. 1C, D). The SAP05 residues binding ZnF and vWA locate at opposite surfaces of SAP05 with the ZnF binding surface comprising predominantly loops (loop surface) and the vWA-binding surface being more structured with p-sheets, a-helices and loops (sheet surface) (Fig. 1B, E).

[0092] Accordingly, in one embodiment, the mutation of one or more loop of the ‘loop surface’ of a SAP05 alters the binding specificity of a ZNF-binding domain therein.

[0093] Accordingly, in one embodiment, the SAP05 comprises a plurality of loop sequences and the at least one mutation is within at least one loop sequence. By loop sequence is meant, a loop sequence preferably of the ZnF binding surface (i.e. the loop surface or transcription factor binding face), and / or optionally the vWA-binding surface (i.e. the sheet surface).

[0094] In a further embodiment, the loop sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten loop sequences. In one embodiment, SAP05 comprises eight loop sequences. In a further embodiment, the at least one mutation is in at least L4 (loop sequence 4). In another embodiment, the at least one mutation is in at least L5 (loop sequence 5). In another embodiment, the at least one mutation is in at least L7 (loop sequence 7). In another embodiment, the at least one mutation is in L4 and / or L5 or within L5 and L7 or within L4, L5 and L7.

[0095] In one embodiment, the sequence of L1 comprises or consists of the following sequence or a variant or fragment thereof.

[0096] EEFVGDM (SEQ ID NO: 29)

[0097] In another embodiment, the sequence of L2 comprises or consists of the following sequence or a variant thereof. SN (SEQ ID NO: 30)

[0098] In another embodiment, the sequence of L3 comprises or consists of the following sequence or a variant thereof.

[0099] HE (SEQ ID NO: 31)

[0100] In another embodiment, the sequence of L4 comprises or consists of the following sequence or a variant thereof.

[0101] FDFTLTGP (SEQ ID NO: 32)

[0102] In another embodiment, the sequence of L5 comprises or consists of the following sequence or a variant thereof.

[0103] GNIAEFAM (SEQ ID NO: 33)

[0104] In another embodiment, the sequence of L6 comprises or consists of the following sequence or a variant thereof.

[0105] KNPPLNL (SEQ ID NO: 34)

[0106] In another embodiment, the sequence of L7 comprises or consists of the following sequence or a variant thereof.

[0107] DDGTRDDEDDKY (SEQ ID NO: 35)

[0108] In another embodiment, the sequence of L8 comprises or consists of the following sequence or a variant thereof.

[0109] IGNGAK (SEQ ID NO: 36)

[0110] As used in any aspect of the invention described herein a “variant” or a “functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,

[0111] 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,

[0112] 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,

[0113] 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

[0114] 96%, 97%, 98%, or at least 99% overall sequence identity to any of the above amino acid sequences.

[0115] In one embodiment, the mutation is the deletion or substitution of one more loop sequences (in its entirety). For example, the mutation is the deletion or substitution of L4, for example as defined above. Alternatively, the mutation is the deletion or substitution of L5, for example as defined above. Alternatively, the mutation is the deletion or substitution of L7, for example as defined above. In a further alternative, the mutation is the deletion or substitution of L4 and / or L5 and / or L7.

[0116] In an alternative embodiment, the mutation is the deletion of one or more residues within the loop sequences.

[0117] In a further alternative embodiment, the substitution of one or more residues within the loop sequences. For example, the mutation is the substitution of one or more residues in L4, for example as defined above. Alternatively, the substitution of one or more residues in L5, for example as defined above. Alternatively, the mutation is the substitution of one or more residues in L7, for example as defined above. In a further alternative, the mutation is the substitution of one or more residues in L4 and / or L5 and / or L7.

[0118] The substitution is preferably the substitution of one or more negatively charged residues for positively charged residues or neutral residues. Alternatively, the substitution is one or more negatively charged residues for positively charged residues or neutral residues. Alternatively, the substitution is one or more neutral residues for a charged residue, such as a positively charged residue or a negatively charged reside.

[0119] Positively charged amino acids are Lysine (K), Arginine (R) and Histidine (H).

[0120] Negatively charged amino acids are: Aspartic Acid (D) and Glutamic acid (E). Neutral amino acids are: Glycine (G), Alanine (A), Valine (V), Cysteine (C), Proline (P), Leucine (L), Isoleucine (I), Methionine (M), Tryptophan (W), Phenylalanine (F), Serine (S), Threonine (T), Tyrosine (Y), Asparagine (N) and Glutamine (Q).

[0121] As shown in the Examples (Figure 1 and 7), the mutation may be selected from one or more of the following:

[0122] - A substitution at position 66 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. In one embodiment, the substitution is a D for a neutral amino acid. In one embodiment the neutral amino acid is A. and / or

[0123] - A substitution at position 77 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. In one embodiment, the substitution is a N for a charged amino acid. In one embodiment the charged amino acid is R. Alternatively, the charged amino acid is D. and / or

[0124] - A substitution at position 80 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. In one embodiment, the substitution is an E for a positively charged amino acid or a neutral amino acid. In one embodiment the positively charged amino acid is R. In another embodiment, the neutral amino acid is A. and / or

[0125] - A substitution at position 104 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. In one embodiment, the substitution is a R for a neutral amino acid or a negatively charged amino acid. and / or - A substitution at position 106 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. In one embodiment, the substitution is a D for a neutral amino acid. In one embodiment the neutral amino acid is A. Alternatively, the substitution is D for a positively charged amino acid. In one embodiment, the positively charged amino acid is R.

[0126] In one embodiment, the mutation is a substitution at position 66 of SEQ ID NO: 1 or a corresponding position in a homologous sequence. In one embodiment, the substitution is a D for a neutral amino acid. In one embodiment the neutral amino acid is A.

[0127] In one embodiment, the modified SAP05 protein has a sequence selected from SEQ ID NO: 25, 26, 27 and 28 or a functional variant thereof.

[0128] In another embodiment, the homologous sequence of SEQ ID NO: 1 may be selected from one or more of the following sequences: SEQ ID NO: 7, 9, 11 , 12, 14 and 16.

[0129] In another aspect of the invention, there is provided a fusion compound comprising an Rpn10 binding moiety and a target-binding moiety. In one embodiment, the Rpb10 targeting moiety is a vWA-domain binding moiety.

[0130] By By “vWA binding moiety” is meant any structure capable of binding to a vWA domain for example, as shown in SEQ ID NO: 18 or a homologue thereof, or portion or fragment of the vWA domain, and in particular a portion of fragment that is not involved in binding to the proteasome. Such a targeting or binding moiety may include peptides, cyclic peptides, binding domains, small molecules / chemicals, t-cell receptors, antibodies, functional fragments of an antibody, nanobodies, monobodies, macrocycleor aptamers. DARPins, affibodies, adnectins, affimers, anticalins or aptamers. Preferably the vWA binding moiety binds the vWA domain at a position and / or at a level of binding (i.e. binding affinity) that is sufficient to permit delivery of a target protein to the proteasome for degradation.

[0131] In one embodiment, the vWA binding moiety binds to one or more of the following amino acid positions: Position 71 (preferably K71) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0132] Position 27 (preferably Q27) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0133] Position 77 (preferably H77) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0134] Position 31 (preferably E31) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0135] Position 34 (preferably N34) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0136] Position 42 (preferably Q42) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0137] Position 68 (preferably D68) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0138] Position 70 (preferably G70) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0139] Position 24 (preferably S24) on SEQ ID NO: 5 or a corresponding position in a homologous sequence.

[0140] In one embodiment, the vWA binding moiety is a modified SAP05 peptide as described above.

[0141] By “target-binding moiety” is meant any structure capable of binding to a target protein or a portion or fragment of a target protein. This includes peptides, cyclic peptides, binding domains, small molecules / chemicals, t-cell receptors, antibodies, functional fragment of an antibody, nanobodies, monobodies, (peptide) macrocycles, DARPins, affibodies, adnectins, affimers, anticalins or aptamers. Such target-binding moieties are routinely diversified and screened using phage display, yeast display, mRNA display or ribosome display, to design specific high affinity moieties. Additionally, protein-protein interaction domains can be designed and optimised using computational models, such as machine learning models. For example, using the open source and freely accessing RF Diffusion allows for the design and optimisation of target-binding moieties. Accordingly, computer-generated proteins or fragments are include within the scope of, “target-binding moiety”. Use of these computational approaches are now only possible due to our work on the SAP05 crystal structure. In one embodiment, the target-binding moiety is a modified PROTAC, preferably wherein the E3 ligase portion of the PROTAC is replaced with SAP05. A particular advantage of this fusion compound is that neither SAP05 nor PROTAC is degraded with the target protein and thus the fusion compound can be recycled for further cycles of target protein degradation.

[0142] In a further embodiment, the fusion compound may further comprise a RPN10 protein or a VWA domain. In an alternative embodiment of the methods and uses described herein a fusion compound comprising a vWA targeting moiety and a target-binding moiety may be administered in combination with a (separate) RPN10 peptide or vWA domain peptide. Preferably, RPN10 or vWA is administered concurrently with the fusion compound or SAP05 protein. In one embodiment, RPN10 or vWA is selected from one of the following sequences.

[0143] In one embodiment, the RPN10 is a non-plant RPN10, for example an insect RPN10, an animal RPN10 or a human RPN10.

[0144] In one embodiment, the human RPN10 may comprise a sequence selected from SEQ ID NO: 21 to 24 or a functional variant or homologue thereof

[0145] Alternatively, a non-plant RPN10, such as a human RPN10 (e.g. those defined in SEQ ID NO: 21 and 23), can be modified to comprise one or more of the following mutations:

[0146] Mutation at position 71 (preferably K71) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0147] Mutation at position 27 (preferably Q27) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0148] Mutation at position 77 (preferably H77) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0149] Mutation at position 31 (preferably E31) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0150] Mutation at position 34 (preferably N34) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or Mutation at position 42 (preferably Q42) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0151] Mutation at position (preferably D68) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0152] Mutation at position (preferably G70) on SEQ ID NO: 5 or a corresponding position in a homologous sequence; and / or

[0153] Mutation at position (preferably S24) on SEQ ID NO: 5 or a corresponding position in a homologous sequence.

[0154] .By “fusion compound” is meant a compound, often but not exclusively a protein, that comprises portions (for example, subunits, motifs or domains) from two or more proteins or compounds (i.e. small molecules) or a combination of a protein and a small molecule. As described above, a fusion compound comprising a target-binding moiety and a vWA binding moiety. For example, a fusion compound may comprise a SAP05 protein and an immunoglobulin domain. Alternatively, the fusion compound may comprise a bispecific antibody or nanobody (i.e. an antibody with two binding specificities), which can recognise both a vWA domain, or portion thereof and a target protein. The two or more proteins are typically heterologous proteins, but in some circumstances a fusion compound may comprise multiple portions from the same protein. A fusion compound may also comprise a monobody, a synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold. A fusion compound may comprise an engineerable protein-protein binder for example a (peptide) macrocycle or a synthetic computer-generated protein. A fusion compound may be encoded by a nucleic acid generated through the joining of two or more genes or motifs or domains from genes that originally coded for separate proteins. Fusion compounds are also known as chimeric proteins.

[0155] Suitable homologues, as well as corresponding positions in homologous sequences referred to above, can be identified by sequence comparisons and identification of conserved domains using databases such as NCBI and alignment programmes known to the skilled person.

[0156] In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known amino acid sequence is used as a probe that selectively hybridizes to other corresponding amino acid sequences present in a population of cloned amino acid sequences from a chosen organism. The hybridization probes may be labelled with a detectable group, or any other detectable marker.

[0157] In some embodiments, the protein further comprises a linker, linking the vWA targeting moiety and target-binding moiety and / or linking the vWA targeting moiety and the vWA domain. The linker may be cleavable or non-cleavable. An example of a linker is a GS linker.

[0158] In further embodiments, the fusion compound is for use as a medicament. By “medicament” is meant any substance used for medical treatment.

[0159] In another aspect of the invention, there is provided an expression vector comprising a nucleic acid encoding the modified SAP05 of the invention. In one embodiment, the nucleic acid sequence encodes an amino acid sequence as defined in SEQ ID NO: 25 to 28 or a functional variant thereof. In a preferred embodiment, the nucleic acid sequence encoding the modified SAP05 of the invention is operably linked to a regulatory sequence such as a promoter, as described below.

[0160] Suitable vectors for the expression of the fusion compound can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. RNA delivery may also be appropriate, for example via lipid nanoparticles. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. Vectors may comprise an inducible promoter, so that the vector (and therefore the fusion compound, and in some embodiments the RPN10 protein in some cases) is expressed only in certain conditions. This can allow for the vector only to be expressed in certain cells or tissues. For example, inducible promoters can be controlled by chemicals or environmental stimuli. Vectors may alternatively comprise a tissue specific promoter so that the expression occurs in specific cell or tissue types.

[0161] In some embodiments, the expression vector further comprises a nucleic acid encoding a plant RPN10 protein or vWA domain or a fragment thereof, preferably wherein the nucleic acid comprises the nucleic acid sequence as defined in SEQ ID NO: 7 or 18 or a functional variant thereof. In a preferred embodiment, the RPN10 fragment comprises or consists of the vWA domain. Accordingly, in one embodiment, the expression vector further comprises a nucleic acid encoding a vWA domain as defined in SEQ ID NO: 18 or a functional variant thereof. In an alternative embodiment of the methods and uses described herein, RPN10 or vWA are expressed from a second separate expression construct to the SAP05 expression vector.

[0162] C-expression of an expression vector expressing the fusion compound of the invention and RPN10 or a RPN10 fragment as described above (either in the same or different constructs) is particularly helpful in applications using non-plant cells or organisms, for example, such as animals, including humans. Accordingly, in one embodiment, there is provided a method of targeted protein degradation in humans and animals. For example, as shown in Figure 12, co-expression of SAP05 and a vWA domain can be used to degrade proteins through the human 26S proteasome. Having to deliver plant RPN10 together with SAP05 may increase the safety of any therapeutic application if leaky production of SAP05 in non-target tissues has lethal consequences. As a result, having to deliver ‘plant’ RPN10, or a modified non-plant RPN10 (e.g. a human RPN10, such as defined in SEQ ID NO: 21 or 23) with changes in amino acids detailed above will allow for engineering higher specificity.

[0163] Our work will also enable the engineering of modified SAP05 proteins capable of binding directly to PSMD4 (human Rpn10). Whilst L3 (loop 3) of SAP05 may clash with H38 and S40 of PSMD4 to preclude a SAP05-PSMD4 interaction without a vWA domain, our work has identified the regions and residues that are the key determinants of SAP05 interactions with alternative RpnIOs. Based on the data from our crystal structure experiments, we have identified that the region of (approx, residue) 40 to 70 and positions 120 to 135 of SAP05 is important for mediating the interaction with Rpn10. By mutating these key residues, the natural preclusion of SAP05 and alternative RpnIOs, including those of humans and other animals, can be achieved. Particularly, it is understood that the residues R43 to F61 and another region from N125 to Y132 in A.thaliana will be particularly important in achieving the interaction. Specifically, there are two regions important is mediating the interaction with alternative RpnIOs. The key resid ues / targets for mutation within these regions are as follows:

[0164] Region 1: R43, I44, V45, N46, V47, N48, L49, S50, N51, I52, D53, I54, L55, K56, K57, H58, E59, T60, F61 , K62, K63, Y64

[0165] Region 2: K124, N125, M126, Y127, 1128, F129, W130, Q131 , Y132

[0166] These mutation positions are shown in Figure 1.

[0167] Of course, corresponding residues in alternative or variant Phytoplasma SAP05’s or proteins capable of binding the vWA domain will also be able to be mutated to facilitate interactions with alternative RpnIOs.

[0168] Accordingly, in another aspect of the invention, a SAP05 protein (e.g. those defined in SEQ ID NO: 1, 7, 9, 11, 12, 14 or 16), can be alternatively or additionally modified to comprise one or more mutations within the region of position 40 to 70 and / or positions 120 to 135 of the amino acid sequence. The modification allows binding to a non-plant vWA domain.

[0169] As described herein, binding to a non-plant vWA domain can be determined using any known technique in the art, including but not limited to, yeast two hybrid assays (the method of performing which is described below), other alternative assays In another embodiment the mutants may be introduced into a yeast line that expresses human Rpn10 (or derivative) and a ZnF-fused enzyme, which converts a component in the yeast growth medium into a compound that is toxic to yeast. The yeast line will be transformed with the SAP05 mutant library or targeted SAP05 mutants. If a SAP05 variant degrades the enzyme, the yeast will survive. A similar method is used to remove plasmids from yeast lines.

[0170] In one embodiment the non-plant vWA domain is a human vWA domain, for example as described in SEQ ID NO: 21 or 23 or a functional fragment thereof. Alternatively, or additionally, the mutation may comprise at least one mutation in the ‘sheet surface’ of SAP05. As shown in Figure 1 , the sheet surface is more structured with p-sheets, a-helices and loops. Accordingly, in one embodiment, the mutation may comprise at least one mutation in at least one p-sheet of the sheet surface and / or a- helices and / or a loop of the sheet surface.

[0171] In one embodiment, the sheet surface comprises five p-sheets, namely pi, p2, p3, p4 and p5. In one embodiment, the at least one mutation is within at least one of the p- sheets of the sheet surface.

[0172] In one embodiment, the sequence of pi comprises or consists of the following sequence or a variant or fragment thereof:

[0173] RIVNVNL (SEQ ID NO: 43)

[0174] In one embodiment, the sequence of p2 comprises or consists of the following sequence or a variant or fragment thereof.

[0175] RYN

[0176] In one embodiment, the sequence of p3 comprises or consists of the following sequence or a variant or fragment thereof.

[0177] IWKI (SEQ ID NO: 44)

[0178] In one embodiment, the sequence of p4 comprises or consists of the following sequence or a variant or fragment thereof.

[0179] LGVFF (SEQ ID NO: 45)

[0180] In one embodiment, the sequence of p5 comprises or consists of the following sequence or a variant or fragment thereof.

[0181] NMYIFWQY (SEQ ID NO: 46) In one embodiment, the at least one mutation of the sheet surface alters the binding specificity and / or affinity of SAP05 for a vWA-domain.

[0182] In an even more preferred embodiment, a SAP05 protein can be modified to comprise one or more mutations in the region of 43 to 61 and / or 125 to 132 of SEQ ID NO: 1.

[0183] In a further embodiment, the SAP05 protein can be modified to comprise one or more mutations at the following amino acid positions:

[0184] Position 43 (preferably R43) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation from an R to an A or E; and / or

[0185] Position 44 (preferably I44) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0186] Position 45 (preferably V45) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0187] Position 46 (preferably N46) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0188] Position 47 (preferably V47) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0189] Position 48 (preferably N48) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0190] Position 49 (preferably I44) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0191] Position 50 (preferably S50) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation from an S to an A; and / or

[0192] Position 51 (preferably N51) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0193] Position 52 (preferably I52) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0194] Position 53 (preferably D53) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or Position 54 (preferably I54) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0195] Position 55 (preferably L55) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0196] Position 56 (preferably K56) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0197] Position 57 (preferably K57) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0198] Position 58 (preferably H58) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation from an H to an A or W; and / or

[0199] Position 59 (preferably E59) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0200] Position 60 (preferably T60) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation from a T to a W; and / or

[0201] Position 61 (preferably F61) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0202] Position 62 (preferably K62) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0203] Position 63 (preferably K63) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or

[0204] Position 64 (preferably Y64) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation.

[0205] Additionally, or alternatively, in one embodiment, the SAP05 protein can be modified to comprise one or more mutations at the following amino acid positions:

[0206] Position 124 (preferably K124) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation;

[0207] Position 125 (preferably N125) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation from a N to an A or D; and / or

[0208] Position 126 (preferably M126) of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and is preferably a substitution mutation; and / or Position 127 (preferably Y127) of SEQ ID NO: 1 or a corresponding position in a homologous sequence and is preferably a substitution mutation from a Y to a W; and / or

[0209] Position 128 (preferably 1128) of SEQ ID NO: 1 or a corresponding position in a homologous sequence and is preferably a substitution mutation; and / or

[0210] Position 129 (preferably F129) of SEQ ID NO: 1 or a corresponding position in a homologous sequence and is preferably a substitution mutation; and / or

[0211] Position 130 (preferably W130) of SEQ ID NO: 1 or a corresponding position in a homologous sequence and is preferably a substitution mutation; and / or Position 131 (preferably Q131) of SEQ ID NO: 1 or a corresponding position in a homologous sequence and is preferably a substitution mutation; and / or Position 132 (preferably Y132) on SEQ ID NO: 1 or a corresponding position in a homologous sequence and is preferably a substitution mutation.

[0212] As described in Figure 7D, these are the residues that have been identified to interact at the interface between SAP05 and vWA_Rpn10.

[0213] The substitution is preferably the substitution of one or more negatively charged residues for positively charged residues or neutral residues. Alternatively, the substitution is one or more negatively charged residues for positively charged residues or neutral residues. Alternatively, the substitution is one or more neutral residues for a charged residue, such as a positively charged residue or a negatively charged reside.

[0214] Accordingly, as described herein a SAP05 protein (e.g. those defined in SEQ ID NO: 1, 7, 9, 11 , 12, 14 and 16), can be modified to comprise one or more mutations of amino acid residue(s) that allow interaction or binding with the vWA region of a human or animal Rpn10. In an additional or alternative embodiment, a SAP05 protein can be modified to comprise one or mutations that reduces or abolishes the binding of SAP05 to a GATA or SPL transcription factor.

[0215] In another aspect of the invention, there is provided a fusion compound comprising the above-described modified SAP05 and a target-binding moiety.

[0216] Our work has also identified that when the ternary complex is accommodated on the

[0217] 26S proteasome, the interaction of SPL5 with the Rpt4 and Rpt5 coiled-coil dimer could potentially instigate the switch of the proteasome to the active stage, leading to the degradation of the transcription factor. This finding indicates that any mutation within SAP05 or a RPN10 will benefit from any effect that increases association with the coiled-coil domains of Rpt4 and Rpt5, for it is these coiled coil domains that are responsible for physically linking substrate recruitment and processing to the unfolding machinery. Accordingly, this invention has identified that mutation which result in an increased association with the coiled-coil domains, i.e. the a-helices of Rpt4 and Rpt5, show enhanced degradation.

[0218] Accordingly, in one embodiment, the modified SAP05 of the invention additionally or alternatively comprises at least one mutation that increases the association with or localisation to the region comprising the Rpt4 and Rpt5 coiled-coil domains of the 26S proteasome. Again, association with or localisation to the Rpt4 and Rpt5 coiled-coil domains of the 26S proteasome can be determined using known binding assays, as described above (e.g. the yeast two hybrid assay).

[0219] In a preferred embodiment of any aspect of the invention, the plant RPN10 protein is derived from Arabidopsis thaliana or the protein comprises the amino acid sequence of SEQ ID NO: 5. In a preferred embodiment of any aspect of the invention, the plant vWA domain is derived from Arabidopsis thaliana or the protein comprises the amino acid sequence of SEQ ID NO: 18.

[0220] To allow two proteins to be expressed as individual proteins from a single mRNA molecule, ribosomal skipping sequences may be added to the 5’ and / or 3’ end of the nucleic acid encoding the fusion compound and / or nucleic acid encoding the plant RPN10 or vWA protein. During translation, when the ribosome encounters a ribosomal skipping sequence it is prevented from creating the peptide bond with the last proline in the ribosomal skipping sequence. As a result, translation is stopped, the nascent polypeptide released and translation is re-initiated to produce a second polypeptide. This results in the addition of a C-terminal ribosomal skipping sequence (or the majority of such a sequence) to the first polypeptide chain, and a N-terminal proline to the next polypeptide.

[0221] Accordingly, in a further embodiment, the nucleic acid construct comprises at least one ribosomal skipping sequence. In one example, the ribosomal skipping sequence may be selected from one of the following:

[0222] F2A; A 2A DNA sequence variant used between two CDS.

[0223] F2A: GGACAACTTCTCAACTTTGACTTGCTAAAGTTAGCTGGTGATGTTGAATCTAA TCCTGGACCA (SEQ ID NO: 37).

[0224] Use of the F2A sequence results in the addition of the F2Aaa1-20 polypeptide sequence to the C-terminus of the protein upstream of the ribosomal skipping site and a proline residue (F2Aaa21) to the downstream protein.

[0225] F2Aaa1-20: GQLLNFDLLKLAGDVESNPG (SEQ ID NO: 38) F2Aaa21: P

[0226] F2A30; A 2A DNA sequence variant used between two CDS.

[0227] F2A30: CACAAACAGAAAATTGTGGCACCGGTGAAGCAGACTCTCAACTTTGACTT GCTAAAGTTAGCTGGTGATGTTGAATCTAATCCTGGACCA (SEQ ID NO: 39).

[0228] Use of the F2A30 sequence results in the addition of the F2A30aa1-29 polypeptide sequence to the C-terminus of the protein upstream of the ribosomal skipping site and a proline residue (F2A30aa30) to the downstream protein.

[0229] F2Aaa1-20: HKQKIVAPVKQTLNFDLLKLAGDVESNPG (SEQ ID NO: 40) F2Aaa21: P

[0230] In one embodiment, the nucleic acid encoding the fusion compound includes a C- terminal skipping sequence, preferably F2A30(aa1-29).

[0231] In a further embodiment, the nucleic acid encoding the plant RPN10 or vWA domain includes a N-terminal skipping sequence and F2A30(aa30), i.e. a proline amino acid residue.

[0232] In a further alternative embodiment, an internal ribosomal entry site (IRES), tRNA sequence, a ribozyme (such as a Hammerhead (HH) ribozyme unit and / or a hepatitis delta virus (HDV) ribozyme unit) or direct repeat (DR) sequence could be used instead of a ribosomal skipping sequence. Again, such sequences may be added to the 5’ and / or 3’ end of the nucleic acid encoding the fusion compound and / or the nucleic acid encoding the plant RPN10 or vWA protein and allows two proteins to be expressed as individual proteins from a single mRNA transcript and from a single regulatory sequence (promoter).

[0233] In a further aspect of the invention, there is provided a cell comprising the fusion compound or the expression vector described above. The cell is preferably a eukaryotic cell. Preferably the cell is a mammalian or plant cell. In some embodiments the cell may be a fungal or yeast cell.

[0234] In another aspect of the invention, there is provided a transgenic organism expressing the expression vector or comprising the cell described above, wherein the organism is not a human. The organism is preferably eukaryotic. The organism may be a mammal (but not a human) or a plant. Where the organism is a plant, the invention also covers seeds obtained or obtainable from the plant, as well as progeny obtained from the seed, and further generations of progeny. Preferably the seed and progeny comprise and express the expression vector. Where the organism is a mammal, the mammal may be for example a rodent (such as a mouse, rat), a lagomorph (such as a rabbit), a primate (such as a monkey for instance Macaca spp). In other embodiments the mammal may be a bovid, caprid, ovid, porcid. In other embodiments the organism may be avian; for example, a galloanserine such as chicken, duck. In one embodiment, the expression vector is stably incorporated into the organism’s genome.

[0235] In another aspect of the invention, there is provided a genetically altered organism, wherein the organism comprises and expresses the expression vector or the fusion compound of the present invention. In one embodiment, the expression vector or the nucleic acid encoding the fusion compound is stably incorporated into the genome. This may be achieved using genome editing techniques such as CRISPR. In one embodiment the organism is not a human.

[0236] In a further aspect of the invention, there is provided the use of the fusion compound or expression construct of the invention in targeted protein degradation. By “targeted protein degradation” is meant reduction or elimination of levels of a target protein. Reduction may mean a decrease of at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the protein level before use of the fusion compound. Elimination means that the protein level is reduced to an undetectable level.

[0237] In some embodiments, the target protein degradation is ubiquitin-independent protein degradation, meaning that the protein degradation occurs without the need for ubiquitination.

[0238] In another aspect of the invention, there is provided a method of targeted protein degradation, the method comprising applying or administering the fusion compound or the expression vector described above to an organism or to a sample. In an alternative embodiment, the method comprises applying a modified SAP05 protein as described above (or another vWA targeting moiety, as described above) and a vWA protein as described above to an organism or to a sample. In a further aspect of the invention, there is provided a method of controlling the (protein) level of a target protein, the method comprising applying the fusion compound described above or the expression vector described above to an organism or to a sample. In another alternative embodiment, there is provided a method of controlling the (protein) level of a target protein, the method comprising a modified SAP05 protein as described above (or another vWA targeting moiety) and a vWA protein as described above to an organism or to a sample.

[0239] The sample can be any form of sample that contains the target protein, for example, it may be a cell culture or a cellular sample derived from an organism or tissue. For example, where the organism is a plant, the sample may be a protoplast sample. In another example where the organism is a human, the sample may be a blood sample (or another other bodily sample). Applying or administering (such terms may be used interchangeably) may comprise simply adding the fusion compound or expression vector to the sample, but may also require one or more extra steps to deliver the fusion compound or expression vector into a cell. For example, it may be necessary to carry out methods such as electroporation, transfection, viral particle transfer (such as that disclosed in US 2012 / 0015899), or gene gun or RNA delivery (for example lipid nanoparticle) to cause uptake of the fusion compound or expression vector into a cell. Controlling may mean reducing the level of a target protein to a level found in a steady state or a healthy state. Controlling may also mean eliminating levels of a certain protein.

[0240] In one embodiment of any of the methods described above a fluorescently tagged antibody (or other target binding protein or fragment thereof) may be first applied to the sample, where the antibody is specific to the target protein. As such, application of the fluorescently tagged antibody labels the target protein. In this embodiment, the targetbinding moiety of the fusion compound is specific for the fluorescent tag. As a result, degradation of the labelled target protein by the modified SAP05 of the invention (or another vWA targeting moiety) in the sample can be detected by a decrease in fluorescent signal. This method allows a target protein to be detected and visualised using a fluorescently tagged antibody (e.g. with GFP), and then the degradation of the target protein to be detected and visualised using a fusion compound of the invention that specifically targets this fluorescent tag. Such a fusion compound can therefore be used to target any protein that is bound to a fluorescent antibody (or other targetbinding protein). In other words, a single fusion compound can be used in combination with any other tagged target-binding protein providing a valuable research tool. Suitable fluorescent tags include GFP, YFP, CFP, or BFP, and any other fluorescent tag known to the skilled person. Accordingly, in a further aspect of the invention, there is provided a kit comprising a tagged target binding protein (e.g. an antibody), preferably a fluorescently tagged antibody and a fusion compound as described above, wherein the target-binding moiety of the fusion compound is specific for the tag.

[0241] In some embodiments of any of the methods described above, the target protein is a cytosolic protein. By “cytosolic protein” is meant any protein that is present in the cytosol. Examples of cytosolic proteins include hormones, cytokines, signalling proteins, transcription factors, structural proteins, effector proteins and enzymes, but many other examples will be known by the skilled person. In another embodiment, the protein may be an antibody; for example, an autoantibody.

[0242] In some embodiments, the target protein is a nuclear protein, such as but not limited to histone proteins. We believe that, in general, SAP05 fusion compounds will be sufficiently small to migrate into the cell nuclei, and the 26S proteasome is also active within the nuclei. In alternative embodiments, the target protein is a cell surface protein. By “cell surface protein” is meant any protein that is present on the cell surface. Cell surface proteins often play a vital role in the communication between the cell and its environment. Examples of cell surface protein include receptors, transporters, channels, and celladhesion proteins, but many other examples will be known by the skilled person. Preferably, in this embodiment the modified SAP05 of the invention or fusion compound comprises a localisation signal that causes such a protein to be located to the endoplasmic reticulum and / or golgi apparatus. A suitable localisation signal includes KKXX or KDEL). Alternatively, the modified SAP05 of the invention or fusion compound may be able to mediate degradation of cell surface proteins through an interaction between RPN10 or vWA and the autophagosome (a component of the autophagy pathway).

[0243] In another embodiment, the target protein is a species-specific protein, the targeting of which for degradation by the methods or fusion compounds of the invention can be used as a pesticide including insecticides, herbicides, fungicides and nematicides. Examples include proteins involved in producing the chitin skeletons of insects, cytochrome P450 proteins that are involved in insecticide resistance and degradation of plant defence molecules, proteins involved in spore and appressorium development (and plant infection organelles) of fungi and oomycetes, specific KAI proteins to controls weeds (e.g. https: / / www.biorxiv.Org / content / 10.1101 / 376939v2) and proteins involved in mucus production of slugs.

[0244] In other embodiments, the target protein is an exogenous protein, i.e. a protein that has been introduced into a cell or organism that is not usually expressed by that cell or organism. In one example, this exogenous protein is a CRISPR enzyme, such as Cas or Cpf1. In one example, Cas nuclease from a CRISPR-Cas system that has been introduced into a cell or organism, such as Cas9, can be selectively or conditionally degraded by the fusion compound or expression vector of the invention to limit off- target effects and / or unwanted persistence of the CRISPR-Cas system once the desired gene editing has been obtained. In particular, where the expression vector of the invention is operably linked to an inducible or tissue-specific promoter, targeted degradation of the CRISPR enzyme can be controlled either spatially or temporally. In one example, this may be desirable where gene knock-out at the embryonic stage or at a certain developmental stage would be lethal. Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.

[0245] Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNA, the pre- crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre- crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick basepairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a doublestranded break within the protospacer. The specificity of the system can be programmed by selecting appropriate spacer sequences; for ease of use, the tracrRNA, spacer, and crRNA regions can be combined into a single RNA molecule, referred to as a single guide RNA or sgRNA.

[0246] One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted (Wiles et al., 2015).

[0247] Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.

[0248] The single guide RNA (sgRNA) is the second component of the CRISPR / Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3.

[0249] Accordingly, in a further aspect of the invention, there is provided a method of gene editing, the method comprising introducing a CRISPR-Cas system comprising a CRISPR enzyme to a cell or organism, allowing the CRISPR-Cas system to edit a gene, and delivering a fusion compound according to the invention comprising a target moiety that is specific for said CRISPR enzyme, and allowing the fusion compound to degrade the CRISPR enzyme so as to inhibit any further activity of the CRISPR-Cas system. The fusion compound may be delivered together with the CRISPR-Cas system, or separately; and may be delivered simultaneously or sequentially; depending on the desired timing of cleavage and degradation.

[0250] In other embodiments of any of the methods described above, the target protein can cause pathology in a target organism, and / or the target protein may be a drug target.

[0251] In another aspect of the invention, there is provided a method of modulating a biochemical or physiological response in an organism, the method comprising administering the fusion compound or the expression vector described above. Modulating may mean increasing or decreasing depending on the desired outcome. For example, there are situations where an increase in immune response is desired (e.g. to boost the immune response against cancer) or a decrease in immune response is desired (e.g. to limit the immune response during auto-immunity). In this example, it would be clear to the skilled person that a response can be increased by degrading an inhibitory protein, and a response can be decreased by degrading a stimulatory or effector protein. There may also be an interest in increasing or decreasing a physiological response in research to determine the effect of such a response in a cell, tissue or organism. In some embodiments, the physiological response is selected from a stress response, an immune response, a hormone response, chemical response or a light response.

[0252] A stress response is the response of a cell or organism to a stressor such as an environment condition. A stressor may be abiotic or biotic. An “abiotic stressor” includes drought, salinity, wind, high or low temperature or high light. A “biotic stressor” refers to harmful effects caused by another (living) organism such as by the secretion of a toxin or an effector. Accordingly, in one embodiment, the method increases the resistance of an organism to a stressor.

[0253] An immune response is the response of an organism to a foreign or harmful antigen. In some cases, it is desirable to increase the immune response, for example in the context of cancer or infection where there is a suboptimal immune response raised which causes pathology. In other cases, it is desirable to decrease immune response, for example in the context of autoimmunity or a cytokine storm, where self-recognition or and / or excessive responses by the immune system can cause inflammation and damage to the host. In certain embodiments, the target-binding moiety is specific against an autoantibody such as those raised in autoimmune conditions like rheumatoid arthritis, multiple sclerosis, type 1 diabetes, lupus, inflammatory bowel disease or psoriasis and related conditions, or in infections such as anti-IFN antibodies during SARS-CoV-2 infection.

[0254] A hormone response is the response of a cell or organism to a hormone, wherein the hormone may be secreted by another cell or organism and is sensed by the cell or organism of the invention. Hormones may be eicosanoids, steroids or amino acid / protein derivatives. Hormones may affect a variety of processes including digestion, metabolism, respiration, tissue function, sensory perception, sleep, excretion, lactation, stress induction, growth and development, movement, reproduction and mood.

[0255] A chemical response is the response of a cell or organism to a chemical, such as to a toxin or volatile compound. A light response is a response of a cell or organism to a light stimulus, and this may be a light stimulus of a specific wavelength. A light response includes phototropism, which is the growth of an organism in response to a light stimulus, and photoperiodism, which is the flowering or change in other development processes in response to the photoperiod (i.e. the length of time a light stimulus is received). A light response may cause an effect on circadian rhythm.

[0256] In a further aspect of the invention, there is provided a method of treating a condition in a patient in need thereof, the method comprising administering the fusion compound or the expression vector described above. The condition may be any condition that is characterised or caused by the expression or increased expression of a protein that causes pathology in the host organism.

[0257] In another aspect of the invention, there is provided the fusion compound or expression vector of the invention for use in the treatment of a condition that is characterised or caused by the expression or increased expression of a protein that causes pathology in the host organism. In a further aspect of the invention, there is provided the use of the fusion compound or expression vector of the invention in the manufacture of a medicament for the treatment of a condition that is characterised or caused by the expression or increased expression of a protein that causes pathology in the host organism.

[0258] As used herein, increased expression refers to a level of expression that is higher than the level of expression in a host organism that lacks the given pathology.

[0259] In one embodiment, the condition may be cancer, infection, a neurodegenerative disorder and / or a proteopathy. Alternatively, the condition may be an autoimmune disease or a metabolic bone disease.

[0260] The cancer may be a carcinoma, sarcoma, lymphoma, leukemia, germ cell tumour or blastoma. Examples of cancer include bone and muscle sarcomas, brain and nervous system cancers, breast cancers, endrocrine system cancers, eye cancer, gastrointestinal cancer, genitourinary and gynecologic cancers, head and neck cancers, hematopoietic cancers, skin cancers, thoracic and respiratory cancers. Other specific cancers will be known to the skilled person. Where the condition is a cancer, the binding moiety of the fusion compound may be specific for an oncogene protein, a growth factor, an extracellular matrix protein, a cytoskeleton protein, a cell cycle protein, a checkpoint protein, or a receptor.

[0261] Examples of infection include infection with viruses, viroids, bacteria, fungi, prions, parasites and arthropods. An infection may include an antibiotic-resistant bacterial infection, a hospital acquired infection, a superinfection, or a biofilm; which are all recognised as difficult to treat.

[0262] Examples of neurodegenerative disorders include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, fatal familial insomnia, and Huntington's disease.

[0263] Proteopathies are also known as protein conformational disorders or protein misfolding diseases, and cause disease due to proteins becoming structurally abnormal leading to toxicity or lack of normal function of the protein. Examples of proteopathies include: Creutzfeld-Jakob disease and other prion diseases, Alzhemier’s disease, Parkinson’s disease, amyloidosis, multiple system atrophy, cystic fibrosis, tauopathies and other aggregate-prone disorders, amyotrophic lateral sclerosis, type II diabetes, and sickle cell disease. Where the condition is a proteopathy, the binding moiety of the fusion compound may be specific for tau or beta-amyloid.

[0264] In some embodiments, the condition is characterised by increased expression or activity of a target protein, and the target-binding moiety is specific for said target protein.

[0265] In another aspect of the invention, there is provided a method of treating an infection caused by a microorganism, the method comprising administering the fusion compound or the expression vector described above, wherein the target binding moiety is specific for a protein expressed by a microorganism. By “microorganism” is meant a bacteria, archaea, fungi, protozoa, algae, nematodes or virus.

[0266] In some embodiments, the microorganism is pathogenic (i.e. is a pathogen). In certain embodiments, the target protein is a protein that is essential for the survival and / or replication of the microorganism, and therefore degrading this protein causes death or non-replication of the microorganism. In other embodiments, the target protein is an effector molecule expressed by the microorganism (i.e. a molecule that causes pathology). In further embodiments, this effector molecule is a microbial toxin, and preferably the method is treating an infection which has caused sepsis or toxic shock in a subject. Microbial toxins may be produced by gram-positive or gram-negative bacteria, or fungi, and examples include cholera toxin, diphtheria toxin, pertussis toxin, mycoplasma toxin, E.coli toxin, Shiga toxin, Pseudomonas exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin, Bordetella pertussis AC toxin, Bacillus anthracis EF toxin, S. aureus exofolitin B, perfringiolysin O, hemolysin, listeriolysin, alpha toxin, pneumolysin, streptolysin O, leucocidin and pyrogenic exotoxins.

[0267] In certain embodiments, a pathogen or pest excretes a toxin or an effector into the cytosol of cells. In this case, an expression vector comprising an inducible promoter which is only induced during infection with said pathogen drives expression of an expression vector containing a fusion compound according to the invention (and RPN10 or vWA in some cases) targeting a toxin or effector from said pathogen. Such pathogen inducible promoters include PPP1, hsr203J, gst1, PR1, PR5 promoters, and also includes synthetic pathogen inducible promoters. Therefore, specific fusion compound according to the invention (and optionally RPN10 or vWA) expression can be delivered only in cells and / or tissues affected by the pathogen.

[0268] In some embodiments, the pathogen or pest excretes a toxin or effector in a plant. Such pathogens or pests affecting include plant pathogenic bacteria like Pseudomonas, Xanthomonas, Ralstonia, Erwinia, Pantoea, Liberibacter, Xylella fastidiosa, Clavibacter, and Streptomyces', plant pathogenic fungi like Fusarium and Colletotrichunr, oomycetes such as Phytophthora infestans, Phytophthora palmivora, Phytophthora ramorum, Phytophthora capsica, Pythium, and Powdery mildews; Protozoa like Plasmodiophora brassicae, and Polymyxa betae nematodes like Potato cyst nematodes; pests like aphids, leafhoppers, psyllids, whiteflies, planthoppers, Phylloxera, mealybugs, sharpshooters and froghoppers.

[0269] In some embodiments, the pathogen or pest excretes a toxin or effector in a fish. Such pathogens or pests affecting fish include viruses like rhabdovirus, Esocid lymphosarcoma and Saprolegnia sp. In a further aspect of the invention, there is provided a method of increasing the immunogenicity of a protein, the method comprising administering the fusion compound or the expression vector described above, wherein the target-binding moiety is specific for the protein, and wherein proteasome degradation of the protein results in increased antigen presentation of peptides degraded from the protein.

[0270] Immunogenicity is the ability of an antigen to trigger an immune response in a host. Degradation of proteins into shorter peptides allows for presentation of these peptides by antigen-presenting immune cells to the rest of the immune system in order to trigger an immune response against the antigen. Accordingly, this method can be particularly useful in the context of cancer or infection, where a sub-optimal immune response occurs due to a lack of immunogenicity to a protein. For example, protein degradation of cancer cell surface markers may allow for an improved cytotoxic T cell response against cancer cells expressing this marker due to increased immune recognition. Therefore, in some embodiments, the fusion compound or the expression vector described above is administered to a subject suffering from an infection or from cancer.

[0271] In another aspect of the invention there is provided a method for creating a protein knockout model, the method comprising administering the fusion compound or the expression vector described above to a cell or an organism. Such models may be used to study protein function in development, homeostasis and / or disease. In some embodiments, the model is a disease model and the disease is caused or characterised by a dysfunction or absence of a target protein, and the target moiety is specific for the target protein.

[0272] In a further aspect of the invention, there is provided a method of identifying a degradation effect of a target protein in a biological system, the method comprising applying the fusion compound described above, or the expression vector described above to the biological system. The biological system may be a cell, a tissue or an organism. The target protein may have many downstream effects. In some embodiments, where the target protein is a transcription factor or a regulatory protein, and the method further comprises performing RNA sequencing. By “regulatory protein” is meant any protein that controls the rate of transcription of DNA to RNA through specific DNA binding, and includes transcription factors. RNA sequencing, also known as RNA-Seq, is a next-generation sequencing method that allows for measurement of RNA in a biological sample, known as the transcriptome. Methods for carrying out RNA sequencing are known to the skilled person and are well described in the art. RNA sequencing can be helpful in this context to identify the degradation effect of a target protein, particular of regulatory proteins, in a biological system, as RNA sequencing can be used before and after applying the fusion compound or expression vector described above and the results can be compared.

[0273] In another aspect of the invention, there is provided a method of increasing yield in a plant, the method comprising introducing the fusion compound or the expression vector described above to the plant, wherein the target-binding moiety is specific for a target protein the expression or activity of which is negatively correlated with yield. The plant may be selected from rice, maize, wheat, barley, sorghum, potato, tomato, cotton, soybean, Brassicas, such as B. napus, coconut, papaya, oil palms, grape, apple, oranges, sugarcane, citrus (such as lime, citrus, orange, grapefruit), egg plant, elm, ash, willow, elm, sesame, alfalfa, pea, plantain, birch, cassava, peanut, loofah, cocoa, date or date palm, sweet potato, lettuce, chrysanthemum, poinsettia, sunflower, phlox, hortensia, tulips, gladiolus and other bulbs, onion, garlic, cabbage tree, pine, trees, stone fruit trees, palm, carrot, strawberry, blueberry, cranberry and other berry plants. Alternatively, the plant may be a non-vascular plant, for example: liverworts (e.g. Marcanthia), mosses, hornworts, or verns. The term "plant" as used herein encompasses whole plants and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned carry at least one of the herein described mutations. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the mutations as described herein.

[0274] The invention also extends to harvestable parts of a plant of the invention as described herein, including but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. In another aspect of the invention, there is provided a product derived from a plant as described herein or from a part thereof.

[0275] The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and / or weight. The actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres.

[0276] Thus, according to the invention, yield comprises one or more of and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased viability / germination efficiency, increased number or size or weight of seeds or pods or beans or grain, increased growth or increased branching, for example inflorescences with more branches, increased biomass, increased fresh weight or grain fill. Preferably, increased yield comprises at least one of an increased number or weight of seeds, beans or pods per plant, increased thousand kernel weight (TKW), increased biomass, increased fresh weight and increased growth. Yield is increased relative to a control or wild-type plant. For example, the yield is increased by 2%, 3%, 4%, 5%-50% or more compared to a control plant, for example by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0277] In another aspect of the invention, there is provided a method of reducing or removing glutens in a plant, the method comprising introducing the fusion compound or the expression vector described above to the plant, wherein the target-binding moiety is specific for gliadin or glutenin. Gliadin is the water-soluble component of gluten and gliadin is the water-insoluble component of gluten. It is particularly advantageous to reduce or remove gliadins in a plant, as this is the principal toxic component of gluten for celiac patients.

[0278] Methods of the invention can also be used in fungi, for example to degrade a specific target protein which may be responsible for an unwanted effect arising from fungal growth, eg, food spoilage, infection, toxicity. In some embodiments of any of the methods described above, the targeted protein degradation is ubiquitin-independent protein degradation as described above.

[0279] In a further aspect of the invention, there is provided a pharmaceutical composition comprising the modified SAP05 of the invention, the fusion compound or the expression vector described above, and a pharmaceutically acceptable diluent, carrier or excipient. In some embodiments, the at least one pharmaceutically acceptable diluent, carrier or excipient is physiological saline, phosphate buffered saline (PBS) and / or sterile water. In some embodiments the pharmaceutical composition essentially consists of the fusion compound or the expression vector described above. In some embodiments, the pharmaceutical composition is for use in treatment of cancer, infection, a neurodegenerative disorder or proteopathy, as described above.

[0280] In another aspect of the invention there is provided a pharmaceutical composition for use in therapy. In another aspect of the invention there is provided a method of treating a condition, the method comprising administering the pharmaceutical composition of the invention to a patient in need thereof.

[0281] In another aspect of the invention, there is provided the pharmaceutical composition of the invention for use in the treatment of a condition that is characterised or caused by the expression or increased expression of a protein that causes pathology in a host organism. In a further aspect of the invention, there is provided the use of the pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a condition that is characterised or caused by the expression or increased expression of a protein that causes pathology in a host organism. In a further aspect of the invention there is provided a method of treating a condition that is characterised or caused by the expression or increased expression of a protein that causes pathology in a host organism, the method comprising administering the pharmaceutical composition of the invention to a patient in need thereof.

[0282] In one embodiment, the condition may be cancer, infection, a neurodegenerative disorder and / or a proteopathy. Alternatively, the condition may be an autoimmune disease or a metabolic bone disease. In another aspect of the invention there is provided a kit comprising the pharmaceutical composition of the invention and preferably, instructions for use.

[0283] In another aspect of the invention, there is provided a screening library comprising a plurality of fusion compounds as described above, wherein each fusion compound comprises a different target-binding moiety.

[0284] In some embodiments, each target-binding moiety is specific for a different protein. This may be particularly helpful in drug discovery, whereby a variety of targets can be evaluated as binding moieties can be screened against a disease model (e.g. a cancer cell line) to find fusion compounds containing target-binding moieties that are effective in a given disease. In some embodiments the disease model may be a model organism (eg, a mouse), or may be a transgenic or non-transgenic cell line (e.g. a transgenic yeast or other fungal cell line, or a transgenic mammalian cell line) which expresses a protein of interest for screening.

[0285] In other embodiments, each target-binding moiety is specific for the same protein, but for a different epitope or binding region on the protein. Therefore, this screening library can be used to find the most effective target-binding moiety against a given target. This may be particularly helpful where e.g. there is need to screen a variety of antibodies against a known disease protein to find a fusion compound comprising the most effective antibody against the known disease protein.

[0286] Therefore, in a related aspect of the invention, there is provided a method for screening target-binding moieties using the screening library described above.

[0287] While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0288] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0289] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0290] The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0291] The invention is now described in the following non-limiting example.

[0292] EXAMPLE 1 : Phytoplasma SAP05 interacts with the ZnF domain of SPL5 and the vWA domain of RPN10.

[0293] SAP05 forms a ternary complex with the ZnF domain of SPL5 (ZnF_SPL5) and vWA domain of RPN10 (vWA_RPN10). To investigate how SAP05 binds two larger proteins, we generated crystal structures ZnF_SPL5 and vWA_RPN10 complexes. We separately expressed constructs containing SAP05 residues 33 to 155 that correspond to the entire mature part of SAP05 (without signal peptide), residues 60 to 127 of Arabidopsis thaliana SPL5 (accession number AT3G15270, SEQ ID NO: 27) corresponding to the ZnF domain and residues 1 to 195 comprising the vWA domain of A. thaliana RPN10 (accession number AT4G38630, SEQ ID NO: 21) (Fig. 6) in Escherichia coli. The three proteins were successfully purified at high purity via 6* His tag immobilized metal-affinity chromatography (IMAC) followed by tags removal and gel filtration (Superdex 7526 / 60) (Fig. 6B).

[0294] Subsequently, the binding affinities of the interaction using isothermal titration calorimetry (ITC) were determined. Titration of SAP05 into a solution of ZnF_SPL5 showed an exothermic binding isotherm with a fitted dissociation equilibrium constant (Kd) of 0.51 pM and stoichiometry of 1:1 (Fig. 2C).

[0295] EXAMPLE 2: Crystal structures of the SAP05-ZnF and SAP05-vWA complexes.

[0296] The molecular bases of SAP05 and ZnF_SPL5 and SAP05 and vWA_RPN10 interactions were resolved by co-expressing the protein combinations in E. coli and purifying both complexes.

[0297] After crystallization screens and optimizations, we successfully obtained the crystals of two complexes We obtained crystals in multiple conditions and optimization of some conditions produced needle like crystals of the SAP05 - ZnF_SPL5 and SAP05 - vWA_Rpn10 mixtures. The structure of SAP05-ZnF complex was solved via the singlewavelength anomalous diffraction method due to the presence of zinc ions bound to ZnF. Subsequently, the structure of SAP05-VWA complex was solved using molecular replacement method using SAP05 as a template from the SAP05-ZnF structure.

[0298] Both the SAP05 - ZnF_SPL5 and SAP05 - vWA_RPN10 structures comprise 1:1 complexes (Fig. 1 C, D). The SAP05 residues binding ZnF and vWA locate at opposite surfaces of SAP05 with the ZnF binding surface comprising predominantly loops (loop surface) and the vWA-binding surface being more structured with a p-sheet, a-helices and loops (sheet surface) (Fig. 1 B, E).

[0299] Comparing the SAP05 structures between the SAP05 - ZnF_SPL5 and SAP05 - vWA_RPN10 complexes revealed no changes in SAP05 configuration between the complexes (Fig. 1C, D). Moreover, modeling of the ternary structure revealed that there is no steric hindrance of the ZnF and vWA domains indicating that ZnF and vWA have the capacity to bind SAP05 simultaneously to form a ternary complex (Fig. 1 F), consistent with the isolation of ternary complexes containing ZnF, SAP05 and vWA by gel filtration analyses (Fig. 6C). These data indicate that the SAP05 structure consists of distinct surface regions (multiple faces). One face of SAP05 mediates the interaction with ZnF and the other face with vWA (Fig. 1). These data provide evidence that SAP05 has a bimodular architecture with opposite loop and sheet surfaces that enable interactions with ZnF of TFs and vWA of Rpn10, thereby acting as a molecular glue to link SPL and GATA TFs to Rpn10.

[0300] EXAMPLE 3: Identification of SAP05 amino acids that mediate binding specificity to ZnF of SPLs and GAT As.

[0301] We further examined the binding surfaces of the SAP05 - ZnF_SPL5 interface and investigated the role of specific residues involved. The interaction of SAP05 and ZnF occurs at a binding interface involving predominantly charged and some polar residues in three loop structures of SAP05 that interact with oppositely charged or polar residues in alpha-helices of ZnF (Fig. 2B). Hence, the SAP05-ZnF complex is dominated by electrostatic interactions.

[0302] The SAP05 amino acids that interact with the ZnF_SPL5 include N77 and D106, which each bind three amino acids, and D66, G76, E80, and R104, which each bind one amino acid of the ZnF_SPL5 (Fig. 2A, 3A, 3B). These residues locate in the central region of the linear SAP05 sequence and position at loops region. We generated 10 structure-guided SAP05 mutants by replacing each residue with a neutral, similarly charged, or oppositely charged residue (Fig. 7). The binding of these mutants to SPLs and GATAs transcription factors and RPN10 was tested in yeast two-hybrid assays, and their ability to mediate the degradation of these transcription factors in N. benthamiana leaves was assessed (Fig. 2D, E, F; Fig 3D, 3F).

[0303] Mutations in SAP05 residues N77 (N77A, N77D, N77R), E80 (E80A, E80R), D106 (D106A, D106R), D66A retained binding to RPN10, but lost binding to one or both SPL11 and SPL15 (Fig. 30, Fig. 7). N77R, D106A, D106R, D66A and G76W mutants lost the ability to mediate degradation of SPL5 in N. benthamiana leaves, though G76W was not detected in leaves (Fig. 2D; Fig. 3D). However, N77A, N77D, E80A, and E80R mediated degradation of SPL5, indicating that these mutants still retained binding to SPLs. Yeast two-hybrid (Y2H) assays confirmed that these mutants lost the ability to bind SPL TFs, and except for G76W, retained their affinity for Rpn10 (Fig. 2E). G76W, N77R, D106A and D106R also retained the ability to bind GATA TFs unlike D66A that lost the ability to bind both SPLs and GATAs (Fig. 2E). SAP05 mutant D66A has a weak interaction with RPN10, and still mediated the degradation of SPL5, while mutant G76W lost interaction with RPN10, but did not mediate SPL5 degradation. The finding that SAP05 G76W lost binding to both SPLs and Rpn10 suggest that this mutant has more profound structural changes, in agreement with its instability in N. benthamiana leaves. Moreover, SAP05 D66A, retained binding to RPN10 and lost binding to both SPL and GATA transcription factors in yeast two-hybrid assays (Fig. 30). Consistent with these binding activities, D66A did not mediate degradation of SPL5 or GATA19 in leaves, whereas D66E and N77R, which binds to GATA19 in yeast two-hybrid assays, mediated GATA19 degradation. As discussed above, a mutated SAP05 that has reduced or abolished SPL and GATA transcription factor binding and / or degradation is highly beneficial for use in agriculture and within animal systems (including humans).

[0304] These results suggest that the loop structure containing D66 is important for SAP05 interactions with both SPL and GATA transcription factors, whereas the two loop structures containing N77 and D106 are predominantly involved in SPL, but not GATA, binding activity. Additionally, the change of G76 to the more bulky residue W may induce structural changes that impact SAP05 interactions with RPN10. Of significant note, these findings suggest that residues engaged in binding SPLs and GATAs do not impact SAP05 binding to RPN10. D66 is part of L4, N77 of L6 and D106 of L7 indicating that all three loop structures of the SAP05 loop surface are involved in binding ZnF of SPLs, thereby validating the crystal structure.

[0305] Our finding that D66 of one of the loops (L4) is involved in binding GATA prompted us to use AlphaFold-Multimer modeling (Richard et al., 2022) to assess the SAP05 - ZnF_GATA19 complex structure. The AlphaFold-Multimer predictions of the SAP05 and ZnF_SPL5 structures and their interactions were consistent with the crystal structure (Fig. 8), thereby validating the modeling approach. We therefore proceeded with using AlphaFold-Multimer to model the SAP05-ZnF_GATA19 structure and obtained a higher prediction confidence score for this model than for that of SAP05- ZnF_SPL5 (Fig. 8A, B - Middle panels). For the interacting residues, AlphaFold-Multimer predicted the interactions of SAP05 D66 and ZnF_SPL5 Q77 as well as SAP05 D106 and ZnF_SPL5 K90 correctly (Fig. 80).

[0306] ZnF_GATA19 interacts with the SAP05 loop surface in the model (Fig. 8B). Moreover, SAP05 D66 is one of the residues involved in the interaction with GATA19 unlike N77 (Fig. 80), in agreement with the finding that SAP06 D66A doesn’t degrade GATA19 in N. benthamiana leaves, whereas SAP06 N77R does (Fig. 2F). SAP05 E80 and I84 of loop 5 and D106 and D108 of loop 7 were also predicted to mediate interactions with GATA19 (Fig. 80). E80 and D106 both interact with R111 of GATA19 (Fig. 80), in agreement with the observation that mutation of D106 alone doesn’t disrupt the binding of the SAP05-ZnF_GATA19 interaction (Fig. 2E).

[0307] Interestingly, the ZnF region that interacts with SAP05 also mediates the binding of SPL transcription factors to DNA (Yamasaki et al., 2004). Further modelling revealed that the SAP05 binding surface with ZnF has a similar configuration as the major grooves in the transcription-binding regions of DNA. Therefore, SAP05 loop-rich binding surface with SPLs may mimic DNA.

[0308] Taken together, these data demonstrate that SAP05 interactions with SPL and GATA TFs involves structures of the SAP05 ‘loop surface’. Most SAP05 residues involved in binding SPLs and GATAs do not play a role in SAP05 binding of Rpn10 consistent with the bimodular architecture of SAP05.

[0309] EXAMPLE 4: The SAP05 ‘sheet surface’ forms polar interactions with vWA_Rpn10

[0310] We further investigated the role of specific residues involved in the SAP05 - vWA_Rpn10 interface. The SAP05 ‘sheet surface’ comprises a p-sheet composed of two p-strands that run in opposite directions of which one is located at the N-terminus and the other at the C-terminus of the linear SAP05 sequence as well as two loop structures separated by an a-helix and an a-helix at the N-terminus of SAP05. The SAP05 and vWA_RPN10 interaction is mediated by eight SAP05 amino acids that includes R43 and N48 located on pi, S50 on L2, H58 on L3, T60 on a2, and N125, Y127 and Y132 on p5 of SAP05 (Fig. 3A; Fig. 7C). Each of these residues interact with one residue of vWA-Rpn10, except for N48 that interacts with two (Fig. 3A; Fig. 7C). All vWA-Rpn10 residues that interact with SAP05 locate on a-helices (Fig. 3A). The SAP05-vWA interaction is largely mediated by polar forces. Thus, the SAP05 ‘sheet surface’ contains rigid secondary p-sheets and a-helix structures that are held in place by hydrogen bonds within SAP05, as opposed to the ZnF-binding loop surface that involves protruding loop structures that are more flexible.

[0311] We introduced single amino acid mutations in the SAP05 ‘sheet surface’ by replacing each with neutral, non-polar or oppositely charged residues generating 9 single amino acid SAP05 mutants (Fig. 7). All retained the ability to degrade SPL5 in N. benthamiana leaves, though some of the SAP05 mutants were not detected in leaves and hence may have high turnover rates (Fig. 9). However, several SAP05 double mutants and one triple mutant had reduced or no ability to mediate degradation of SPL5 (Fig. 3B), consistent with their loss of binding to Rpn10 in Y2H (Fig. 3C). SAP05 H58A T60W and S50A H58W retained affinity to SPL and GATA TFs, indicating that these double mutations had minimal impacts on the overall structure of SAP05, unlike the other double mutants that lost affinity to SPLs and the triple mutant that did not bind any of the targets (Fig. 30). These data indicate that multiple SAP05 residues mediate interactions with vWA, thereby validating the crystal structure. S50, H58 and T60 contribute to this interaction without an obvious impact on SAP05 interactions with the TFs, in line with the bimodular architecture of SAP05.

[0312] EXAMPLE 5: Conservation of interacting residues among SAP05, SPL and vWA homologs

[0313] We previously identified SAP05 homologs in phytoplasmas, including ones that bind both SPLs and GATAs, and ones that bind only SPLs or only GATAs. SAP05 amino acids that interact with SPL5 and vWA are conserved among all or most SAP05 homologs, including D66 that mediates binding with SPL and GATA TFs. The SAP05 homologs that bind only SPLs (PnWBa, WBDLa and P. mali) and that bind only GATAs (PnWBb, WBDLb) had the most sequence differences in the interacting residues among the SAP05 homologs. We noticed that regions corresponding to the 7 amino acids FTLTGPR (SEQ ID NO: 1) that form L4 and connects to P2 in SAP05_AYWB (Fig. 1A-B) were different in sequence among the SPL versus GATA-binding SAP05 homologs (Fig. 10A). L4 starts with the conserved F65 and D66 amino acids (Fig. 1A- B; Fig. 10A). Given our finding herein that D66 is essential for both SPL and GATA binding and degradation (Fig. 2), we investigated if L4 is involved in determining SAP05-binding specificity for SPLs and GATAs. Swapping corresponding L4 sequences from SAP05 homologs PnWBa and WBDLa to PnWBb and WBDLb resulted in gain of SPL binding, and conversely, swapping these from PnWBb and WBDLb to PnWBa and WBDLa resulted in gain of GATA binding (Fig. 10B). Therefore, L4 contributes to SAP05 binding-specific to SPLs and GATAs.

[0314] Next, we determined in how far interacting residues are conserved among the ZnF domains of SPL TFs. The Zinc-binding domain of SPL proteins contains two zinc- binding sites formed by eight conserved Cys or His residues. SAP05-binding residues locate in both ZnF domains and the majority of these are conserved among the SPL TFs. However, SAP05 D66 interacts with Q in SPL5 and this amino acid is not conserved. Given the importance of SAP05 D66 in mediating interactions with both SPL and GATAs, we compared electrostatic surface areas of SAP05 D66 and D66A. The surface area in the D66 environment is electronegative and the D66A mutation changed the interaction surface potential to neutral for the interface with ZnF_SPL5 (Fig. 11 A) and to predominantly positive with GATA19 (Fig. 11 A). These results indicate that D66 contribution to the electronegative surface potential is an important factor for binding SPL and GATA TFs.

[0315] Among the SAP05 residues that bind vWA, particularly H58, in combination with S50 or T60, have essential roles (Fig. 3). These locate in L3 that connect to a2 in the SAP05 structure (Fig. 1A-B). SAP05 H58 is conserved among the majority of SAP05 homologs (Fig. 10) and interacts with N34 that is conserved in vWA domains of most Rpn10 homologs. SAP05 S50 interacts with A. thaliana vWA E31 that is also an acidic amino (D) in other vWA sequences, and SAP05 T60 with Q42 that is conserved among plant vWA. The three amino acids locate in the first a-helix at the N-terminus of A. thaliana vWA (Fig. 4).

[0316] Rpn10 is positioned in the 19S RP where it forms an important component in the interface of the lid and base structure. The cryo-EM structure of the spinach (Spinacia oleracea) 26S proteasome was recently resolved (Kandolf et al., 2022). The vWA residues that interact with SAP05 are conserved in the vWA domains of spinach and A. thaliana Rpn10 homologs. A structural model that superimposes the SAP05 - vWA_RPN10 complex onto the spinach 26S proteasome did not reveal obvious steric clashes with proteasome components (Fig. 5A). Notably, SAP05 interacts with two parallel a-helices that locate on the free surface away from the vWA interactive surface with the 19S subunit (Fig. 5B). This indicates that SAP05 has minimal disadvantageous effects on Rpn10 interaction with the 19S RP. Nonetheless, ZnF_SPL5 of the ternary structure clash with two a-helices that protrude from the 26S proteasome.

[0317] We previously reported that SAP05 does not interact with insect and human Rpn10 (the latter is also known as PSMD4) (Huang et al., 2021). Structural modelling of SAP05 interactions with vWA domains of plant and PSMD4 revealed that L3 of SAP05 clashes with H38 and S40 of PSMD4 precluding a SAP05-PSMD4 interaction (Fig. 4). Similarly, Q38 of vWA of yeast Rpn10 clashes with L3 of SAP05 precluding SAP05 from binding yeast Rpn10 (Fig. 4). This corroborates data presented herein that H58 of the SAP05 L3 region plays an essential role in the SAP05 interaction with A. thaliana vWA.

[0318] Thus, despite the interaction between SAP05 and non-plant RpnIO’s being naturally precluded, our work will enable targeted mutagenesis of SAP05 to achieve these desirable interactions.

[0319] This can be achieved by screening mutant libraries in the regions of approximately residue 40 to 70 and positions 120 to 135 of SAP05, i.e. the key residue(s) anticipated to be a key determinant to the interaction inhibition. By mutating these key residues, the preclusion can be avoided. Particularly, it is understood that the residues R43 to F61 and another region from N125 to Y132 in A.thaliana will be particularly important in achieving the interaction. Specifically, residues R43, N48, S50, H58, T60, N125, Y127 and Y132 are known to interact with the vWA of A. thaliana Rpn10 and so will be key targets for mutation. Of course, corresponding residues in alternative or variant Phytoplasma SAP05’s or proteins capable of binding the vWA domain will also be able to be mutated to facilitate interactions with alternative RpnIO’s.

[0320] The targeting of these residues has only been possible due to the in-depth experiments of the crystal structure of SAP05, as described herein. As such, the identification of the key residues able to overcome the naturally precluded SAP05-PSM4 interaction has required an inventive step. EXAMPLE 6: SAP05 is a prime candidate for engineering a new type of degraders that operate independently of E3 ligases.

[0321] The crystal structure data reported herein demonstrates that the 12.3 kDa bacterial effector SAP05 has a unique globular structure with an internal p-sheet core and interconnecting loops and a-helices. One surface is dominated by rigid p-strands (sheet surface) and the opposite surface by flexible loop structures (loop surface).. The flexible loop surface is however capable of binding multiple transcription factors of the distinct SPL and GATA TF families via their Zinc-finger domains. Therefore, SAP05 appears to have an optimal configuration to act as a molecular glue capable of connecting a conserved proteasome component to multiple members of two TF families.

[0322] Rpn10 vWA has an important function as its deletion causes lethality or severe growth deficiencies in plants, vertebrates and human cell lines. In rpn10 null mutants, deficiencies may be restored upon addition of the vWA domain of Rpn10 and RAD23, and Rpn10 and RAD23 act redundantly (Elsasser et al. 2004). Like Rpn10, RAD23 is a reversible component of the 26S proteasome and bind ubiquitinated substrates. Intriguingly, the phytoplasma effector family SAP54 / phyllogen family hijack RAD23 and mediates plant proteins, the MADS-box TFs for degradation, leading to plant developmental changes. SAP54 bind the RAD23 ubiquitin-associated (UBA) that are shown to covalently bind ubiquitin moieties of ubiquitinated substrates. Therefore, at least two distinct phytoplasma effector families mediate degradation of plant TFs in ubiquitin-independent manner using reversible components of the 19S RP.

[0323] The vWA domain locates at the N-terminus of Rpn10, and the C-terminal part of Rpn10 consists of single-helix ubiquitin interaction motifs (UlMs) connected by a flexible region that bind ubiquitin chains. Due to the flexible C-terminus, only the vWA domain is visible in cryo-EM crystal structures of 26S proteasomes and locates in a central location of the 19S RP, at the interface of its base and lid. There is no evidence that SAP05 interferes with 26S proteasome activity (Huang et al. 2021) and structural information generated herein shows that SAP05 binds to a region of vWA that doesn’t interact with components of the 19S RP. Therefore, SAP05 appears to have evolved to optimally bind plant Rpn10 and is positioned in a central location of the 19S RP. While the SAP05 - vWA_RPN10 complex doesn't appear to cause significant steric hindrance, the ZnF_SPL5 within the ternary structure appears to clash sterically with the a-helices of Rpt4 and Rpt5, as evidenced in the structural model of the spinach 26S proteasome (Kandolf et al., 2022). The coiled-coil (CC) domains of these two Rpt proteins, along with the domains of four others, dimerize to create three CCs (Rpt1 / 2, Rpt6 / 3, and Rpt4 / 5 CCs). These CCs play a vital role in 26S proteasome activity by physically linking substrate recruitment and processing to the unfolding machinery, ultimately leading to substrate degradation. Furthermore, conformational changes within the CCs are essential for transitioning the Rpt1-6 ATPase ring between resting and active stages, the latter of which involves a widening of the central pore to allow substrate entry into the core of the 20S CP. The Rpt4 / 5 CC also binds to Rpn10 upon substrate binding. As such, when the ternary complex is accommodated on the 26S proteasome, the interaction of SPL5 with the CC dimer could potentially instigate the switch of the proteasome to the active stage, leading to the degradation of the transcription factor.

[0324] Thus, our work indicates that any mutation within SAP05 or a RPN10 will benefit from any effect that increases association with the coiled-coil domains of Rpt4 and Rpt5, for it is these coiled coil domains that are responsible for physically linking substrate recruitment and processing to the unfolding machinery. Accordingly, this invention has identified that mutation(s) which result in an increased association with the coiled-coil domains, i.e. the a-helices of Rpt4 and Rpt5, are anticipated to show enhanced degradation.

[0325] We investigated the importance of residues mediating the SAP05 - ZnF_SPL5 and SAP05 - vWA_Rpn10 interfaces. We found that mutations of single amino acids in SAP05 disrupted the SAP05 - ZnF_SPL5 interaction. SAP05 mutant D66A is impaired in the degradation of both SPL5 and GATA19, showing a potential to be further engineered for a useful tool to degrade any protein without causing plant development problems.

[0326] In contrast, multiple amino acid mutations are needed to impair SAP05 interaction with vWA_Rpn10. Even in the double SAP05 H58A T60W and S50A H58W double mutants the ability to mediate degradation has not fully disappeared. This suggest that the SAP05 - vWA interaction is robust. It is striking that SAP05 itself is not degraded, and is highly stable in plant cells, despite its association with the 26S proteasome and its function in degrading substrates that it directly interacts with.

[0327] Impaired functioning of the Ubiquitin Proteasome System (UPS) is linked with a multitude of diseases. However, given the pivotal role of the UPS in orchestrating an array of cellular processes, the SAP05 molecular system presents opportunities for the development of innovative therapeutics. The 26S proteasome has been leveraged to create novel therapeutics such as PROTACs (PROteolysis TArgeting Chimeras), which are small molecules that recruit E3 ligases for ubiquitination of substrates marked for degradation. Several PROTACs and similar systems have shown promising results and are currently advancing through clinical trials. However, their reliance on recruiting E3 ligases has led to challenges associated with side effects and resistance. Since SAP05 does not hamper the ability of the 26S proteasome to degrade substrates, this effector emerges as a prime candidate for engineering a new type of degraders that operate independently of E3 ligases. The structural work presented in this study provides a springboard for bioengineering a new class of degraders that do not depend on E3 ligases.

[0328] Materials and Methods

[0329] Protein production and purification

[0330] Gene cloning, expression, and protein purification for in vitro studies. DNA encoding mature SAP05 excluding signal peptide (Ala33-Lys135) was subcloned to pOPINF vector which contains a N-terminal 6*His tag (Berrow et al., 2007) using Infusion cloning method (Benoit et al., 2006; Bird et al., 2014). DNA encoding the ZnF domain of SPL5 (Ser60-Leu127) was subcloned to pOPINM vector (Berrow et al., 2007) for N-terminal His-MBP tag using Gibson Assembly cloning (Gibson et al., 2009). DNA encoding the vWA domain of RPN10 (Met1-GLy193) was firstly cloned to a Level 0 vector pUAP1-RFP, then assemble with another Level 0 vector (plCSL30028) containing a His-GB1 solubility tag to the Level 1 destination vector pOPIN-F5-RFP-K for a final cassette expressing N-terminal His-GB1 tagged vWA_RPN10, using Golden Gate cloning strategy (Engler et al., 2009; Engler et al., 2008; Weber et al., 2011). The constructs were then transformed into E. coli BL21(DE3) competent cells for expression. Bacterial cultures were grown in LB media (containing relative antibiotics) at 37°C to an ODeoo around 0.5 followed by induction with 1mM Isopropyl-b-D- thiogalactoside (IPTG) at 16°C overnight with shaking at a speed of 220rpm. Cell pellets were resuspended in buffer A1 (50mM Tris-HCI, 50mM glycine, 0.5M NaCI, 20mM imidazole, 5% glycerol, pH 8.0) followed by sonication lysis at 40% amplitude, 5 sec on / 10 sec off pulse for 30min on ice. Cell deris was removed by centrifuge at 17000rpm for 20min. Purifcation of the proteins was performed using an AKTA Xpress purification system comprising initial capture with metal affinity chromatography (IMAC) using Ni-NTA column, which elutes proteins with buffer B1 (50mM Tris-HCI, 50mM glycine, 0.5M NaCI, 0.5M imidazole, 5% glycerol, pH 8.0), followed by gel filtration with the column Superex 75 26 / 600 in buffer A4 (20mM HEPES, 0.15M NaCI, pH 7.5). Fractions with the elution peaks were assessed by SDS-PAGE gels and then pooled and treated with HRV-3C protease overnight to remove His / His-MBP / His-GB1 tags at 4°C overnight. Afterwards, digested protein samples were passed through Ni-NTA column to remove cleaved tags. Then these untagged samples were assessed with SDS-PAGE gels, pooled and concentrated with 3kD cut-off vivaspin concentrators, followed by a second round of gel filtraction with Superex 75 26 / 600 in buffer A4. Eluted samples were assessed with SDS-PAGE gels, then concentrated via 3kD cut-off vivaspin concentrators, to a final concentation of ~10mg / ml for subsequent analysis.

[0331] Gene cloning, expression, and protein purification for crystallization. For crystallization of SAP05 - ZnF_SPL5 complex, SAP05 (Ala33-Lys135) and ZnF_SPL5 (Ser60-Leu127) were separately cloned to pOPINF vector for N-terminal His-SAP05 and His-ZnF_SPL5. The constructs were transformed individually into E. coli BL21(DE3) competent cells, expressed and purified using the same methods mentioned above. Then purified SAP05 and purified ZnF_SPL5 were mixed together in a molar ration of 1 :1 , followed by gel filtration chromatography. Euted fractions were assessed by SDS-PAGE, pooled and concentrated to a final concentration of 15mg / mL, for crystallization studides. For crystallization of SAP05 - vWA_RPN10 complex, SAP05 was cloned to pOPINF vector, vWA_RPN10 (Met1-GLy193) was initially cloned to pOPINM to get a MBP-vWA fusion cassette, then MBP-vWA was amplified and ligated to pOPINA to remove the His tag. Then pOPINF-SAP05 and to pOPINA-MBP-vWA vectors were co-transformed into E. coli BL21(DE3) competent cells. Proteins were co-expressed and co-purified using the IMAC and gel filtration mentioned as above. Eluted fractions from the complex were assesed from gel filtration peaks and SDS-PAGE. Afterwards, the eluted complex was subjected to HRV-3C protease cleavage overnight at 4°C followed by Ni-NTA column to removed tags. After a second round of gel filtration, the purified complex were pooled, and concentrated to a final concentration of 15 mg / mL, for crystallization studies.

[0332] Protein crystallization, structure determination, and refinement

[0333] Crystallization screens were set up in sitting-drop vapor diffusion format in MRC2 96- well crystallization plates with drops comprised of 0.3 pL precipitant solution and 0.3 pL of protein and incubated at 293 K. All crystals were cryoprotected in the crystallization solution supplemented with 25% (v / v) glycerol and mounted in Litholoops (Molecular Dimensions) before flash-cooling by plunging into liquid nitrogen. X-ray data were recorded on beamline I04 at the Diamond Light Source (Oxfordshire, UK) using an Eiger2 XE 16M hybrid photon counting detector (Dectris), with crystals maintained at 100 K by a Cryojet cryocooler (Oxford Instruments). Diffraction data were integrated and scaled using DIALS via the XIA2 expert system then merged using AIMLESS. The majority of the downstream analysis was performed through the CCP4i2 graphical user interface.

[0334] The SAP05 - ZnF_SPL5 complex crystallized from 0.1 M MES pH 6.5, 25% (w / v) PEG 6000 in space group P21, with approximate cell parameters of a = 78.7, b = 165.0, c = 80.9 A, p = 109.7°. All X-ray data were collected from a single crystal, initially recorded at a wavelength of 0.9795 A (2 x 360° passes) and processed to 2.2 A resolution, and then at a wavelength of 1.2770 A (1 x 360° pass), this being close to the K X-ray absorption edge for zinc. The latter data set was processed to 2.4 A resolution and enabled structure solution via the single-wavelength anomalous diffraction method using the CRANK2 pipeline, due to the presence of zinc ions bound to ZnF_SPL5. This produced a partial model corresponding to eight copies of a 1:1 complex of SAP05- ZnF_SPL5 in the crystallographic asymmetric unit, giving an estimated solvent content of 59%, with each copy of ZnF_SPL5 containing two zinc ions. The model was completed through several iterations of model building in COOT and restrained refinement in REFMAC5 against the higher resolution data set at 2.2 A resolution.

[0335] The SAP05 - vWA_Rpn10 complex crystallized from 0.1 M Sodium HEPES pH 7.5, 10.7 % (w / v) PEG 4000 in space group P21, with approximate cell parameters of a = 42.4, b = 68.6, c = 49.9 A, p = 92.8°. X-ray data were recorded from a single crystal at a wavelength of 0.9796 A (2 x 360° passes) and processed to 2.17 A resolution. Analysis of the likely composition of the asymmetric unit suggested that it contained one copy of a 1 :1 complex of SAP05-vWA_Rpn10, giving an estimated solvent content of 44%. The structure was solved via molecular replacement using PHASER (McCoy et al., 2007], One copy of SAP05 was taken from the above SAP05-ZnF_SPL5 complex as the first template, and the second template was derived from a homology model of vWA_Rpn10 produced by Swiss-Model (Waterhouse et al., 2018) and based on PDB entry 5VFT. The model was completed through several iterations of model building in COOT and restrained refinement in REFMAC5.

[0336] In vitro Protein-Protein Interaction studies

[0337] Isothermal titration calorimetry (ITC)

[0338] ITC experiments were performed with MicroCai PEAQ-ITC instrument (Malvern, UK). All the titrations were conducted in Buffer A4 (20mM HEPES, pH 7.5, 0.15M NaCI) at 25°C. For different tests, purified proteins were diluted to required concentrations with Buffer A4 (20mM HEPES, pH 7.5, 0.15M NaCI). Proteins loaded to the calorimetric cell were diluted to a concentration of 20pM, and proteins sucked in the syringe were diluted with a concentration of 200pM. For each interaction, proteins were tested and swapped the loading in the cell and syringe, and with three replicates. Each ITC run included a single injection of 0.4pL for 0.8s followed by 18 injections of 2pL for 4s each. Injections were made with a spacing of 150s and s stirring speed of 750rpm. All data were processed with MicroCai PEAQ-ITC control software.

[0339] SAP05 mutation generation, cloning, yeast two-hybrid (Y2H) assay test and degradation assay

[0340] SAP05 mutation generation and cloning. SAP05 mutations were generated by using overlap PCR (Nelson and Fitch, 2011) or directly synthesized using gBIock from Integrated DNA Technologies company (IDT). Mutaions used for different assays were codon optimized, then cloned to different vectors for tests.

[0341] Yeast two-hybrid assay (Y2H)

[0342] The coding sequences of SAP05 or SAP05 mutants excluding signal peptides were amplified and ligated into Gateway vector pDESTGBKT7 (BD). Full length sequences of RPN10, three SPL11, SPL15, GATA18, GATA19 were amplified and cloned into vector Gateway vector pDESTGADKT7 (AD). Gateway cloning method was used (Hartley et al., 2000; Liang et al., 2013). Constructs used to test protein-protein interactions were co-transformed into the yeast Saccharomyces cerevisiae strain AH 109 using the Matchmaker Gold yeast two-hybrid system (Clontech). Empty vector of pDESTGBKT7 and pDESTGADKT7 were used as negative control. Yeast growth on solid SD medium lacking leucine and tryptophan (SD-LW) indicates presence of AD and BD constructs and on SD medium lacking leucine, tryptophan, histidine and alanine (SD-LWHA) indicates interactions between the AD and BD fusion compounds. Yeast plates were kept in 28°C growth chambers for 5 days before imaging.

[0343] Degradation assay in N. benthamiana

[0344] The coding sequences of SAP05 or SAP05 mutants (excluding signal peptides) were amplified and ligated into Gateway vector pB7WG2. Full length coding sequence of AtSPL5 were amplified and tagged with 3* HA at N-terminal, then ligated into Gateway vector pB7WG2. After sequencing, the constructs were separately transformed to Agrobacterium tumefaciens strain GV3101 and plated on LB solid medium containing rifampicin, gentamicin and spectinomycin and grown at 28 °C for 24-48 hours. Then Colonies were picked and checked by PCR using plasmids extracted from overnight liquid culture and gene-specific primers. Positive colonies were grown at 28 °C overnight, harvested, and resuspended in infiltration buffer (10 mM MgCI2, 10 mM MES, pH 5.6), supplemented with 100 pM acetosyringone. Appropriate combinations of above constructs together with pCB301-P19 were mixed at an GD600 of 0.5 per construct and infiltrated to the abaxial surface of 4 week-old N. benthamiana leaves using 1 ml needleless syringe. Infiltrations were conducted on randomly selected leaves, with triplicates consisting of three leaves each. To minimize variation, each single leaf was infiltrated with four combinations: SAP05 only, one SAP05 mutant only, SAP05 plus HA-SPL5, and SAP05 mutant plus HA-SPL5. After 3 days post-infiltration, the infiltrated leaves were detached, and the infiltrated areas were harvested for total protein extraction. To detect SPL5 and SAP05, Western blots were performed as follows. Total protein extracts were separated on 4-20% gradient gels (Bio-Rad) and transferred to 0.22 pm PVDF membranes using the Bio-Rad mini-PROTEIN Electrophoresis system. Protein loading was visualized with Ponceau S staining solution (Thermo Scientific) and washed off with water. Membranes were then blocked by 5% (w / v) milk powder in Tris-buffered saline (TBS) and 0.1% (v / v) Tween-20 for 2 h at room temperature. Membranes were then overnight incubated at 4°C with the anti- HA primary antibody (OptimAB HA. 11, Eurogentec), which was raised from mouse serum, at a ratio of 1:2000 dilution. Afterwards, the membrane was probed with Alkaline-Phosphatase-conjugated anti-mouse secondary antibody (Thermo Scientific) for 1 h at room temperature and imaged after incubation with NBT / BCIP substrate solution (Thermo Scientific). Membranes were washed after detection, blocked in the same conditions as previously described and incubated overnight at 4°C with rabbit raised anti-SAP05 antibody at a 1:5000 ratio (Huang et al., 2021). The membrane was then probed with HRP conjugated anti-rabbit secondary antibody (Sigma) and imaged with Immobilon Western Chemilum HRP Substrate (Sigma).

[0345] Homology and structural analysis

[0346] Homology analysis

[0347] Sequences of SAP05 homologs from different phytoplasma strains, A. thaliana SPLs and Rpn10 from different organisms were aligned using MUSCLE algorithm on Phylogeny.fr web server (http: / / www.phylogeny.fr / index.cgi; Dereeper et al., 2008). Graphical representation and editing were performed with MEGA11 software.

[0348] Structural analysis

[0349] Structural predictions were conducted using AlphaFold v2.3.2 (Jumper et al., 2021) for single protein and AlphaFold-Multimer v3 (Richard et al., 2022) for protein complexes. Analysis of predicted structure was performed with PyMOL software.

[0350] References

[0351] Elsasser S, Chandler-Militello D, Muller B, Hanna J, Finley D. Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome. J Biol Chem. 2004 Jun 25;279(26):26817-22. doi: 10.1074 / jbc.M404020200. Epub 2004 Apr 26. PMID: 15117949.

[0352] Huang, W., MacLean, A.M., Sugio, A., Maqbool, A., Busscher, M., Cho, S.T., Kamoun, S., Kuo, C.H., Immink, R.G.H., and Hogenhout, S.A. (2021). Parasitic modulation of host development by ubiquitin-independent protein degradation. Cell 784(20):5201- 5214. e12.

[0353] Kandolf, S., Grishkovskaya, I., Belacic, K., Bolhuis, D.L., Amann, S., Foster, B., Imre, R., Mechtler, K., Schleiffer, A., Tagare, H.D., et al. (2022). Cryo-EM structure of the plant 26S proteasome. Plant Commun. 3, 100310. SEQUENCE LISTING

[0354] SEQ ID NO: 1 SAP05 amino acid sequence (Signal peptide underlined)

[0355] >SAP05_AYWB

[0356] MFKIKNNLLKSKIFVFILLGLFVIINNHQAMAAPNEEFVGDMRIVNVN LSN I DI LKKH ETFK

[0357] KYFDFTLTGPRYNGNIAEFAMIWKIKNPPLNLLGVFFDDGTRDDEDDKYILEELKQIGN GAKNMYIFWQYEQK

[0358] SEQ ID NO: 2 SAP05 cDNA sequence (Signal peptide underlined)

[0359] >SAP05_AYWB

[0360] ATGTTTAAAATCAAAAATAATTTATTAAAATCAAAAATATTTGTATTTATTTTATTAGG

[0361] ATTATTTGTAATTATCAATAATCATCAAGCAATGGCTGCCCCTAATGAAGAGTTTGT

[0362] TGGCGACATGAGAATAGTTAATGTAAATTTATCAAATATTGATATTCTTAAAAAACA

[0363] TGAAACATTTAAAAAATATTTTGATTTTACACTAACTGGTCCTCGTTATAATGGAAA CATAGCAGAATTTGCAATGATATGGAAAATTAAAAATCCGCCTCTTAATTTATTAGG TGTTTTTTTTGATGATGGCACCAGAGATGATGAAGATGATAAATATATTTTAGAAGA

[0364] ATTAAAACAAATAGGCAATGGAGCCAAAAATATGTATATTTTTTGGCAATATGAACA AAAATAA

[0365] SEQ ID NO: 3 SAP05-GFP nanobody amino acid sequence

[0366] >SAP05_GFP nanobody

[0367] MAPNEEFVGDMRIVNVNLSNIDILKKHETFKKYFDFTLTGPRYNGNIAEFAMIWKIKNP

[0368] PLNLLGVFFDDGTRDDEDDKYILEELKQIGNGAKNMYIFWQYEQKGDGGSGGGSMD

[0369] QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSA

[0370] GDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQ VTVSS

[0371] SEQ ID NO: 4 SAP05-GFP nanobody cDNA sequence

[0372] >SAP05_GFP nanobody

[0373] ATGGCCCCGAATGAAGAGTTTGTGGGAGACATGCGAATAGTAAATGTCAATCTAA

[0374] GTAATATAGATATACTAAAGAAGCATGAGACGTTTAAGAAGTACTTCGATTTTACAC

[0375] TCACGGGCCCCAGGTACAATGGCAATATAGCCGAATTTGCGATGATCTGGAAAAT

[0376] CAAAAACCCACCACTCAACCTCCTTGGGGTCTTTTTTGATGATGGTACGCGAGAC GACGAAGATGACAAATACATTCTAGAGGAGTTGAAACAGATCGGCAACGGTGCAA AGAACATGTATATATTCTGGCAATATGAGCAAAAGGGCGATGGAGGGAGTGGCGG

[0377] AGGGAGCATGGATCAAGTACAGCTAGTCGAATCTGGGGGTGCTCTCGTGCAGCC

[0378] AGGTGGGTCATTGCGTTTGTCTTGCGCTGCCTCAGGGTTTCCAGTCAATCGATAT

[0379] AGCATGCGATGGTACAGACAAGCTCCGGGTAAGGAACGAGAATGGGTAGCCGGA

[0380] ATGAGTAGCGCAGGTGATCGATCATCATACGAGGACAGCGTTAAGGGACGTTTCA CCATTTCCAGAGACGATGCCCGTAACACGGTCTATCTACAGATGAACTCTCTCAA GCCAGAGGATACAGCCGTATATTATTGTAACGTTAATGTCGGGTTTGAATACTGGG

[0381] GCCAGGGTACGCAGGTAACGGTCTCTAGTTAA

[0382] SEQ ID NO: 5 AtRPNIO amino acid sequence

[0383] MVLEATMICIDNSEWMRNGDYSPSRLQAQTEAVNLLCGAKTQSNPENTVGILTMAGK

[0384] GVRVLTTPTSDLGKILACMHGLDVGGEINLTAAIQIAQLALKHRQNKNQRQRIIVFAGS

[0385] PIKYEKKALEIVGKRLKKNSVSLDIVNFGEDDDEEKPQKLEALLTAVNNNDGSHIVHVP

[0386] SGANALSDVLLSTPVFTGDEGASGYVSAAAAAAAAGGDFDFGVDPNIDPELALALRVS

[0387] MEEERARQEAAAKKAADEAGQKDKDGDTASASQETVARTTDKNAEPMDEDSALLDQ

[0388] AIAMSVGDVNMSEAADEDQDLALALQMSMSGEESSEATGAGNNLLGNQAFISSVLSS LPGVDPNDPAVKELLASLPDESKRTEEEESSSKKGEDEKK SEQ ID NO: 6 AtRPNIO cDNA sequence

[0389] ATGGTTCTCGAGGCGACTATGATATGTATCGACAACTCCGAGTGGATGCGAAACG

[0390] GAGATTACTCTCCGTCTAGGTTACAGGCGCAAACGGAAGCTGTTAATCTTCTTTGC

[0391] GGAGCCAAAACCCAGTCGAATCCGGAGAATACGGTGGGGATTTTGACAATGGCT

[0392] GGCAAAGGAGTTAGAGTATTGACTACTCCTACCTCTGATCTTGGCAAAATTCTGGC

[0393] CTGTATGCACGGCCTTGATGTGGGAGGAGAGATCAACTTAACCGCAGCTATCCAG

[0394] ATCGCCCAGCTAGCTCTTAAGCATCGCCAAAACAAGAATCAACGCCAAAGGATTA

[0395] TTGTTTTTGCTGGAAGTCCAATCAAGTACGAGAAGAAGGCCCTAGAGATAGTTGG

[0396] AAAAAGGCTGAAGAAGAATAGTGTCTCTCTTGATATTGTCAATTTCGGGGAGGATG

[0397] ATGATGAGGAAAAGCCTCAGAAACTCGAGGCGCTCCTTACAGCTGTGAATAACAA

[0398] TGACGGTAGCCACATTGTTCATGTTCCTTCTGGAGCCAATGCTCTCTCAGATGTGC

[0399] TTCTCAGCACACCTGTATTCACGGGTGATGAGGGTGCAAGTGGCTATGTTTCTGC

[0400] GGCAGCTGCTGCAGCGGCCGCAGGTGGGGACTTCGACTTTGGTGTGGACCCAAA

[0401] TATCGATCCAGAACTTGCTCTTGCCCTTCGGGTCTCCATGGAGGAGGAGAGAGCA

[0402] AGACAAGAAGCTGCTGCCAAGAAGGCGGCCGATGAGGCAGGTCAGAAAGACAAA

[0403] GATGGGGACACAGCTTCCGCCTCACAGGAGACAGTTGCTAGGACAACTGACAAG

[0404] AACGCTGAACCAATGGATGAGGACAGTGCGTTGCTAGATCAGGCAATTGCTATGT

[0405] CTGTTGGTGATGTGAATATGTCAGAAGCGGCTGATGAGGACCAGGATCTGGCTTT

[0406] AGCTCTGCAAATGTCAATGAGTGGGGAAGAGTCAAGTGAAGCTACAGGTGCTGGA

[0407] AACAACCTCTTGGGAAATCAAGCCTTCATATCGTCTGTTCTCTCATCGCTTCCTGG

[0408] GGTGGATCCAAATGATCCGGCAGTTAAAGAACTACTAGCGTCTCTGCCAGACGAG

[0409] TCAAAGCGTACCGAGGAGGAAGAGAGTAGTAGCAAAAAAGGCGAGGATGAGAAG

[0410] AAGTGA

[0411] SEQ ID NO: 7 >SAP05_AYWB

[0412] MFKIKNNLLKSKIFVFILLGLFVIINNHQAMAAPNEEFVGDMRIVNVNLS

[0413] NIDILKKHETFKKYFDFTLTGPRYNGNIAEFAMIWKIKNPPLNLLGVFFD

[0414] DGTRDDEDDKYILEELKQIGNGAKNMYIFWQYEQK

[0415] SEQ ID NO: 8 >SAP05_AYWB

[0416] ATGTTTAAAATCAAAAATAATTTATTAAAATCAAAAATATTTGTATTTAT

[0417] TTTATTAGGATTATTTGTAATTATCAATAATCATCAAGCAATGGCTGCCC

[0418] CTAATGAAGAGTTTGTTGGCGACATGAGAATAGTTAATGTAAATTTATCA

[0419] AATATTGATATTCTTAAAAAACATGAAACATTTAAAAAATATTTTGATTT

[0420] TACACTAACTGGTCCTCGTTATAATGGAAACATAGCAGAATTTGCAATGA

[0421] TATGGAAAATTAAAAATCCGCCTCTTAATTTATTAGGTGTTTTTTTTGAT

[0422] GATGGCACCAGAGATGATGAAGATGATAAATATATTTTAGAAGAATTAAA

[0423] ACAAATAGGCAATGGAGCCAAAAATATGTATATTTTTTGGCAATATGAAC

[0424] AAAAATAA

[0425] SEQ ID NO: 9 >SAP05_ATP

[0426] MFEFKKPLQIFKIGFLILLGLFIFNSSDLIAVLPEAEFVGNMRIVNSTVT

[0427] DMGVLEKHASFSEYFDFKQPNACYNGNLGEFGIMWKIKNAPHNLLGVFFD

[0428] DGTRIDEDDRFTLEELKQMGNGAQNMYIFWQYQQK

[0429] SEQ ID NO: 10 >SAP05_ATP

[0430] ATGTTTGAATTCAAAAAACCATTACAAATATTTAAAATTGGATTTTTGAT

[0431] TTTATTGGGATTATTTATCTTCAATAGTTCTGATTTAATAGCAGTCCTTC

[0432] CTGAAGCAGAATTTGTAGGTAACATGAGGATTGTTAATTCAACTGTGACT

[0433] GACATGGGTGTTTTAGAAAAACATGCATCATTTAGTGAATATTTTGATTT

[0434] TAAGCAACCAAACGCTTGTTATAATGGCAATTTAGGTGAATTTGGTATTA

[0435] TGTGGAAAATCAAAAACGCACCGCATAATTTATTAGGAGTTTTCTTCGAT

[0436] GATGGTACTAGAATTGATGAAGATGATAGATTTACTTTAGAAGAATTAAA ACAAATGGGTAATGGTGCTCAAAATATGTATATTTTTTGGCAGTACCAAC

[0437] AAAAATAA

[0438] SEQ ID NO: 11 >SAP05_PnWBa

[0439] MKIYNPNNIIMAAPSQEEIIQGTRIVNVTVSNINVLKTHPSFAQYFDFNQ

[0440] TCPCYNSTVAEFCIMWKIKNPPTNLLGVFFDESTRDDEDDKYSLEELKYM

[0441] ANNSVNMFIFWEHKEK

[0442] SEQ ID NO: 12 >SAP05_PnWBb

[0443] MRSLWYLTFLFIVLNMYNYQNVVVAMPPREEFIGQTRIVHVSIGNINILK

[0444] QHAIFNKYFDWSLQSARYNEDLEDFSMIWTIKDPDPNLLGVFFDGGIRHG

[0445] QDDTYNLQELKHMGNGANNMYCIFLKNN

[0446] SEQ ID NO: 13 >SAP05_PnWBb

[0447] ATGCGAAGTTTATGGTATTTAACTTTTTTATTTATAGTTCTAAATATGTA

[0448] TAACTATCAAAATGTAGTTGTAGCTATGCCGCCAAGAGAGGAATTTATTG

[0449] GCCAAACTAGAATTGTTCATGTATCTATTGGCAATATTAATATTTTAAAA

[0450] CAACATGCCATATTTAATAAATATTTCGATTGGAGTTTACAAAGCGCTCG

[0451] TTATAATGAAGATTTAGAAGATTTCAGCATGATTTGGACAATTAAAGATC

[0452] CAGACCCAAATTTATTAGGTGTTTTTTTTGATGGCGGAATTAGACATGGC

[0453] CAAGATGATACATATAATTTGCAAGAATTAAAACATATGGGTAATGGTGC

[0454] TAATAATATGTATTGTATATTTCTAAAAAATAATTAA

[0455] SEQ ID NO: 14 >SAP05_WBDLa

[0456] MVKIKNNLLLLLNAFFAFTLLGLFLITNNQQVMAAPNEEFVGNMRIVNIT

[0457] VSNINILKNHATFKQYFDFKINRPCYNGNIATFAIMWKIKNPPRNLLGVF

[0458] FDNGTRDDEDDKYNLEDLKKMGNGASNMYIFWQYEQK

[0459] SEQ ID NO: 15 >SAP05_WBDLa

[0460] ATGGTTAAAATAAAAAATAACTTACTATTGTTATTGAATGCATTTTTTGC

[0461] ATTTACTTTATTAGGTTTATTTTTAATTACTAATAATCAACAAGTAATGG

[0462] CCGCCCCTAATGAAGAATTTGTTGGTAATATGAGAATCGTTAATATAACT

[0463] GTATCAAATATTAATATTCTTAAAAATCATGCAACATTTAAACAATACTT

[0464] TGATTTTAAAATAAATAGACCTTGTTATAACGGAAATATAGCAACTTTTG

[0465] CTATTATGTGGAAAATTAAAAATCCACCTCGTAATTTATTAGGTGTTTTT

[0466] TTTGATAATGGCACTAGAGATGATGAAGATGATAAATATAACTTAGAAGA

[0467] TTTGAAAAAAATGGGAAATGGTGCTTCAAATATGTATATTTTTTGGCAAT

[0468] ATGAACAAAAATAA

[0469] SEQ ID NO: 16 >SAP05_WBDLb

[0470] MIKFYFYLYILFIFVKISTLNNIIIAAPPQDEFINGTRIVNVIVASGDIL

[0471] KKHNLFKQYFDWSCEYPSYNSELEEFGMIWKIKNPPENLLGVFFDAGNRD

[0472] DADNKYTLEELKYIANKAKNMYIFWRYKEK

[0473] SEQ ID NO: 17 >SAP05_WBDLb

[0474] ATGATAAAATTTTATTTTTATTTATATATTTTATTTATATTTGTGAAAAT

[0475] TTCTACTTTGAATAATATTATTATAGCTGCACCGCCACAAGATGAATTCA

[0476] TTAATGGAACTAGAATTGTTAATGTAATTGTTGCTAGTGGTGATATTTTA

[0477] AAAAAACATAATTTATTTAAACAATATTTTGATTGGTCTTGCGAATATCC

[0478] TAGTTATAATTCCGAATTGGAAGAATTTGGTATGATTTGGAAAATTAAAA

[0479] ATCCACCCGAAAATTTATTAGGAGTTTTTTTTGATGCAGGTAATAGAGAT

[0480] GATGCAGATAATAAGTATACTTTAGAAGAGTTAAAATATATAGCTAATAA GGCTAAAAATATGTATATTTTTTGGAGATATAAAGAAAAATAA

[0481] SEQ ID NO: 18 >vWA_AtRPN10

[0482] MVLEATMICIDNSEWMRNGDYSPSRLQAQTEAVNLLCGAKTQSNPENTVGILTMAGK

[0483] GVRVLTTPTSDLGKILACMHGLDVGGEINLTAAIQIAQLALKHRQNKNQRQRIIVFAGS

[0484] PIKYEKKALEIVGKRLKKNSVSLDIVNFGEDDDEEKPQKLEALLTAVNNNDGSHIVHVP

[0485] SGANALSDVLLSTPVFTG

[0486] SEQ ID NO: 19 >vWA_AtRPN10

[0487] ATGGTTCTCGAGGCGACTATGATATGTATCGACAACTCCGAGTGGATGCGAAACG

[0488] GAGATTACTCTCCGTCTAGGTTACAGGCGCAAACGGAAGCTGTTAATCTTCTTTGC

[0489] GGAGCCAAAACCCAGTCGAATCCGGAGAATACGGTGGGGATTTTGACAATGGCT

[0490] GGCAAAGGAGTTAGAGTATTGACTACTCCTACCTCTGATCTTGGCAAAATTCTGGC

[0491] CTGTATGCACGGCCTTGATGTGGGAGGAGAGATCAACTTAACCGCAGCTATCCAG

[0492] ATCGCCCAGCTAGCTCTTAAGCATCGCCAAAACAAGAATCAACGCCAAAGGATTA

[0493] TTGTTTTTGCTGGAAGTCCAATCAAGTACGAGAAGAAGGCCCTAGAGATAGTTGG

[0494] AAAAAGGCTGAAGAAGAATAGTGTCTCTCTTGATATTGTCAATTTCGGGGAGGATG

[0495] ATGATGAGGAAAAGCCTCAGAAACTCGAGGCGCTCCTTACAGCTGTGAATAACAA

[0496] TGACGGTAGCCACATTGTTCATGTTCCTTCTGGAGCCAATGCTCTCTCAGATGTGC

[0497] TTCTCAGCACACCTGTATTCACGGGT

[0498] SEQ ID NO: 20 >NM_001330692.2:50-1192 Homo sapiens proteasome 26S subunit ubiquitin receptor, non-ATPase 4 (PSMD4), transcript variant 1, mRNA

[0499] ATGGTGTTGGAAAGCACTATGGTGTGTGTGGACAACAGTGAGTATATGCGGAATG

[0500] GAGACTTCTTACCCACCAGGCTGCAGGCCCAGCAGGATGCTGTCAACATAGTTTG

[0501] TCATTCAAAGACCCGCAGCAACCCTGAGAACAACGTGGGCCTTATCACACTGGCT

[0502] AATGACTGTGAAGTGCTGACCACACTCACCCCAGACACTGGCCGTATCCTGTCCA

[0503] AGCTACATACTGTCCAACCCAAGGGCAAGATCACCTTCTGCACGGGCATCCGCGT

[0504] GGCCCATCTGGCTCTGAAGCACCGACAAGGCAAGAATCACAAGATGCGCATCATT

[0505] GCCTTTGTGGGAAGCCCAGTGGAGGACAATGAGAAGGATCTGGTGAAACTGGCT

[0506] AAACGCCTCAAGAAGGAGAAAGTAAATGTTGACATTATCAATTTTGGGGAAGAGG

[0507] AGGTGAACACAGAAAAGCTGACAGCCTTTGTAAACACGTTGAATGGCAAAGATGG

[0508] AACCGGTTCTCATCTGGTGACAGTGCCTCCTGGGCCCAGTTTGGCTGATGCTCTC

[0509] ATCAGTTCTCCGATTTTGGCTGGTGAAGGTGGTGCCATGCTGGGTCTTGGTGCCA

[0510] GTGACTTTGAATTTGGAGTAGATCCCAGTGCTGATCCTGAGCTGGCCTTGGCCCT

[0511] TCGTGTATCTATGGAAGAGCAGCGGCAGCGGCAGGAGGAGGAGGCCCGGCGGG

[0512] CAGCTGCAGCTTCTGCTGCTGAGGCCGGGATTGCTACGACTGGGACTGAAGGTG

[0513] AAAGAGACTCAGACGATGCCCTGCTGAAGATGACCATCAGCCAGCAAGAGTTTGG

[0514] CCGCACTGGGCTTCCTGACCTAAGCAGTATGACTGAGGAAGAGCAGATTGCTTAT

[0515] GCCATGCAGATGTCCCTGCAGGGAGCAGAGTTTGGCCAGGCGGAATCAGCAGAC

[0516] ATTGATGCCAGCTCAGCTATGGACACATCTGAGCCAGCCAAGGAGGAGGATGATT

[0517] ACGACGTGATGCAGGACCCCGAGTTCCTTCAGAGTGTCCTAGAGAACCTCCCAG

[0518] GTGTGGATCCCAACAATGAAGCCATTCGAAATGCTATGGGCTCCCTGGCCTCCCA GGCCACCAAGGACGGCAAGAAGGACAAGAAGGAGGAAGACAAGAAGTGA

[0519] SEQ ID NO: 21 >NP_001317621.1 26S proteasome non-ATPase regulatory subunit 4 isoform 1 [Homo sapiens]

[0520] MVLESTMVCVDNSEYMRNGDFLPTRLQAQQDAVNIVCHSKTRSNPENNVGLITLAND

[0521] CEVLTTLTPDTGRILSKLHTVQPKGKITFCTGIRVAHLALKHRQGKNHKMRIIAFVGSPV

[0522] EDNEKDLVKLAKRLKKEKVNVDIINFGEEEVNTEKLTAFVNTLNGKDGTGSHLVTVPP

[0523] GPSLADALISSPILAGEGGAMLGLGASDFEFGVDPSADPELALALRVSMEEQRQRQE EEARRAAAASAAEAGIATTGTEGERDSDDALLKMTISQQEFGRTGLPDLSSMTEEEQI AYAMQMSLQGAEFGQAESADIDASSAMDTSEPAKEEDDYDVMQDPEFLQSVLENLP GVDPNNEAIRNAMGSLASQATKDGKKDKKEEDKK

[0524] SEQ ID NO: 22 >NM_002810.4:50-1183 Homo sapiens proteasome 26S subunit ubiquitin receptor, non-ATPase 4 (PSMD4), transcript variant 2, mRNA

[0525] ATGGTGTTGGAAAGCACTATGGTGTGTGTGGACAACAGTGAGTATATGCGGAATG GAGACTTCTTACCCACCAGGCTGCAGGCCCAGCAGGATGCTGTCAACATAGTTTG TCATTCAAAGACCCGCAGCAACCCTGAGAACAACGTGGGCCTTATCACACTGGCT AATGACTGTGAAGTGCTGACCACACTCACCCCAGACACTGGCCGTATCCTGTCCA

[0526] AGCTACATACTGTCCAACCCAAGGGCAAGATCACCTTCTGCACGGGCATCCGCGT GGCCCATCTGGCTCTGAAGCACCGACAAGGCAAGAATCACAAGATGCGCATCATT GCCTTTGTGGGAAGCCCAGTGGAGGACAATGAGAAGGATCTGGTGAAACTGGCT AAACGCCTCAAGAAGGAGAAAGTAAATGTTGACATTATCAATTTTGGGGAAGAGG

[0527] AGGTGAACACAGAAAAGCTGACAGCCTTTGTAAACACGTTGAATGGCAAAGATGG AACCGGTTCTCATCTGGTGACAGTGCCTCCTGGGCCCAGTTTGGCTGATGCTCTC ATCAGTTCTCCGATTTTGGCTGGTGAAGGTGGTGCCATGCTGGGTCTTGGTGCCA GTGACTTTGAATTTGGAGTAGATCCCAGTGCTGATCCTGAGCTGGCCTTGGCCCT

[0528] TCGTGTATCTATGGAAGAGCAGCGGCAGCGGCAGGAGGAGGAGGCCCGGCGGG CAGCTGCAGCTTCTGCTGCTGAGGCCGGGATTGCTACGACTGGGACTGAAGACT CAGACGATGCCCTGCTGAAGATGACCATCAGCCAGCAAGAGTTTGGCCGCACTG GGCTTCCTGACCTAAGCAGTATGACTGAGGAAGAGCAGATTGCTTATGCCATGCA

[0529] GATGTCCCTGCAGGGAGCAGAGTTTGGCCAGGCGGAATCAGCAGACATTGATGC CAGCTCAGCTATGGACACATCTGAGCCAGCCAAGGAGGAGGATGATTACGACGT GATGCAGGACCCCGAGTTCCTTCAGAGTGTCCTAGAGAACCTCCCAGGTGTGGAT CCCAACAATGAAGCCATTCGAAATGCTATGGGCTCCCTGGCCTCCCAGGCCACCA

[0530] AGGACGGCAAGAAGGACAAGAAGGAGGAAGACAAGAAGTGA

[0531] SEQ ID NO: 23 >NP_002801.1 26S proteasome non-ATPase regulatory subunit 4 isoform 2 [Homo sapiens]

[0532] MVLESTMVCVDNSEYMRNGDFLPTRLQAQQDAVNIVCHSKTRSNPENNVGLITLAND CEVLTTLTPDTGRILSKLHTVQPKGKITFCTGIRVAHLALKHRQGKNHKMRIIAFVGSPV EDNEKDLVKLAKRLKKEKVNVDIINFGEEEVNTEKLTAFVNTLNGKDGTGSHLVTVPP GPSLADALISSPILAGEGGAMLGLGASDFEFGVDPSADPELALALRVSMEEQRQRQE

[0533] EEARRAAAASAAEAGIATTGTEDSDDALLKMTISQQEFGRTGLPDLSSMTEEEQIAYA MQMSLQGAEFGQAESADIDASSAMDTSEPAKEEDDYDVMQDPEFLFASQSVLENLP GVDPNNEAIRNAMGSLASQATKDGKKDKKEEDKK

[0534] SEQ ID NO: 24 > AT3G15270. Arabidopsis thaliana SPL5

[0535] MEGQRTQRRGYLKDKATVSNLVEEEMENGMDGEEEDGGDEDKRKKVMERVRGPS TDRVPSRLCQVDRCTVNLTEAKQYYRRHRVCEVHAKASAATVAGVRQRFCQQCSRF HELPEFDEAKRSCRRRLAGHNERRRKISGDSFGEGSGRRGFSGQLIQTQERNRVDR KLPMTNSSFKRPQIR

[0536] SEQ ID NO: 25 SAP05 amino acid sequence with D66A mutation (bold and underlined) MFKIKNNLLKSKIFVFILLGLFVIINNHQAMAAPNEEFVGDMRIVNVNLSNIDILKKHETFK KYFAFTLTGPRYNGNIAEFAMIWKIKNPPLNLLGVFFDDGTRDDEDDKYILEELKQIGN GAKNMYIFWQYEQK

[0537] SEQ ID NO: 26 SAP05 amino acid sequence with N77R mutation (bold and underlined) MFKIKNNLLKSKIFVFILLGLFVIINNHQAMAAPNEEFVGDMRIVNVNLSNIDILKKHETFK

[0538] KYFDFTLTGPRYNGRIAEFAMIWKIKNPPLNLLGVFFDDGTRDDEDDKYILEELKQIGN

[0539] GAKNMYIFWQYEQK

[0540] SEQ ID NO: 27 SAP05 amino acid sequence with D106A mutation (bold and underlined)

[0541] MFKIKNNLLKSKIFVFILLGLFVIINNHQAMAAPNEEFVGDMRIVNVNLSNIDILKKHETFK

[0542] KYFDFTLTGPRYNGNIAEFAMIWKIKNPPLNLLGVFFDDGTRDAEDDKYILEELKQIGN GAKNMYIFWQYEQK

[0543] SEQ ID NO: 28 SAP05 amino acid sequence with D106R mutation (bold and underlined)

[0544] MFKIKNNLLKSKIFVFILLGLFVIINNHQAMAAPNEEFVGDMRIVNVNLSNIDILKKHETFK

[0545] KYFDFTLTGPRYNGNIAEFAMIWKIKNPPLNLLGVFFDDGTRDREDDKYILEELKQIGN

[0546] GAKNMYIFWQYEQK

[0547] SEQ ID NO: 29. Loop 1 of SAP05

[0548] EEFVGDM

[0549] SEQ ID NO: 30. Loop 2 of SAP05

[0550] SN

[0551] SEQ ID NO: 31. Loop 3 of SA P05

[0552] HE

[0553] SEQ ID NO: 32. Loop 4 of SAP05

[0554] FDFTLTGP

[0555] SEQ ID NO: 33. Loop 5 of SAP05

[0556] GNIAEFAM

[0557] SEQ ID NO: 34. Loop 6 of SAP05

[0558] KNPPLNL

[0559] SEQ ID NO: 35. Loop 7 of SAP05

[0560] DDGTRDDEDDKY

[0561] SEQ ID NO: 36. Loop 8 of SAP05

[0562] IGNGAK

[0563] SEQ ID NO: 37: F2A

[0564] GGACAACTTCTCAACTTTGACTTGCTAAAGTTAGCTGGTGATGTTGAATCTAATCC

[0565] TGGACCA

[0566] SEQ ID NO: 38: F2A amino acids 1 to 20

[0567] GQLLNFDLLKLAGDVESNPG

[0568] SEQ ID NO: 39: F2A30 CACAAACAGAAAATTGTGGCACCGGTGAAGCAGACTCTCAACTTTGACTTGCTAAA

[0569] GTTAGCTGGTGATGTTGAATCTAATCCTGGACCA

[0570] SEQ ID NO: 40: F2A30 amino acids 1 to 20 HKQKIVAPVKQTLNFDLLKLAGDVESNPG

[0571] SEQ ID NO: 41. Localisation Signal

[0572] KDEL SEQ ID NO: 42 >SAP05_PnWBa

[0573] ATGAAAATTTATAATCCAAATAATATTATTATGGCTGCGCCATCGCAAGA

[0574] AGAAATCATTCAAGGAACTAGAATTGTTAATGTAACTGTAAGTAATATTA

[0575] ATGTTTTAAAAACACATCCTTCATTTGCGCAATACTTTGATTTTAATCAA

[0576] ACATGTCCTTGTTATAACAGTACTGTAGCAGAATTTTGTATTATGTGGAA AATTAAAAATCCACCTACAAATTTATTAGGTGTTTTTTTTGATGAAAGCA

[0577] CTAGAGATGATGAAGATGATAAATATTCTTTAGAAGAATTAAAATATATG

[0578] GCTAATAATTCTGTTAATATGTTTATTTTTTGGGAACATAAAGAAAAATA

[0579] A

Claims

CLAIMS:

1. A modified secreted AY-WB protein 5 (SAP05) comprising at least one mutation, wherein SAP05 comprises a plurality of loop sequences, and wherein the at least one mutation is within at least one loop sequence and wherein the at least one mutation reduces or abolishes binding of SAP05 to at least one SQUAMOSA promoter binding protein-like protein (SPL) and / or at least one GATA transcription factor.

2. The modified SAP05 protein of claim 1 , wherein the loop sequence comprises an amino acid sequence selected from SEQ ID NO: 29 to 36 or a variant or fragment thereof, and wherein the at least one mutation is within at least one of SEQ ID NO: 29 to 36 or a variant thereof.

3. The modified SAP05 protein of any preceding claim, wherein the at least one mutation is selected from a mutation at positions 66, 76, 77, 80, 104 and 106 of SEQ ID NO: 1 or a corresponding position in a homologous sequence, wherein preferably the homologous sequence is selected from SEQ ID NO: 7, 9, 11, 12, 14 and 16 and functional variants thereof.

4. The modified SAP05 protein of claim 3, wherein the at least one mutation is at position 66 of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and wherein preferably the mutation is a substitution.

5. The modified SAP05 protein of claim 4, wherein the mutation is a substitution of D66 to a positively charged amino acid, more preferably a substitution to D66A.

6. The modified SAP05 protein of any of claims 1 to 6, wherein the SAP05 protein comprises at least one further mutation, wherein the at least one mutation is at positions 40 to 70 or 120 to 135 of SEQ ID NO: 1 or a corresponding position in a homologous sequence, and wherein preferably the at least one mutation allows binding of SAP05 to a non-plant vWA domain7. The modified secreted SAP05 protein of claim 6, wherein the at least one mutation is selected from positions 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 124, 125, 126, 127, 128, 129, 130, 131 and 132 or a corresponding position on a homologous sequence.

8. A modified secreted AY-WB protein 5 (SAP05) comprising at least one mutation, wherein the at least one mutation is at a position selected from positions 40 to 70 or 120 to 135 of SEQ ID NO: 1 or a corresponding position ina homologous sequence, and wherein preferably the at least one mutation allows binding of SAP05 to a non-plant vWA domain.

9. The modified secreted SAP05 protein of claim 8, wherein the at least one mutation is selected from positions 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 124, 125, 126, 127, 128, 129, 130, 131 and 132 a corresponding position in a homologous sequence.

10. The modified SAP05 protein of any preceding claim, wherein preferably the homologous sequence is selected from SEQ ID NO: 7, 9, 11 , 12, 14 and 16 and functional variants thereof.

11. A fusion compound comprising a Rpn10 binding moiety and a target-binding moiety, wherein the Rpn10 binding moiety binds one or more positions selected from positions 24, 27, 31, 34, 42, 68, 70, 71 and 77 on SEQ ID NO: 5 or a corresponding position on a homologous sequence.

12. The fusion compound of claim 11 wherein the Rpn10 binding moiety is a SAP05 peptide, wherein the SAP05 peptide is the modified SAP05 protein of any of claims 1 to 10.

13. The fusion compound of claim 11, wherein the Rpn10 binding moiety is selected from peptides, cyclic peptides, binding domains, small molecules / chemicals, t-cell receptors, antibodies, functional fragment of an antibody, nanobodies, monobodies, (peptide) macrocycles, DARPins, affibodies, adnectins, affimers, anticalins and aptamers.

14. The fusion compound of any one of claims 11 to 13, wherein the fusion compound further comprises a RPN10 protein or a vWA domain, wherein preferably the RPN10 protein comprises an amino acid sequence as defined in SEQ ID NO: 5, 21 or 23 or a functional variant thereof and wherein the vWA domain comprises an amino acid sequence as defined in SEQ ID NO: 18 or a functional variant thereof.

15. The fusion compound of any of claims 11 to 14, wherein the target-binding moiety is selected from peptides, cyclic peptides, binding domains, small molecules / chemicals, t-cell receptors, antibodies, functional fragment of an antibody, nanobodies, monobodies, (peptide) macrocycles, DARPins, affibodies, adnectins, affimers, anticalins and aptamers.

16. The fusion compound of any of claims 11 to 15, wherein the fusion compound is a bi-specific antibody, wherein the bi-specific antibody is capable of binding Rpn10 and a target.

17. An expression vector comprising a nucleic acid sequence encoding the modified SAP05 of any of claims 1 to 10 or the fusion compound of any of claims 11 to 16.

18. A cell comprising the modified SAP05 of any of claims 1 to 10, the fusion compound of any of claims 11 to 16 or the expression vector of claim 17.

19. A transgenic organism expressing modified SAP05 of any of claims 1 to 10, the fusion compound of any of claims 11 to 16 or the expression vector of claim 17, wherein the organism is not a human.

20. The modified SAP05 protein of any of claims modified SAP05 of any of claims 1 to 10, the fusion compound of any of claims 11 to 16 or the expression vector of claim 17, for use as a medicament.

21. Use of the modified SAP05 of any of claims 1 to 10, the fusion compound of any of claims 11 to 16 or the expression vector of claim 17 in targeted protein degradation.

22. The use of claim 21 , wherein the targeted protein degradation is ubiquitin- independent protein degradation.

23. A method of targeted protein degradation, the method comprising applying the modified SAP05 of any of claims 1 to 10, the fusion compound of any of claims 11 to 16 or the expression vector of claim 17 to a sample.

24. A method of controlling the level of a target protein, the method comprising applying the modified SAP05 of any of claims 1 to 10, the fusion compound of any of claims 11 to 16 or the expression vector of claim 17 to a sample.

25. The method of claim 24, wherein a fluorescently tagged antibody is applied to the sample in order to label a protein of interest prior to applying the fusion compound, wherein the target-binding moiety of the fusion compound is specific for the fluorescent tag, and wherein degradation of the labelled protein of interest in the sample can be detected by a decrease in fluorescent signal.

26. The method of any one of claims 24 to 25 wherein the target protein is a cytosolic protein or a cell surface protein.

27. A method of gene editing, comprising: a) introducing a CRISPR-Cas system comprising a CRISPR enzyme to a cell or organism, b) allowing the CRISPR-Cas system to edit a gene,c) delivering a fusion compound according to claims 11 to 16 or the expression vector of claim 17 comprising a target moiety that is specific for said CRISPR enzyme, and d) allowing the fusion compound to degrade the CRISPR enzyme so as to inhibit any further activity of the CRISPR-Cas system.

28. The method of claims 23 or 24 wherein the target protein can cause pathology in a target organism or wherein the target protein is a drug target.

29. A method of modulating a physiological response in an organism, the method comprising administering a fusion compound of any one of claims 11 to 16 or an expression vector claim 17.

30. The method of claim 29, wherein the physiological response is selected from a stress response, an immune response, a hormone response or a light response.

31. A method of treating a condition in a patient in need thereof, the method comprising administering a fusion compound of any one of claims 11 to 16 or an expression vector of claim 17.

32. The method of claim 31 , wherein the condition is characterised by increased expression or activity of a target protein, and the target-binding moiety is specific for said target protein.

33. A method of treating an infection caused by a microorganism, the method comprising administering a fusion compound of any one of claims 11 to 16 or an expression vector of claim 17, wherein the target binding moiety is specific for a protein expressed by a microorganism.

34. A method of increasing the immunogenicity of a protein, the method comprising administering a fusion compound of any one of claims 11 to 16 or an expression vector of claim 17, wherein the target binding moiety is specific for the protein, and wherein proteasome degradation of the protein results in increased antigen presentation of peptides degraded from the protein.

35. The method of claim 34, wherein the fusion compound is administered to a subject suffering from an infection or cancer.

36. A method for creating a protein knockout model, the method comprising administering a fusion compound of any one of claims 11 to 16 or an expression vector of claim 17 to a cell or an organism.

37. The method of claim 36, where the model is a disease model where the disease is caused or characterised by a dysfunction or absence of a target protein, and the target moiety is specific for the target protein.

38. A method of identifying a degradation effect of a target protein in a biological system, the method comprising applying the fusion compound of any of claims 11 to 16 or an expression vector of claim 17 to the biological system.

39. The method of claim 38, wherein the target protein is a regulatory protein, and the method further comprises performing RNA sequencing.

40. A method of increasing yield in a plant, the method comprising introducing the fusion compound of any of claims 11 to 16 or an expression vector of claim 17, to the plant, wherein the target-binding moiety is specific for a target protein the expression or activity of which is negatively correlated with yield.

41. A method of reducing or removing glutens in a plant, the method comprising introducing the fusion compound of any of claims 11 to 16 or an expression vector of claim 17 to the plant, wherein the target-binding moiety is specific for gliadin or glutenin.

42. A pharmaceutical composition comprising the fusion compound of any of claims 11 to 16 or an expression vector of claim 17 and a pharmaceutically acceptable diluent, carrier or excipient.

43. The pharmaceutical composition of claim 42 for use in treatment of cancer, infection, a neurodegenerative disorder or a proteopathy.

44. A screening library comprising a plurality of fusion compounds according to any one of claims 11 to 16, wherein each fusion compound comprises a different target-binding moiety.

45. A method for screening target-binding moieties using the screening library of claim 44.

46. A kit comprising a fluorescently tagged antibody and a fusion compound of any one of claims 11 to 16 or the expression vector of claim 17, wherein the targetbinding moiety of the fusion compound is specific for the fluorescent tag.

47. A method of identifying a SAP05 variant capable of binding to a non-plant RPN10, the method comprising screening a library of SAP05 variants, wherein the variants comprise at least one mutation, and identifying variants with binding to non-plant RPN10.

48. An expression vector comprising a nucleic acid encoding the SAP05 of the invention, wherein the nucleic acid sequence encodes an amino acid sequence as defined in SEQ ID NO: 25 to 28 or a functional variant thereof.

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