Modified fusion proteins and nucleic acid constructs

Abstract

The present invention relates to modified fusion proteins and nucleic acid constructs suitable for protein degradation in cells. The fusion protein comprises an RING structural domain; and an adaptor domain, wherein the adaptor domain is capable of positioning the RING domain together with a substrate. The fusion protein is incapable of N-terminal self ubiquitination and has an increased cell half-life. The invention also relates to compositions comprising these fusion proteins and nucleic acids, and the use of the fusion proteins and nucleic acid constructs in therapy.

Description

Technical Field

[0001] The present invention relates to modified fusion proteins and nucleic acid constructs suitable for protein degradation in cells. The fusion proteins are unable to undergo N-terminal autoubiquitination and have an increased cell half-life. The present invention also relates to compositions comprising these fusion proteins and nucleic acids, and the use of the fusion proteins and nucleic acid constructs in therapy. Background Art

[0002] Protein degradation occurs naturally in cells and provides an endogenous mechanism to prevent the occurrence of misfolded proteins and mediate cellular responses. The main pathway for protein degradation is through the ubiquitin-proteasome system (UPS). The ability to manipulate the UPS to redirect the system and provide targeted protein degradation in cells has great potential for application in research, drug discovery and therapeutics.

[0003] Selective depletion of target proteins enables the study of protein function and dynamic protein interactions at the cellular level. Such selective depletion is particularly useful in drug discovery, where small molecules called “proteolysis-targeting chimeras” (PROTACs) can be used to redirect protein degradation to induce selective depletion of target proteins (Schapira et al., 2019). Similarly, “Trim-Away TM ” and other techniques, which utilize a specific component of the UPS, an E3 ubiquitin ligase called TRIM21, to selectively deplete antibody-bound target proteins (Clift et al. 2017; Zeng et al. (2021); Castro-Dopico, T. et al. 2019; Chen, X et al. 2019). Another strategy is also disclosed in WO 2022175549, which utilizes constructs containing the RING domain of an E3 ubiquitin ligase. These emerging tools and drug discovery platforms enable the study of protein interactions in a post-translational context and avoid many of the limitations associated with genetic manipulation, which often fails to provide insight into the phenotype and can be expensive and time-consuming.

[0004] Targeted protein degradation has the potential for therapeutic applications (Wu, T et al. 2020), especially for diseases associated with excessive protein production or abnormal protein aggregation. Using targeted protein degradation as a therapeutic strategy can minimize off-target effects of drugs and avoid or reduce systemic drug exposure.

[0005] TRIM proteins are the largest family of E3 ligases in mammals. They include TRIMs that inhibit viral infection (TRIM5 (Stremlau et al., 2004), TRIM21 (Mallery et al., 2010), TRIM22 (Pagani et al., 2021), TRIM25 (Galao et al., 2022)), TRIMs that activate innate immunity (TRIM32 (Zhang et al., 2012), TRIM56 (Tsuchida et al., 2010), TRIM65 (Kato et al., 2021), RIPLET (Cadena et al., 2019)), and TRIMs that inhibit transcription (TRIM4 (Herquel et al., 2011), TRIM28 (Robbez-Masson et al., 2018)). In particular, the intracellular antibody receptor TRIM21 is responsible for the Trim-Away TM TRIM ligases mediate targeted protein degradation during ubiquitination. Despite the importance of TRIM ligases, their ubiquitination mechanism remains elusive. TRIM ligases contain both substrate targeting domains and catalytic domains in one polyprotein. However, how TRIM catalyzes ubiquitination is not fully understood, especially in terms of activation, ubiquitin priming, and chain elongation.

[0006] The mechanism of TRIM catalysis is currently mainly obtained through experiments on two antiviral proteins, TRIM5 and TRIM21. Both proteins are dimers containing RING, B-box, coiled coil and PRYSPRY domains. Each RING domain is arranged at both ends of an extended antiparallel coiled coil (Sanchez et al., 2014b). Although ubiquitination of monomeric RING can be detected in vitro, dimerization is required for full cellular activity (Dickson et al., 2018; Zeng et al., 2021). TRIM21 also undergoes supramolecular aggregation (Zeng et al., 2021), including aggregation on the surface of viral capsids (McEwan et al., 2012), but TRIM21 is anchored to its substrate through an intermediate antibody molecule (Mallery et al., 2010). The Fab of each antibody binds to the substrate, while the Fc binds to TRIM21 PRYSPRY (James et al., 2007).

[0007] TRIM ligases are degraded together with their substrates. This has been demonstrated for TRIM5 during HIV infection (Rold and Aiken, 2008) and TRIM21 with multiple substrates during Trim-Away (Clift et al., 2017). Furthermore, TRIM21 and its substrates are degraded with matching kinetics, suggesting that they are processed together as a complex (Clift et al., 2017). In support of TRIM ligase self-degradation, light-induced aggregation of the TRIM21 RING-cryptochrome 2 fusion is sufficient to lead to ligase degradation (Zeng et al., 2021). Meanwhile, TRIM5 self-degradation can be induced simply by ectopic overexpression (Diaz-Griffero et al., 2006), which results in the formation of large oligomers called ‘cytoplasmic bodies’, a phenomenon that is likely driven by B-box trimerization (Diaz-Griffero et al., 2009; Wagner et al., 2016).

[0008] It has been proposed that ubiquitination of the ligase alone can drive proteasome recruitment, leading to degradation of the entire TRIM: substrate complex (Diaz-Griffero et al., 2006; Kiss and James, 2022; Mallery et al., 2010; Towers, 2007). Due to the degradation of the entire TRIM: substrate complex, the turnover of TRIM-containing proteins in cells may be rapid.

[0009] Therefore, it is necessary to develop more fusion proteins and corresponding nucleic acid constructs for selectively degrading proteins in cells, wherein the fusion proteins and nucleic acid constructs have improved cell half-lives. Such fusion proteins will be particularly suitable for both therapeutic and research fields. Summary of the invention

[0010] The present invention relates to fusion proteins that are suitable for degrading proteins in cells and do not undergo N-terminal autoubiquitination, and nucleic acid constructs encoding such proteins. Specifically, a fusion protein comprising at least one RING domain and an adapter sequence is suitable for degrading proteins in cells. The inventors surprisingly found that modification of the N-terminus of the fusion protein can decouple ligase and substrate degradation. The inventors provide a fusion protein that can be used to degrade proteins, wherein the target substrate is degraded, while the fusion protein is not degraded.

[0011] In a first aspect, the present invention provides a fusion protein comprising:

[0012] - at least a first RING domain; and

[0013] - an adaptor domain capable of positioning the RING domain and the substrate together, and

[0014] The fusion protein is incapable of N-terminal autoubiquitination.

[0015] The fusion protein comprising the RING domain cannot undergo N-terminal autoubiquitination. The fusion protein cannot serve as a substrate for the E2 enzyme Ube2W. The N-terminus of the fusion protein is modified to inhibit the ubiquitination of the fusion protein itself by the E2 enzyme, especially Ube2W. The E2 enzyme Ube2W is still able to bind to the fusion protein. The fusion protein can ubiquitinate the substrate of the fusion protein using Ube2W, but the N-terminal ubiquitination of the fusion protein itself is inhibited (e.g., autoubiquitination). The inventors have found that inhibiting the ability of the fusion protein comprising RING to undergo N-terminal autoubiquitination slows down the self-renewal of the fusion protein, i.e., increases the cell half-life, allowing it to last longer in the cell without hindering substrate degradation. The fusion protein will not degrade with its target substrate. Compared with the equivalent unmodified fusion protein, the modified fusion protein has an increased half-life in the cell while still maintaining the ability to degrade the substrate protein in the cell.

[0016] The second aspect of the present invention provides a nucleic acid construct encoding the fusion protein according to the first aspect.

[0017] The third aspect of the present invention provides a nucleic acid construct, comprising a first nucleic acid sequence encoding a first RING domain and a second nucleic acid sequence encoding an adaptor domain, wherein the nucleic acid construct encodes a fusion protein that is incapable of N-terminal autoubiquitination.

[0018] The fourth aspect of the present invention provides a pharmaceutical composition, comprising the fusion protein according to the first aspect or the nucleic acid according to the second aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0019] A fifth aspect of the present invention provides a fusion protein according to the first aspect or a nucleic acid construct according to the second aspect for use as a medicament.

[0020] The sixth aspect of the present invention provides a method for degrading a target protein in a cell, the method comprising introducing the fusion protein according to the first aspect or the nuclear construct according to the second aspect into the cell.

[0021] The seventh aspect of the present invention provides a method for increasing the cellular half-life of a fusion protein comprising a RING domain and an adaptor domain, wherein the adaptor domain is capable of positioning the RING domain together with a substrate, and the method comprises modifying the fusion protein so that the fusion protein cannot undergo N-terminal autoubiquitination.

[0022] The eighth aspect of the present invention provides a method for producing the fusion protein according to the first aspect, the method comprising:

[0023] (a) culturing a host cell comprising a first vector encoding a fusion protein under conditions allowing expression of the fusion protein, wherein the fusion protein comprises a RING domain and an adaptor domain; and

[0024] (b) obtaining the expressed protein from the host cell, wherein the fusion protein is incapable of N-terminal autoubiquitination.

[0025] Additional aspects and embodiments of the invention are described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Dimeric Ube2W and RING aggregation is required for ligase autoubiquitination. (A) TRIM21 RING (blue) in complex with Ube2W (pink). X-ray structure. (B) Close-up of the E2:E3 interface. (C) Structural model of the RING:Ube2W~Ub complex based on the RING:Ube2W structure and superposition of the Ub-RING:Ube2N~Ub:Ube2V2 structure (7BBD) (Kiss et al., 2021). Ube2N~Ub is superimposed on Ube2W. (D) Close-up of the RING:Ube2W~Ub model showing a potential salt bridge between TRIM21 E13 and ubiquitin K11. (E) Schematic model of the catalytic RING topology for N-ubiquitination of TRIM21 by Ube2W dimers. (F) Ube2W-mediated TRIM21 RING(R) monoubiquitination experiments performed using 10 μM T21-R-PS or RR-PS and 0.25 μM Ube2W WT or monomeric V30K / D67K. Representative examples from n = 2 independent experiments. (G) Schematic model of antibody-induced recruitment of two RINGs or two RING dimers. Only the latter satisfies the ‘two plus one’ model of RING autoubiquitination (Fletcher et al. 2018; Kiss et al., 2021). (H) Antibody-induced N-ubiquitination of 100 nM T21-R-PS or RR-PS in the absence or presence of 1 molar equivalent of anti-GFP antibody. Ube2W was serially diluted (25, 50, 100, 200 nM). Representative examples from n = 3 independent experiments. (I) Quantification of monoubiquitination of (H). The figure shows the mean and sem from n = 3 independent experiments. (J) RPE-1CAV1-mEGFP cells were electroporated with PBS or anti-GFP antibody ± the indicated E2 protein, and CAV1-mEGFP fluorescence was quantified using the IncuCyte system 24 hours later. Values ​​are normalized to PBS control conditions. The figure shows the mean and sem from n = 3 independent experiments. Statistical significance in (I) and (J) was based on two-tailed Student's t-test.

[0027] Figure 2:Ligase N-terminal ubiquitination is not required for substrate degradation and antiviral activity. (A) shows a schematic model of N-terminal acetylation of TRIM21 by AcCoA and NAT, and then incubating the acetylated or unmodified ligase with ubiquitin (Ub) and Ube2W in the ubiquitination reaction. (B) Protein staining gel image of the ubiquitination reaction described in (A) using TRIM21 RR-PS ligase. Monoubiquitination of the ligase incubated with AcCoA and NAT was inhibited. Representative examples from n=3 independent experiments. (C) shows a schematic model of the Trim-Away experiment, in which the antibody is electroporated into cells together with RR-PS (±N-acetylated). Once in the cell, a ternary complex is formed with the target protein. If degradation is driven by ligase N-terminal ubiquitination, the RR-PS activity of the N-terminal acetylation will be inhibited. (D) The results of the Trim-Away experiment described in (C) show that N-terminal acetylation of the ligase does not change the kinetics of substrate (CAV1-GFP) degradation. RPE-1CAV1-mEGFP cells were electroporated with PBS or anti-GFP antibody (1.67 μM) ± RR-PS protein (2.1 μM), and CAV1-mEGFP fluorescence was quantified using the IncuCyte system. "Time" indicates the number of hours (hour, h) after electroporation. Values ​​are normalized to PBS control conditions. The figure shows the mean and sem from n = 4 technical replicates. Representative examples from n = 2 independent experiments. Note that anti-GFP alone degrades CAV1-mEGFP due to the presence of endogenous cellular TRIM21. (E) A schematic model showing the electroporation of N-acetylated ligase (Ac-RR-PS) into cells and then infection with Adv5 in the presence of anti-hexon antibody 9C12. If ligase N-terminal ubiquitination is required for the antiviral function of TRIM21, neutralization of infection and immune signaling will be inhibited. (F, G) Neutralization of AdV5 infection by increasing 9C12 concentrations (F) and AdV5-9C12-induced NFkB activation (G) in TRIM21 KO cells after electroporation with PBS or RR-PS±N-acetylation. 9C12H433A does not bind to TRIM21PRYSPRY. Graphs show mean and sem from n=3 independent experiments. Statistical significance is based on two-way ANOVA (F) and Student's t-test (G).

[0028] Figure 3: N-terminal ubiquitination regulates ligase turnover in cells. (A) Schematic model showing electroporation and ubiquitin-proteasome-dependent self-degradation of RR-PS±N-acetylated. (B) Western blot of the experiment described in (A) 1 hour after electroporation. RR-PS protein levels were restored by adding 10 μM proteasome inhibitor epoxomicin. Acetylated RR-PS protein persists in cells regardless of proteasome inhibition. Representative examples from n=2 independent experiments. (C) Schematic model showing electroporation of RR-PS±N-acetylated into cells followed by delayed Trim-Away or Adv5 neutralization experiments. (D) For delayed Trim-Away experiments, mRNA (80 nM) encoding an antibody construct (vhhGFP4-Fc) responsible for recruiting RR-PS to a substrate (CAV1-mEGFP) was co-electroporated into NIH3T3-CAV1-mEGFP cells with PBS or RR-PS ± N-acetylation (2.4 μM); the Trim-away experiment was delayed for approximately 2 hours until the translation of the vhhGFP4-Fc protein was completed. The figure shows the mean and sem of 4 technical replicates of CAV1-mEGFP fluorescence quantified using the IncuCyte system. Values ​​are normalized to PBS control conditions (no vhhGFP4-Fc). "Time" indicates hours (hour, h) after electroporation. Representative examples from n = 2 independent experiments. Note that NIH3T3 cells do not contain endogenous TRIM21, and expressing vhhGFP4-Fc in the absence of TRIM21 activity stabilizes GFP. (E) For delayed Adv5 neutralization experiments, HEK293T TRIM21 KO cells were infected with AdV5±9C12 two hours after electroporation of PBS or RR-PS±N-acetylated. Graphs show mean and sem from n=3 independent experiments. Statistical significance was based on two-way ANOVA.

[0029] Figure 4: TRIM21 ubiquitinates itself and its substrates independently. (A) Schematic diagram of an in vitro ubiquitination reaction is shown, in which the ligase (RR-PS), substrate (GFP), and anti-GFP antibody are incubated with various E2 enzymes to promote monoubiquitination or polyubiquitination. (B) Western blot of the experiment described in (A). The top panel is the GFP blot, the middle panel is the IgG blot, and the bottom panel is the TRIM21 blot. Depending on the E2 enzyme present, monoubiquitinated species or higher molecular weight tailing bands indicative of polyubiquitination can be observed. Representative examples from n = 2 independent experiments. (C) Western blot of an experiment similar to (B), but here it is a Western blot from an experiment comparing RR-PS to Ac-RR-PS. Note that while RR-PS ubiquitinates itself, the antibody heavy chain, and the substrate, Ac-RR-PS only ubiquitinates the antibody and the substrate. Representative examples from n = 2 independent experiments. See also Fig. 9 .

[0030] Figure 5 :Substrate ubiquitination is synchronized with substrate degradation during Trim-Away. (A) shows a scheme for a Trim-Away experiment, in which antibodies are electroporated into cells expressing endogenous TRIM21. Ubiquitination and degradation are then monitored in the presence or absence of the proteasome inhibitor MG132. (B, C) RPE-1 cells were electroporated with PBS or (B) anti-ERK1 antibody or (C) anti-IKKα antibody, and whole cell lysates were collected at the specified time after electroporation for immunoblotting analysis. Short-term exposure shows degradation of TRIM21 and substrates. Long-term exposure reveals substrate ubiquitination, followed by degradation of ubiquitinated species. (D) RPE-1 cells were electroporated with PBS or anti-IKKα antibody ± MG132, and whole cell lysates were collected 3 hours after electroporation for immunoblotting analysis. (E) RPE-1TRIM21 KO cells reconstituted with TRIM21-HA or empty vector (EV) were electroporated with PBS or anti-ERK1 antibody ± MG132, and whole-cell lysates were collected 1 hour after electroporation for immunoblot analysis. Representative examples from 3 independent experiments.

[0031] Figure 6:Trim-Away mediates protein depletion in the absence of lysine ubiquitination of the ligase or substrate. (A). Schematic diagram of the T21R-vhhGFP4 fusion protein and the substitution to remove all lysines is shown. (B, C) RPE-1CAV1-mEGFP-Halo cells were electroporated with PBS or T21R-vhhGFP4 protein ± lysine ± N-acetylation. (B) CAV1-mEGFP-Halo fluorescence was quantified using the IncuCyte system. "Time" indicates the number of hours (hour, h) after electroporation. Values ​​are normalized to PBS control conditions. The figure shows the mean and sem from n=4 technical replicates. (C) Whole cell lysates were collected 3 hours after electroporation for immunoblot analysis. Representative examples (B, C) from 3 independent experiments. (D) shows a schematic diagram of a model of a lysine-free substrate consisting of a vhhGFP4 nanobody with 12 copies of the native lysine-free ALFAtag epitope (with lysine substitutions in the box). (E) shows a scheme for a Trim-Away experiment, in which an anti-ALFAtag antibody (NbALFA-Fc) is electroporated into a cell expressing endogenous TRIM21 and a lysine-free substrate (12xALFAtag-vhhGFP4 K0 ) in cells. (FH) PBS or NbALFA-Fc was used to express substrates with lysine (12xALFAtag-vhhGFP4) or substrates without lysine (12xALFAtag-vhhGFP4 K0) RPE-1WT or TRIM21 KO cells were electroporated, and whole cell lysates were collected 8 hours after electroporation for capillary-based immunoblotting analysis. Lane views (F) and quantitative analysis (G, H) of substrate (G) and TRIM21 (H) protein levels normalized to PBS conditions. The figure shows the mean and sem from 2 independent experiments. Statistical significance is based on two-way ANOVA. Note that in the absence of TRIM21, the binding of NbALFA-Fc stabilizes the substrate. (I) Schematic diagram of the Trim-Away experiment without lysine. The substrate is composed of vhhGFP4 nano antibodies with 12 copies of the ALFAtag epitope. The ligase is composed of T21 RING fused to the anti-ALFAtag nano antibody. 12xALFAtag allows the aggregation of multiple T21 RINGs, thereby triggering degradation. In the experiment, the substitution required to remove lysine from each domain is shown in a box. Note that ALFAtag and HA epitopes do not naturally contain lysine. (J, K) Expression of substrates with lysine (12xALFAtag-vhhGFP4) or without lysine (12xALFAtag-vhhGFP4) in water (control) or mRNA encoding the indicated constructs. K0 ) of RPE-1TRIM21 KO cells were electroporated, and whole cell lysates were collected 8 hours after electroporation for capillary-based immunoblotting analysis. Substrate protein levels are normalized to control conditions in lane views (J) and quantitative analysis (K). Figure shows the mean value and sem from 2 independent experiments. Statistical significance from control conditions is based on two-way ANOVA. Note that the combination of independent NbALFA stabilizes the substrate.

[0032] Figure 7 :Interaction between Ube2W and TRIM21 RING. (A) Targeting TRIM21 RING M10E (R M10E ) sequence shows the histogram of chemical shift perturbations (CSPs). These CSPs were generated by converting Ube2W V30K / D67K / C91K In a 1:1 molar ratio 15 NMR titration of the TRIM21 triionic mutants labeled with N. Blue circles indicate proline residues and white circles indicate unidentified assignments. (B) In the absence (blue) or presence of 1:1 molar equivalent of Ube2W V30K / D67 / C91K In the case of R M10E of 15 Partial overlay of N-HSQC spectra. In addition, the presence of 1:1 molar equivalent of Ube2W is also shown. V30K / D67 / C91KSpectra of TRIM21 mutants (light green for E12A, dark green for E12R, and orange for E13A) in the case of . (C) The histograms shown in (A) are shown here as overlays. (D) Ube2W-mediated TRIM21RING monoubiquitination experiment. Shown are time course graphs, where error bars represent sem from n = 3 independent experiments. Western blots are representative results of all repeated experiments. (E) RING dimers of different TRIM21 complexes are shown (RING:Ube2W, RING-box (5OLM) (Dickson et al., 2018), RING:Ube2N~Ub (two RING dimers in the asymmetric unit, 6S53) (Kiss et al., 2019), Ub-RING:Ube2N~Ub:Ube2V2 (7BBD) (Kiss et al., 2021)). Zn 2+ -Atoms are shown as grey spheres, isopeptide bonds are marked with arrows, and polar interactions are represented by black dashed lines.

[0033] Figure 8 :The TRIM21 RING can be N-terminally acetylated with NAT and AcCoA to block N-terminal autoubiquitination. (A) LC-MS / MS spectra of TRIM21 RR-PS after 4 hours of acetylation show that N-acetylated TRIM21 N-terminal peptides appear after digestion with the protease N-Asp. (B) Instant-Blue stained gel shows Ube2W-mediated monoubiquitination of TRIM21 with RR-PS. Acetylation reactions were performed for the indicated times before ubiquitination reactions. Gel images are representative results of n=2 independent experiments. (C) Similar to (B), but showing the results of ubiquitination reactions after 4 hours of N-acetylation and incubation with Ube2W and Ube2N / V2 for 1 hour. In contrast to N-terminal monoubiquitination, incubation with NAT and AcCoA does not affect the ubiquitin tailing characteristics that are unique to free K63 chain formation. Gel images are representative results of n=2 independent experiments.

[0034] Fig. 9 : Ubiquitination of ligase and substrate in vitro. (A) Western blot of ubiquitination reactions using RR-PS, anti-GFP antibody, GFP, Ube2W, and combinations of Ube2N / Ube2V2. Substrate and antibody heavy chain were ubiquitinated only in the presence of RR-PS, and substrate was ubiquitinated only in the presence of both antibody and RR-PS. Western blots are representative results of n=2 independently performed experiments.

[0035] Fig.10: Substrate ubiquitination during Trim-Away in living cells. (A) Western blots of Trim-Away time course experiments performed with anti-IKKα antibodies in the presence or absence of MG132. Proteasome inhibition rescues IKKα degradation and leads to accumulation of ubiquitinated proteins. Western blots are representative results of n = 2 independent experiments. (B) Ubiquitination of CAV1-mEGFP by T21RBCC-vhhGFP4 or T21R-vhhGFP4 fusions in the presence or absence of MG132. Western blots are representative results of n = 2 independent experiments. Ponceau staining indicates consistent loading. (C) Schematic diagram showing electroporation of T21R-vhhGFP4 protein fusions into cells expressing CAV1-mEGFP. Aggregation of RING fusions may lead to ubiquitination and complex degradation on substrates or ligases. (E) Expression of WT T21R-vhhGFP4 or a mutant that cannot catalyze ubiquitination (T21R-vhhGFP4 I18R / M72E ) Western blot of Trim-Away experiment. Represents representative results of n=3 independent experiments.

[0036] Fig.11 : List of plasmids used in the examples and their sequences (where applicable).

[0037] Fig.12 : List of proteins and their sequences (where applicable) used in the Examples. DETAILED DESCRIPTION

[0038] The inventors have discovered that inhibition of N-terminal autoubiquitination by the E2 enzyme Ube2W of a fusion protein comprising at least one RING domain and an adaptor domain increases the cellular half-life of the fusion protein while still maintaining its cellular activity.

[0039] Therefore, the present invention provides a fusion protein, the fusion protein comprising:

[0040] - at least a first RING domain; and

[0041] - an adaptor sequence capable of positioning the RING domain and the substrate together, and

[0042] The fusion protein is incapable of N-terminal autoubiquitination.

[0043] The fusion protein of the present invention has E3 ubiquitin ligase activity, but it cannot perform N-terminal self-ubiquitination. In some embodiments, the fusion protein cannot be ubiquitinated. The fusion protein can bind to the E2 enzyme Ube2W and use it to ubiquitinate the substrate, but after being modified, it will prevent its own ubiquitination (self-ubiquitination). By "cannot perform N-terminal self-ubiquitination", "cannot perform N-terminal self-ubiquitination", "inhibited N-terminal self-ubiquitination", or "cannot perform N-terminal self-ubiquitination" or the like, it means that the fusion protein cannot perform self-ubiquitination on itself, but can perform ubiquitination on other proteins that exist. In other words, the present invention provides a fusion protein containing RING, which cannot perform N-terminal ubiquitination on itself, but still has catalytic activity and can perform N-terminal ubiquitination on other proteins.

[0044] RING must be active to mediate degradation of target substrates, however, active RING itself will also degrade unless its autoubiquitination is blocked. By blocking the N-terminus of the fusion protein, it is possible to extend the half-life of the fusion protein in the cell. The inventors have found that it is possible to block the autoubiquitination of a fusion protein containing RING without affecting its ability to mediate substrate degradation. Modifying the N-terminus of the fusion protein to inhibit the autoubiquitination of the fusion protein means that once it is delivered to the cell, it will survive longer, thus lasting long enough to degrade multiple copies of the substrate. The fusion protein is no longer degraded with its target, thereby achieving more efficient and longer-lasting protein depletion.

[0045] Even in the absence of target substrate, the RING may have some residual activity and can be targeted by other ligases. Therefore, blocking the N-terminus allows RING-containing fusion proteins to persist longer in cells.

[0046] The N-terminus of the fusion protein is modified to inhibit the autoubiquitination of the fusion protein. The modification of the N-terminus of the fusion protein prevents the N-terminus ubiquitination of the fusion protein by an E2 enzyme such as Ube2W. This is achieved by making the reactive N-terminus of the fusion protein incapable of being covalently modified with ubiquitin by an E2 enzyme such as Ube2W. The fusion protein is still able to bind to Ube2W. The E2 enzyme, especially Ube2W, is still able to bind to the E2 enzyme binding site of the RING domain. However, the bound Ube2W is unable to conjugate ubiquitin to the N-terminus of the fusion protein.

[0047] The E2 enzyme binding site of the RING domain retains its ability to bind to the E2 enzyme, preferably retains its ability to bind to Ube2W.

[0048] In one embodiment, the first RING domain is located at the N-terminus of the fusion protein, and the adapter domain is located at the C-terminus of the RING domain. In other embodiments, the adapter domain is included at the N-terminus of the fusion protein.

[0049] In one embodiment, the fusion protein is N-terminally acetylated, i.e., the fusion protein comprises an acetyl group at its N-terminal residue. Capping the N-terminus of the fusion protein with an acetyl group can prevent ubiquitination of the N-terminus and thus avoid degradation of the fusion protein, such as during Trim-Away.

[0050] In some embodiments, the N-terminus of the fusion protein is capped by other chemical moieties that prevent the N-terminus from being ubiquitinated. The chemical moiety is covalently coupled to the N-terminus of the fusion protein. The chemical moiety inhibits Ube2W ubiquitination of the fusion protein. In addition to the acetyl group, the chemical moiety that can be conjugated to the N-terminus also includes other amine reactive moieties, such as a methyl group. Therefore, other modifications include when the fusion protein is N-terminally methylated, i.e., the fusion protein includes a methyl group at its N-terminal residue.

[0051] In some embodiments, the N-terminal of the fusion protein comprises an N-acetyltransferase recognition site. The N-acetyltransferase recognition site is a short amino acid sequence that the N-acetyltransferase can recognize. Preferably, the recognition site comprises the sequence DDDI (SEQ ID NO:14) or EEEI (SEQ ID NO:15), more preferably the sequence DDDI. The presence of these sites allows the acetylation of the fusion protein, so that the fusion protein comprises an acetyl group at its N-terminal.

[0052] In some embodiments, the N-terminus of the fusion protein is capable of N-terminal cyclization, preferably the fusion protein is capable of N-pyroglutamate cyclization. In order to promote the N-terminal cyclization of the fusion protein, the fusion protein may contain glutamic acid or glutamine as the N-terminal residue of the fusion protein. In some embodiments, the N-terminal glutamine is part of the sequence GFA located at the N-terminus of the fusion protein.

[0053] Once the fusion protein is N-terminally cyclized, the resulting fusion protein will contain an N-terminal pyroglutamine as the N-terminal residue. Thus, in some embodiments, the fusion protein contains an N-terminal pyroglutamic acid residue. Pyroglutamic acid as used herein refers to an amino acid derivative that cyclizes the free amino group of glutamic acid or glutamine to form a lactam. Without being bound by theory, it is believed that the presence of pyroglutamic acid at the N-terminus of the fusion protein can protect the fusion protein from N-terminal autoubiquitination by the E2 enzyme Ube2W.

[0054] In some embodiments, the N-terminal amino acid of the fusion protein is replaced or modified by an amino acid or an amino acid sequence, and the amino acid or amino acid sequence inhibits the ability of the E2 enzyme, such as Ube2W to ubiquitinate the fusion protein. In some embodiments, the N-terminal amino acid can be replaced by an amino acid sequence, and the amino acid sequence inhibits the Ube2W ubiquitination of the fusion protein, preferably inhibiting the ubiquitination of the fusion protein by the E2 enzyme. In some embodiments, the amino acids at positions 1, 2, 3, 4, and 5 are modified or replaced to form a sequence that inhibits the ubiquitination of the fusion protein by Ube2W. Preferably, at least the amino acids at positions 1, 2, and 3, more preferably at least the amino acid at position 1 are replaced or modified. For example, in one embodiment, the N-terminal amino acid can be replaced by a polyproline sequence, that is, the amino acids at positions 1, 2, 3, 4, and 5 can be replaced by a polyproline sequence, or by other sequences that can block the Ube2W ubiquitination of the fusion protein (preferably ubiquitination of the E2 enzyme). The amino acid segment can replace the equivalent amino acids at the beginning of the fusion protein. In some embodiments, the N-terminus of the fusion protein is modified by adding a sequence to the N-terminus that blocks ubiquitination of the fusion protein by Ube2W, preferably blocking the E2 enzyme, such as adding the sequence to the N-terminus of the first RING domain or the adapter domain. Therefore, in some embodiments, the present invention provides a fusion protein comprising a RING domain and an adapter domain, which fusion protein cannot be ubiquitinated.

[0055] The fusion protein of some embodiments of the present invention can be expressed as:

[0056] Ac-RING-RING-AD; NATRS-RING-RING-AD; Q-RING-RING-AD; E-RING-RING-AD; PCA-RING-RING-AD; Ac-RING-AD; NATRS-RING-AD; Q-RING-AD; E-RING-AD; or PCA-RING-AD.

[0057] Wherein RING=ring domain, AD=adaptor domain, Ac=acetyl group; NATRS=N-acetyltransferase recognition site, E=glutamic acid, Q=glutamine, PCA=pyroglutamic acid, and wherein a linker sequence may optionally be present between domains. Although when representing the fusion protein, the adaptor domain is located at the N-terminus of the RING domain, the adaptor domain and the RING domain may be in any order as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal autoubiquitination, such as making Ube2W unable to ubiquitinate the fusion protein.

[0058] The RING domain of the fusion protein may be derived from any suitable polypeptide. The RING domain is known in the art and was described by Freemont PS et al. in 1991, which functions as an E3 ligase (Meroni G and Roux G, 2005).

[0059] The RING domain used in the fusion protein of the present invention has E3 ubiquitin ligase activity. The RING domain of TRIM21 is an E3 ubiquitin ligase and targets the ubiquitin conjugating enzyme to a substrate. Members of the RING (Really Interesting New Gene) domain family generally have a consensus sequence Cys-X2-Cys-X( 9-39 )-Cys-X( 1-3 )-His-X( 2-3 )-(Ans / Cys / His)-X2-Cys-X( 4-48 )-Cys—X2-Cys (Deshaies RJ and JoazeiroC, 2009). The RING E3 ligase domain is present in a variety of proteins. Other RING domains include the RING domain from X-linked mammalian inhibitor of apoptosis protein (XIAP) and the RING domain of DER3 / Hrd1. Therefore, RING domains from other protein families used in the fusion protein are also included. The present invention is particularly suitable for RING domains that are capable of self-ubiquitination, i.e., have self-ubiquitination activity.

[0060] Preferably, the RING domain of the fusion protein is derived from a TRIM polypeptide. The TRIM family includes a large number of RING E3 ligases (Marin, I. et al., 2012). In a preferred embodiment, the RING domain is derived from a TRIM21 polypeptide, preferably from human TRIM21. The sequence of human TRIM21 is shown in SEQ ID NO: 1 (Uniprot: P19474).

[0061] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRK HRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCSS ALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVC RDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY(SEQ ID NO:1)

[0062] The RING domain of human TRIM21 comprises at least amino acids 3 to 81 of the human TRIM21 sequence as shown in SEQ ID NO: 1, preferably amino acids 1 to 85 of the human TRIM21 amino acid sequence as shown in SEQ ID NO: 1. The RING domain comprising amino acids 1 to 85 of human TRIM21 comprises the following sequence:

[0063] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEARE(SEQ ID NO:2)

[0064] Therefore, in one embodiment of the present invention, the RING domain comprises amino acids 3-81 of SEQ ID NO: 2, preferably amino acid residues 1-81 of SEQ ID NO: 2 or a variant thereof. In one embodiment, the RING domain comprises the sequence of SEQ ID NO: 2 or a variant thereof, preferably the RING domain of the fusion protein consists of the sequence of SEQ ID NO: 2 or a variant thereof.

[0065] The amino acids 3-81 of human TRIM21 comprise the following sequence:

[0066] SAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQ(SEQ ID NO:3)

[0067] The amino acids 1-81 of human TRIM21 comprise the following sequence:

[0068] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQ(SEQ ID NO:4)

[0069] In some embodiments, the RING domain comprises amino acid residues 2-81 of human TRIM21. In some embodiments, the RING domain consists of amino acid residues 2-81 of human TRIM21.

[0070] The amino acids 2-81 of human TRIM21 comprise the following sequence:

[0071] ASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQ(SEQ ID NO:5)

[0072] Preferably, using the default parameters of the BLAST computer program (Atschul et al., 1990. Provided by HGMP (Human Genome Map Project)), at the amino acid level, the variant sequence has at least 60% identity with the reference sequence. More preferably, the variant sequence of SEQ ID NO: 2 may have at least 65%, 70%, 75%, 80%, 85%, 90%, even more preferably 95% (still more preferably at least 99%) identity with the sequence of SEQ ID NO: 2 at the amino acid level.

[0073] "Identity (identity)" known in the art refers to the relationship between two or more polypeptide sequences determined by comparing sequences, or between two or more polynucleotide sequences. In the art, identity also means the sequence correlation (homology) between polypeptides or polynucleotide sequences as appropriate, as determined by the matching situation between such sequence strings. Although there are many methods to measure the identity between two polypeptides or two polynucleotide sequences, the method generally used to determine identity has been incorporated into a computer program. Preferred computer programs for determining the identity between two sequences include but are not limited to GCG program packages (Devereux et al., 1984, BLASTP, BLASTN and FASTA (Atschul et al., 1990)).

[0074] The N-terminal methionine of the expressed fusion protein may be cleaved from the expressed protein during or after translation, for example, by methionine aminopeptidase. Therefore, when referring to the N-terminal amino acid of the fusion protein as being modified, it also includes such a fusion protein: after the methionine residue is removed and the expressed fusion protein undergoes other translational or post-translational processing, the N-terminal residue of the modified fusion protein, such as the second residue of SEQ ID NO:2. Therefore, in some embodiments, if the RING domain is located at the N-terminus of the fusion protein, it is the arginine residue of the RING domain that is modified, for example, the residue will be acetylated or otherwise modified to inhibit the ability of Ube2W to ubiquitinate the fusion protein.

[0075] In some embodiments, a RING domain from a TRIM polypeptide other than TRIM21, such as a RING domain from TRIM5, TRIM7, TRIM 19, TRIM25, TRIM28, and / or TRIM32, can be used. Preferably, a RING domain from TRIM5 can be used.

[0076] The fusion protein comprises at least one RING domain, ie 1, 2, 3 or more RING domains, preferably the fusion comprises 2 or 3 RING domains, more preferably 2 RING domains.

[0077] In some embodiments, the fusion protein comprises a second RING domain, wherein the second RING domain is located between the first RING domain and the adapter sequence. By providing a fusion protein comprising at least two RING domains and an adapter domain, the fusion protein can form a part of a catalytic RING topology, thereby achieving degradation of the target protein.

[0078] Fusion proteins comprising two RING domains may degrade target proteins more effectively than corresponding fusion proteins comprising only one RING domain, however, double RING fusion proteins are constitutively active even in the absence of substrate (e.g., under steady-state conditions). This means that such fusion proteins act faster and more effectively, but their half-life is shorter. Therefore, inhibiting N-terminal autoubiquitination, for example by blocking the N-terminus of a fusion protein comprising a RING-RING format, is particularly beneficial to the RING-RING fusion protein format. Compared with single RING domain fusion proteins that are constitutively N-terminal ubiquitinated, such RING-RING fusion proteins that are not capable of N-terminal autoubiquitination have longer cellular half-lives and improved functions, and compared with corresponding RING-RING fusion proteins with autoubiquitination activity, their half-lives are also longer.

[0079] When the fusion comprises two RING domains, the adapter domain is preferably located at the C-terminus of the first RING domain and the second RING domain. The various domains of the fusion protein can be arranged in the order from N-terminus to C-terminus as RING domain-RING domain-adapter domain. In such embodiments, the amino acid sequence of the first RING domain is connected to the N-terminus of the second RING domain, and the adapter domain is connected to the C-terminal domain of the second RING domain. Such fusion proteins comprising at least two RING domains are described in WO2022 / 175549, wherein the adapter protein is described as a protein targeting domain, and the contents of the document are incorporated herein by reference.

[0080] When the fusion protein comprises two RING domains, the sequences of the RING domains are capable of dimerizing with each other to form a RING dimer. Preferably, the RING domains comprise the same sequence. In one embodiment, both the first RING domain and the second RING domain comprise the sequence of SEQ ID NO:2. If the RING domains comprise different sequences, at least the sequences of the first RING domain and the second RING domain should be capable of dimerizing with each other to form a RING dimer. In one embodiment, the first RING domain comprises the sequence of SEQ ID NO:2, and the second RING domain comprises a variant sequence of SEQ ID NO:2, or vice versa. The variant sequence may have at least 65%, 70%, 75%, 80%, 85%, 90% and preferably 95% (still more preferably at least 99%) identity with the sequence of SEQ ID NO:2.

[0081] The fusion protein comprises an adapter domain. The adapter domain helps to locate the fusion protein near the target substrate (i.e., the target protein to be degraded). The adapter domain is a polypeptide sequence that can locate the fusion protein and the target substrate together. The adapter domain can directly associate with the target protein, or can bind to an antibody or its antibody that binds to the target protein. The adapter protein can be located at the C-terminus or N-terminus of the construct, and preferably the adapter protein is located at the C-terminus of the fusion protein.

[0082] In one embodiment, the adaptor domain is referred to as a protein targeting domain. The protein targeting domain guides the fusion protein to the target protein (substrate) to be degraded, also referred to as the protein of interest. The protein targeting domain binds to the target protein or an antibody or fragment thereof or an antibody mimetic that binds to the target protein, and may be referred to as a "protein binding domain". The protein targeting domain may directly bind to the target protein to form a fusion protein-target protein complex, or bind to an antibody, an antibody fragment thereof or an antibody mimetic that binds to the target protein to form a fusion protein-antibody-target protein complex. The protein targeting domain is preferably connected to the C-terminus of the RING domain.

[0083] In one embodiment, the protein targeting domain is a PRYSPRY domain. In one such embodiment, the fusion protein comprises a first RING domain and a PRYSPRY domain. In another embodiment, the fusion protein comprises a first RING domain; a second RING domain; and a PRYSPRY domain. In another embodiment, the fusion protein comprises a first RING domain; a second RING domain; and a PRYSPRY domain. The PRYSPRY domain is positioned at the C-terminus or N-terminus of the RING domain (e.g., RING-PRYSPRY, RING-RING-PRYSPRY, PRYSPRY-RING-RING, or PRYSPRY-RING).

[0084] The N-terminus of the fusion protein is modified to prevent autoubiquitination of the fusion protein. For example, as discussed above, the N-terminus of the first RING is N-acetylated (Ac), the fusion protein comprises an N-acetyltransferase recognition site (NATRS), or the fusion protein comprises an N-terminal glutamic acid (E), glutamine (Q) or pyroglutamic acid (PCA) residue, such constructs can be represented as:

[0085] AC-RING-RING-PRYSPRY; NATRS-RING-RING-PRYSPRY; Q-RING-RING-PRYSPRY; E-RING-RING-PRYSPRY; PCA-RING-RING-PRYSPRY;

[0086] AC-RING-PRYSPRY; NATRS-RING-PRYSPRY; Q-RING-PRYSPRY; E-RING-PRYSPRY; PCA-RING-PRYSPRY.

[0087] Although the adaptor domain is located at the N-terminus of the RING domain when expressing a fusion protein, the adaptor domain and the RING domain can be in any order as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal autoubiquitination, such as making Ube2W unable to ubiquitinate the fusion protein.

[0088] The PRYSPRY domain may be derived from a TRIM polypeptide, preferably from TRIM21, more preferably from human TRIM21. The PRYSPRY domain comprises the PRY region and SPRY region of the amino acid sequence at positions 286-337 and 339-465 of human TRIM21 as shown in SEQ ID NO:1.

[0089] The amino acids 286-337 of human TRIM21 are:

[0090] AVHITLLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHF(SEQ ID NO:7)

[0091] The amino acids 339-465 of human TRIM21 are:

[0092] SGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPL(SEQ ID NO:8)

[0093] Preferably, the PRYSPRY domain comprises the following sequence:

[0094] AVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCR

[0095] DSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPL(SEQ ID NO:9)

[0096] In one embodiment of the present invention, the PRYSPRY domain comprises the sequence of SEQ ID NO: 9 or a variant thereof. Preferably, the variant sequence has at least 60% identity with the reference sequence at the amino acid level using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403-410 (1990), provided by HGMP (Human Genome Map Project). More preferably, the variant sequence of SEQ ID NO: 9 may have at least 65%, 70%, 75%, 80%, 85%, 90% and preferably 95% (even more preferably at least 99%) identity with the sequence of SEQ ID NO: 9 at the amino acid level.

[0097] The PRYSPRY domain of the fusion protein binds to the Fc region of an antibody or an antibody fragment thereof, for example, to the Fc region of human IgG1. The fusion protein binds to an antibody that binds to a target protein.

[0098] The Fc is a dimer and can therefore be bound by two PRYSPRY domains. The PRYSPRY domain of the first fusion protein binds to one monomer of the Fc, while the PRYSPRY domain of the second fusion protein binds to the second monomer of the Fc. This allows the two fusion proteins to co-localize, bringing the RING dimers of each fusion protein into close proximity, so that one RING dimer of one fusion protein can be used to mediate ubiquitination of the RING dimer of the other fusion protein.

[0099] In an additional embodiment of the present invention, the protein targeting domain is an antibody, an antibody fragment thereof or an antibody mimetic. Preferably, the antibody fragment molecule is selected from the group consisting of: Fab, Fab', F(ab')2, scFab, Fv, scFV, dAB, its VL fragment, its VH fragment and sdAb (i.e., nanobody), such as its VHH fragment. Preferably, the antibody fragment molecule is scFV or VHH.

[0100] In one embodiment, the fusion protein comprises a RING domain; and a VHH domain, wherein the RING domain is derived from a TRIM polypeptide, preferably from TRIM21, wherein the VHH binds to a protein of interest. Preferably, the VHH is located at the C-terminus of the RING domain. Preferably, the fusion protein does not comprise a coiled-coil domain and / or a B-box domain derived from TRIM, and the TRIM21 is located between the VVH domain and the RING domain, and more preferably, the fusion protein does not comprise any coiled-coil domain or B-box domain sequence. The N-terminus of the fusion protein is modified to prevent autoubiquitination of the fusion protein. For example, as discussed above, the N-terminus of the first RING is N-acetylated (Ac), the fusion protein comprises an N-acetyltransferase recognition site (NATRS), or the fusion protein comprises an N-terminal glutamic acid (E), glutamine (Q) or pyroglutamic acid (PCA) residue, and such constructs can be represented as:

[0101] AC-RING-VHH; NATRS-RING-VHH; Q-RING-VHH; E-RING-VHH; PCA-RING-VHH.

[0102] Although the VHH is located at the N-terminus of the RING domain when expressing the fusion protein, the VHH and RING domains may be in any order as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal autoubiquitination, such as making Ube2W unable to bind to the fusion protein. When the order of the fusion protein is VHH-RING, the N-terminus of the VHH may be N-acetylated.

[0103] In a preferred embodiment, the fusion protein comprises a first RING domain; a second RING domain; and a VHH domain, wherein the RING domain is derived from a TRIM polypeptide, preferably from TRIM21, wherein the VHH binds to a protein of interest. Preferably, the VHH is located at the C-terminus of the first RING domain and the second RING domain. Preferably, the fusion protein does not include a coiled-coil domain and / or a B-box domain derived from TRIM, and the TRIM21 is located between the VVH domain and the second RING domain, and more preferably, the fusion protein does not include any coiled-coil domain or B-box domain sequence. The N-terminus of the fusion protein is modified to prevent autoubiquitination of the fusion protein. For example, as discussed above, the N-terminus of the first RING is N-acetylated (Ac), the fusion protein comprises an N-acetyltransferase recognition site (NATRS), or the fusion protein comprises an N-terminal glutamic acid (E), glutamine (Q) or pyroglutamic acid (PCA) residue, and such constructs can be represented as:

[0104] AC-RING-RING-VHH; NATRS-RING-RING-VHH; Q-RING-RING-VHH; E-RING-RING-VHH; PCA-RING-RING-VHH.

[0105] Although the VHH is located at the N-terminus of the RING domain when expressing the fusion protein, the VHH and RING domains may be in any order as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal autoubiquitination, such as making Ube2W unable to bind to the fusion protein. When the order of the fusion protein is VHH-RING-RING, the N-terminus of the VHH may be N-acetylated.

[0106] The antibody, antibody fragment or antibody mimetic (e.g., VHH) of the fusion protein specifically binds to the target protein. The fusion protein directly binds to the target protein to be degraded at the target sequence of the target protein. Many proteins are oligomers (or at least dimers), or are part of a protein complex, so the antibody domain of the first fusion protein can bind to one monomer of the oligomer or protein complex, while the antibody domain of the second fusion protein binds to the second monomer of the oligomer or protein complex.

[0107] In one embodiment, the target protein can be a protein having a pathogenic form and a non-pathogenic form. The protein targeting domain binds to the pathogenic form of the protein but not to the non-pathogenic form of the protein. The pathogenic form of the target protein can contain a repeating domain or be a multimeric form of the protein.

[0108] The target protein may be an intracellular protein selected from the group consisting of Huntingtin and tau. In one embodiment, if the intracellular protein is Huntingtin, the protein target domain of the fusion protein binds to the polyglutamic acid sequence of Huntingtin.

[0109] In one embodiment, the adaptor domain encodes a protein or fragment thereof that is capable of localizing the RING domain to the substrate.

[0110] Preferably, the fusion protein does not include the B-box domain and the coiled-coil domain of TRIM21, and the TRIM21 is located between the first RING domain and the adapter domain. In one embodiment, if the fusion comprises two RING domains, the fusion protein may not include the B-box domain and the coiled-coil domain derived from any protein, and the protein is located between the second RING domain and the adapter domain, or between the first RING domain and the second RING domain. In one embodiment, the fusion protein does not include a B-box domain, such as a B-box domain derived from TRIM21, and preferably does not include a B-box domain derived from any protein. In one embodiment, the fusion protein does not include a coiled-coil domain derived from TRIM21, and preferably does not include a coiled-coil domain derived from any protein.

[0111] The B-box domain of human TRIM21 comprises amino acids 91 to 128 of the amino acid sequence of human TRIM21 as shown in SEQ ID NO: 1. The coiled-coil domain of human TRIM21 comprises amino acids 128 to 238 of the amino acid sequence of human TRIM21 as shown in SEQ ID NO: 1.

[0112] The B-box domain may comprise the following sequence:

[0113] RCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVPL(SEQ ID NO:10)

[0114] Therefore, in one embodiment, the fusion protein does not comprise the sequence of SEQ ID NO: 10 or a variant thereof.

[0115] The coiled-coil domain may comprise the following sequence:

[0116] EEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHS(SEQ ID NO:11)

[0117] Therefore, in one embodiment, the fusion protein does not comprise the sequence of SEQ ID NO: 11 or a variant thereof.

[0118] Preferably, the fusion protein does not comprise the sequences of SEQ ID NO: 10 and SEQ ID NO: 11 or their functional variants. Preferably, the variant sequence has at least 60% identity with the reference sequence at the amino acid level using the default parameters of the BLAST computer program (Atschul et al., 1990). More preferably, the variant sequence of SEQ ID NO: 10 or 11 may have at least 65%, 70%, 75%, 80%, 85%, 90% and preferably 95% (even more preferably at least 99%) identity with the sequence of SEQ ID NO: 10 or 11 at the amino acid level.

[0119] In an embodiment having two RING domains, not including the coiled-coil domain and the B-box domain between the second RING domain and the adaptor domain helps to allow the RING dimer of the fusion protein to be in close proximity with the RING dimer of the second fusion protein co-localized on the target protein (or an antibody bound to the target protein).

[0120] However, in some embodiments, the fusion construct may include a coiled-coil domain, a B-box domain, or a coiled-coil domain and a B-box domain. If a coiled-coil domain and / or a B-box is present in the fusion protein comprising two RING domains, it should be kept at a sufficient distance from the adapter domain and the RING domain so that the RING dimer of the first fusion protein can still be in close proximity with the RING dimer of the second fusion protein co-localized on the target protein (or an antibody bound to the target protein), for example, when both are bound to the same Fc.

[0121] Linker sequences may be provided between the RING domain and the adaptor domain and between each RING domain present in the fusion protein.

[0122] The linker sequence may be a sequence derived from a TRIM polypeptide, wherein the linker sequence does not encode the coiled-coil domain and / or the B-box domain of the TRIM polypeptide.

[0123] In other embodiments, standard linker sequences known in the art may also be used, for example, polyglycine or polyserine amino acid sequences, or linker sequences comprising a combination of glycine and serine residues, such as a linker having the sequence GSGGGGS (SEQ ID NO: 12). The length of the linker may vary.

[0124] However, in embodiments comprising two RING domains, the linker sequence between the two RING domains should be long enough to allow the fusion protein to be flexible and to allow the two RING domains present to dimerize. In one embodiment, the linker sequence between the RING domains is 1 to 50 amino acids long, preferably 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15 or 1 to 10 amino acids long. Most preferably, the linker sequence is 1 to 6 amino acids long, for example, the linker sequence is 1, 2, 3, 4, 5 or 6 amino acids long. In some embodiments, there may be no linker between the first RING domain and the second RING domain.

[0125] In embodiments where the fusion comprises two RING domains, the linker sequence between the RING domain and the adapter domain should be of sufficient length to allow close proximity when the RING dimer of the first fusion protein and the RING dimer of the second fusion protein are co-localized on a target protein (or an antibody bound to a target protein). The linker should be long enough to form a catalytic RING topology with the RING domain of the second protein.

[0126] In one embodiment, the length of the linker sequence between the adapter domain and the RING domain is between 5 and 50 amino acids, preferably the length of the linker sequence is 5 to 40, 5 to 30, 5 to 25, 10 to 25, 15 to 25, 15 to 20 or 10 to 20 amino acids. More preferably, the length of the linker sequence is 10 to 20 amino acids.

[0127] In one embodiment, the linker sequence may be a sequence derived from a TRIM polypeptide, wherein the linker sequence does not encode the coiled-coil domain and / or the B-box domain of a TRIM polypeptide. For example, the linker sequence provided between the RING domain and the adapter domain may comprise the sequence GTQGERGLKKMLRTC (SEQ ID NO: 13). In one embodiment, the sequence consists of the sequence GTQGERGLKKMLRTC (SEQ ID NO: 13).

[0128] Therefore, one embodiment of the present invention comprises a fusion protein, the fusion protein comprising a first RING domain; a second RING domain; an adapter domain located at the C-terminus of the first RING domain and the second RING domain, and a linker sequence located between the RING domain and the adapter domain, wherein the RING domain is derived from a TRIM polypeptide, preferably from TRIM21, and preferably wherein the fusion protein does not comprise a coiled-coil domain or a B-box domain, and wherein the fusion protein is not capable of N-terminal autoubiquitination. Preferably, the first RING domain is N-acetylated (Ac), the fusion protein comprises an N-acetyltransferase recognition site (NATRS) at its N-terminus, or the fusion protein comprises an N-terminal glutamic acid (E), glutamine (Q) or pyroglutamic acid (PCA) residue.

[0129] Another embodiment of the present invention comprises a fusion protein, the fusion protein comprising a RING domain; an adapter domain positioned at the C-terminus of the RING domain, and a linker sequence between the RING domain and the adapter domain, wherein the RING domain is derived from a TRIM polypeptide, preferably from TRIM21, and preferably wherein the fusion protein does not comprise a coiled-coil domain or a B-box domain, and wherein the fusion protein cannot undergo N-terminal autoubiquitination. Preferably, the N-terminus of the fusion protein is modified to prevent autoubiquitination of the fusion protein. Preferably, the RING domain is N-acetylated (Ac), the fusion protein comprises an N-acetyltransferase recognition site (NATRS) at its N-terminus, or the fusion protein comprises an N-terminal glutamic acid (E), glutamine (Q) or pyroglutamic acid (PCA) residue.

[0130] "Fusion protein" and "fusion polypeptide" refer to a polypeptide covalently linked by two or more parts, each of which is a polypeptide with a specific property, and the parts can be the same or different. The property can be a biological property, such as in vitro or in vivo activity. The property can also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two parts can be directly linked by a single peptide bond, or connected by a peptide linker containing one or more amino acid residues. Typically, the two parts and the linker are located in the reading frame with each other.

[0131] In this article, the term "fusion protein" generally means one or more proteins linked together by chemical means (including hydrogen bonds or salt bridges), or by peptide bonds of protein synthesis, or both. Typically, fusion proteins will be prepared by standard DNA recombinant technology in the art, and may be referred to as recombinant fusion proteins in this article.

[0132] The present invention also provides a nucleic acid construct encoding the fusion protein of the present invention. The nucleic acid construct may include a first nucleic acid sequence encoding a first RING domain; and a second nucleic acid sequence encoding an adapter domain. In some embodiments, the nucleic acid construct may include a third nucleic acid sequence encoding a second RING domain, and the second RING domain is positioned between the first RING domain and the adapter domain. The nucleic acid may encode a fusion protein that cannot be ubiquitinated by itself, and includes an N-terminus that inhibits the ubiquitination of the fusion protein by Ube2W. Although the fusion protein encoded by the nucleic acid construct inhibits the ubiquitination of the fusion protein by Ube2W, the fusion protein encoded by the core construct is able to bind to Ube2W.

[0133] In some embodiments, the nuclear construct comprises a sequence encoding an N-acetyltransferase recognition site at the N-terminus of the fusion protein. In some embodiments, the nucleic acid construct encodes a fusion protein having a sequence DDDI (SEQ ID NO: 14) or EEEI (SEQ ID NO: 15) at its N-terminus.

[0134] In some embodiments, the nuclear construct encodes a fusion protein comprising a glutamine or glutamic acid residue as the N-terminal residue. In some embodiments, the nucleic acid construct may further comprise a sequence encoding glutamine cyclase.

[0135] In some embodiments, the nucleic acid construct encodes a fusion protein having an amino acid sequence at its N-terminus that inhibits the fusion protein from being ubiquitinated by Ube2W. The amino acid sequence may be in addition to the RING domain and the adapter domain. Alternatively, the amino acid sequence may replace the N-terminal sequence of the RING or adapter domain (depending on which domain is located at the N-terminus). For example, when the nucleic acid construct encodes a fusion protein sequence having a RING domain at the N-terminus, the core construct encodes the RING domain, wherein the N-terminal residue of the RING domain has been replaced by an amino acid sequence that inhibits the fusion protein from being ubiquitinated by Ube2W, for example, the N-terminal residue of the RING domain may be replaced by a polyproline sequence. In another embodiment, wherein the nucleic acid construct encodes a fusion protein sequence having the RING domain at the N-terminus, the nuclear construct comprises an additional nucleic acid sequence encoding an amino acid sequence that inhibits ubiquitination of the fusion protein by Ube2W, wherein the amino acid sequence that inhibits ubiquitination of the fusion protein by Ube2W is located at the N-terminus of the RING domain, and the amino acid sequence at the N-terminus of the RING domain has been replaced by an additional sequence.

[0136] In some embodiments, the nucleic acid construct does not encode a coiled-coil domain; or does not encode a B-box domain, or does not encode both a coiled-coil domain and a B-box domain.

[0137] The nucleic acid construct may be provided in the form of a vector, such as an expression vector, and in addition, may also include chromosomal vectors, free-type and viral-derived vectors, such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papovaviruses such as SV40, vaccinia viruses, adenoviruses, lentiviruses, fowlpox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations of such vectors, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Generally, any vector suitable for maintaining, propagating or expressing nucleic acids in a host to express polypeptides can be used for expression in this regard. The vector may include multiple nucleic acid constructs as defined above, such as two or more. Preferably, the vector is a viral delivery vector, and more preferably, the vector is an adenoassociated virus (AAV) vector or a lentiviral vector.

[0138] Described nucleic acid construct of the present invention preferably comprises the promoter or other regulating and controlling sequence of described nucleic acid expression.Described promoter or other regulating and controlling sequence can be operably connected with the described nucleotide sequence of the described domain of described fusion protein of encoding.Promoter and other regulating and controlling sequence of the expression of control nucleic acid have been identified, and are known in the art.Those skilled in the art will note that, may not need to utilize complete promoter or other regulating and controlling sequence.May only need minimum necessary regulating and controlling element, and in fact, this type of element can be used to make up chimeric sequence or other promoter.

[0139] The term "nucleic acid construct" generally refers to a nucleic acid of any length, which may be DNA, cDNA or RNA, such as mRNA obtained by cloning or produced by chemical synthesis. The DNA may be single-stranded or double-stranded. The single-stranded DNA may be a coding sense strand, or it may be a non-coding strand or an antisense strand. For therapeutic use, the nucleic acid construct is preferably in a form that can be expressed in a subject to be treated.

[0140] The present invention also provides a host cell comprising such a nucleic acid construct. The fusion protein can be expressed in a variety of cells.

[0141] The present invention also provides a method for preparing the fusion protein of the present invention, the method comprising culturing or maintaining a host cell comprising the nuclear construct or vector described above under conditions such that the host cell produces the fusion protein, and optionally further comprising isolating the fusion protein.

[0142] In one aspect of the present invention, a method for producing the fusion protein of the present invention is provided. The method comprises:

[0143] (a) culturing a host cell comprising a first vector encoding a fusion protein under conditions allowing expression of the fusion protein, wherein the fusion protein comprises a RING domain and an adaptor domain; and

[0144] (b) obtaining the expressed protein from the host cell, wherein the fusion protein is incapable of N-terminal autoubiquitination.

[0145] In some embodiments, the first vector further encodes:

[0146] - an N-acetyltransferase recognition site at the N-terminus of the fusion protein, preferably wherein the N-acetyltransferase recognition site is the sequence DDDI (SEQ ID NO: 14) or EEEI (SEQ ID NO: 15);

[0147] - a glutamic acid residue or glutamine at the N-terminus of the fusion protein; or

[0148] - the sequence QFA located at the N-terminus of the fusion protein;

[0149] - an amino acid sequence located at the N-terminus of the fusion protein that inhibits the ubiquitination of the fusion protein by Ube2W, such as a polyproline sequence.

[0150] In some embodiments, the first vector further encodes glutamine cyclase, and the expressed fusion protein comprises an N-terminal glutamic acid residue or an N-terminal glutamine residue, and the method comprises culturing the host cell under conditions that allow expression of the fusion protein and the glutamine cyclase.

[0151] For example, in one embodiment, the first vector also expresses a fusion protein with a tev cleavage site, and the second vector expresses a tev protease. The fusion protein can be expressed on a polycistronic vector that also expresses a glutamine cyclase. The first vector is co-transfected with the second vector. During expression, the tev protease cleaves the RING fusion protein to expose the N-terminal glutamine that can be cyclized by the glutamine cyclase. The cyclized RING fusion protein with pyroglutamic acid at its N-terminus is then purified, for example, based on a C-terminal His tag. In such embodiments, the following fusion proteins are expressed during the production of the fusion protein of the present invention.

[0152] 1.T7P-RBS-ENLYVQQFA-RR-PY-6His-Stop-RBS-bQC(E45Q)-Stop-T7T

[0153] 2.T7P-RBS-MBP-TEVP(S219V)

[0154] Wherein T7P=T7 promoter, RBS=ribosome binding site, ENLYVQ (SEQ ID NO:16) is the tev site, QFA is the site of glutamine cyclase (QC), R=RING, PY=PRYSPRY domain, 6His=6 histidines, Stop=stop codon, T7T=T7 terminator, MBP=maltose binding protein, TEVP=TEV protease.

[0155] In some embodiments, the method includes the step of modifying the N-terminus of the expressed fusion protein. The modification of the N-terminus of the fusion protein can be performed in a variety of ways.

[0156] In some embodiments, the first vector also encodes an N-acetyltransferase recognition site at the N-terminus of the expressed fusion protein, and the method further comprises the step of incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA. The NAT can add an acetyl group to the N-terminus of the fusion protein.

[0157] The N-terminus of the fusion protein is capped with an acetyl group to prevent the N-terminus from being ubiquitinated and the fusion protein from being degraded, for example during Trim-Away.

[0158] In some embodiments, the first vector encodes a fusion protein having a glutamic acid residue or a glutamine residue at the N-terminus of the expressed fusion protein, and the method further comprises the step of incubating the fusion protein with a glutamine cyclase. In some embodiments, the fusion protein encoded by the vector has the sequence QFA as its N-terminal sequence. The glutamine cyclase cyclizes the free amino group of glutamic acid or glutamine to form a lactam, thereby providing pyroglutamic acid for the N-terminal residue of the fusion protein. Capping the N-terminus of the fusion protein with a pyroglutamic acid residue can prevent the N-terminus from being ubiquitinated and the fusion protein from being degraded, for example during Trim-Away.

[0159] In some embodiments, the fusion protein can be expressed in the form of an N-terminal methionine. Such residues can be cleaved from the expressed protein after translation, for example by methionine aminopeptidase. Therefore, when it is mentioned that the N-terminal amino acid of the fusion protein is modified, it also includes such a fusion protein, that is, what is modified is the N-terminal residue of the fusion protein after the methionine residue is removed from the expressed fusion protein, such as the second residue of SEQ ID NO:2. Therefore, in some embodiments, if the RING domain is located at the N-terminus of the fusion protein, it is the arginine residue of the RING domain that will be modified, for example, it will be acetylated or otherwise modified to inhibit autoubiquitination by Ube2W, or it can be replaced by an amino acid that can inhibit the ability of Ube2W to ubiquitinate the fusion protein.

[0160] In some embodiments, the method further comprises:

[0161] - methylating the N-terminal amino acid of the expressed fusion protein; or

[0162] - coupling a chemical moiety to the N-terminal amino acid of the expressed fusion protein, wherein the chemical moiety reduces the ability of the E2 enzyme Ube2W to ubiquitinate the fusion protein.

[0163] Thus, in some embodiments, the methods provide fusion proteins that are not ubiquitinated.

[0164] The present invention also provides a pharmaceutical composition comprising the fusion protein or nucleic acid construct of the present invention. The pharmaceutical composition may contain a variety of pharmaceutically acceptable carriers and / or excipients. Suitable pharmaceutically acceptable carriers and / or excipients are well known in the art. The pharmaceutical composition of the present invention may be administered by any suitable method known in the art, including but not limited to intravenous, intramuscular, oral or intraperitoneal. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule or foam.

[0165] The fusion proteins and nucleic acid constructs of the present invention can be used as drugs for therapy. In one embodiment, the present invention also provides treatment of neurological diseases, such as Alzheimer's disease or Huntington's disease. In other embodiments, the present invention provides treatment of infections, such as viral infections such as HIV. In other embodiments, the present invention provides treatment of trinucleotide repeat diseases, particularly trinucleotide repeat diseases in which the trinucleotide repeat sequence is located in the coding sequence of a gene. Trinucleotide repeat diseases that can be treated with the fusion proteins or nucleic acid constructs of the present invention include Huntington's disease, dentatorubral pallidum Lewy body atrophy, and spinocerebellar ataxia.

[0166] Treatment of a neurological disease, infection, or trinucleotide repeat disease comprises administering to a subject a fusion protein, nucleic acid, or pharmaceutical composition of the invention.

[0167] In one embodiment, the treatment involves administering a fusion protein comprising: a first RING domain; an adapter domain; wherein the fusion protein is incapable of N-terminal autoubiquitination. The adapter domain is located at the C-terminus of the first RING domain and the second RING domain. Preferably, the administered fusion protein does not comprise a coiled-coil domain or a B-box domain. In some embodiments, the fusion protein comprises a second RING domain positioned between the first RING domain and the adapter domain.

[0168] In one embodiment of the invention, the treatment involves administering a nucleic acid construct comprising a first nucleic acid sequence encoding a first RING domain; and a second nucleic acid sequence encoding a protein targeting domain, wherein the nucleic acid encodes a fusion protein that cannot undergo N-terminal autoubiquitination. The nucleic acid construct encodes a fusion protein, wherein the protein targeting domain is located at the C-terminus of the RING domain. Preferably, the administered nucleic acid construct does not comprise a sequence encoding a B-box domain or a coiled-coil domain. In some embodiments, the nucleic acid construct comprises a third nucleic acid sequence encoding a second RING domain, wherein the second RING domain is located between the first RING domain and the adapter domain.

[0169] When the condition to be treated is a neurological disease such as Alzheimer's disease, the adapter protein may encode a sequence that targets tau. In one embodiment, the adapter domain may encode an antibody, an antibody fragment thereof, or an antibody mimetic that specifically binds to tau. When the condition to be treated is Huntington's disease, the adapter domain may encode a sequence that targets huntingtin protein. In one embodiment, the adapter domain may encode an antibody, an antibody fragment thereof, or an antibody mimetic that specifically binds to the polyglutamic acid sequence of huntingtin protein.

[0170] The nucleic acid construct according to the present invention can also be administered by a delivery vector. These delivery vectors include viral delivery vectors, such as adenoviral delivery vectors, retroviral delivery vectors or slow viral delivery vectors known in the art. Other non-viral delivery vectors include lipid delivery vectors, including liposome delivery vectors known in the art.

[0171] Treatment includes prophylactic (preventive) and therapeutic treatment. The term "treat", "treating" or "treatment" (or equivalent terms) refers to a reduction in the severity of a condition of an individual or at least a partial improvement or alleviation, and / or at least one clinical symptom is alleviated, mitigated or reduced to a certain extent, and / or the progression of a condition is inhibited or delayed, and / or the onset of a disease or disorder is prevented or delayed.

[0172] The terms "patient," "individual," or "subject" include human and other mammalian subjects receiving prophylactic or therapeutic treatment with the fusion proteins or nucleic acid constructs described herein. Mammalian subjects include primates, such as non-human primates. Mammalian subjects also include laboratory animals commonly used in research, such as, but not limited to, rabbits and rodents, such as rats and mice.

[0173] The fusion proteins and nucleic acid constructs of the invention can also be used as research tools, for example, for the degradation of proteins in cells or samples.

[0174] Therefore, in one embodiment of the present invention, a method for degrading a protein in a cell is provided, the method comprising administering the fusion protein or nucleic acid of the present invention. The cell may be an in vitro cell.

[0175] Additional embodiments of the present invention provide methods for degrading proteins in a sample, the methods comprising introducing the fusion protein or nucleic acid construct of the present invention into the sample.

[0176] In one embodiment, the method for degrading proteins in cells or samples involves administering a fusion protein comprising: a first RING domain; an adapter domain; wherein the fusion protein is incapable of N-terminal autoubiquitination. The adapter domain is preferably located at the C-terminus of the RING domain. Preferably, the administered fusion protein does not comprise a coiled-coil domain or a B-box domain. In some embodiments, the fusion protein comprises a second RING domain positioned between the first RING domain and the adapter domain.

[0177] In one embodiment of the present invention, the method for degrading proteins in cells or samples involves the administration of a nucleic acid construct comprising a first nucleic acid sequence encoding a first RING domain; and a second nucleic acid sequence encoding an adapter domain, wherein the nucleic acid encodes a fusion protein that cannot undergo N-terminal autoubiquitination. Preferably, the administered nucleic acid construct does not comprise a sequence encoding a B-box domain or a coiled-coil domain. In some embodiments, the nucleic acid construct comprises a third nucleic acid sequence encoding a second RING domain, the second RING domain being located between the first RING domain and the adapter domain. The nucleic acid construct may have other features as described above.

[0178] Antibodies, antibody fragments or antibody mimetics thereof targeting proteins of interest, or nucleic acids encoding such antibodies, antibody fragments or antibody mimetics thereof, can also be administered to cells or samples. "Protein of interest" refers to a protein selected for targeted degradation. The antibody, antibody fragment or antibody mimetics thereof can specifically bind to the protein of interest.

[0179] The method is particularly useful for degrading proteins in cells that do not endogenously express TRIM21. The method is particularly useful in degrading intracellular proteins. However, in some embodiments, the antibody will bind to the protein of interest outside the cell, for example when targeting pathogens such as viruses. The antibody-target will be internalized into the cell, where the fusion protein will bind to the antibody-target, thereby degrading the protein.

[0180] The fusion protein or nucleic acid can be introduced into the cell by transfection, for example, by injection, including microinjection or by electroporation, or transduction, for example, by using a viral delivery vector, such as an AAV vector. Other suitable delivery techniques for introducing the fusion protein and nucleic acid construct into the cell are known in the art.

[0181] Compared with the corresponding fusion protein capable of self-ubiquitination, the fusion protein provided by the present invention has an increased cellular half-life.

[0182] In one aspect of the present invention, the present invention provides a method for increasing the cellular half-life of a fusion protein comprising a RING domain and an adaptor sequence, wherein the adaptor sequence is capable of positioning the RING domain together with a substrate, the method comprising modifying the fusion protein so that the fusion protein does not undergo N-terminal autoubiquitination.

[0183] By "increased cellular half-life" is meant that the cellular half-life of the modified fusion protein is increased relative to the unmodified corresponding fusion protein.

[0184] Compared to an unmodified fusion protein that is capable of N-terminal autoubiquitination, rendering the fusion protein incapable of N-terminal autoubiquitination involves modifying the N-terminus of the fusion protein. The modification of the N-terminus will produce a fusion protein that cannot be ubiquitinated by Ube2W. This is achieved by rendering the reactive N-terminus of the fusion protein incapable of covalent modification with ubiquitin by Ube2W. The modification of the N-terminus of the fusion protein can be performed in a variety of ways.

[0185] In one embodiment, the method comprises incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA to acetylate the N-terminus of the fusion protein. The N-terminus of the fusion protein is capped with an acetyl group to prevent the N-terminus from being ubiquitinated and the protein from being degraded, such as during Trim-Away.

[0186] The fusion rotein can also be modified by introducing the N-acetyltransferase recognition site into the N-terminal of the fusion rotein. The N-acetyltransferase recognition site comprises sequences such as DDDI and EEEI. In some embodiments, the method can further comprise incubating the fusion rotein with N-acetyltransferase (NAT) and acetyl-CoA to carry out N-terminal acetylation to the fusion rotein. The existence of the N-acetyltransferase recognition site also makes the fusion rotein become the substrate of cytoplasm N-acetyltransferase. The cytoplasm N-acetyltransferase can add acetyl group to the N-terminal of fusion rotein.

[0187] Other techniques include methylating the N-terminal amino acid of the fusion protein or coupling a chemical moiety to the N-terminal amino acid of the fusion protein, wherein the chemical moiety inhibits the ability of the E2 enzyme, particularly Ube2W, to ubiquitinate the fusion protein.

[0188] The fusion protein may also be modified such that the fusion protein is capable of undergoing N-terminal cyclization. Preferably, the method comprises modifying the fusion protein such that the fusion protein is capable of undergoing N-terminal pyroglutamate cyclization.

[0189] To provide a fusion protein that can undergo N-terminal pyroglutamate cyclization, the method may include introducing a glutamic acid or glutamine residue into the N-terminus of the fusion protein. Preferably, the method includes encoding an N-terminal glutamic acid residue into the fusion protein.

[0190] Proteins that begin with glutamine or glutamic acid can undergo an N-terminal cyclization reaction to form pyroglutamic acid at their N-terminus. Without being bound by theory, it is believed that in fusion proteins with an N-terminal RING domain, the presence of pyroglutamic acid at the N-terminus of the fusion protein protects the RING domain from N-terminal autoubiquitination by Ube2W.

[0191] The reaction can occur spontaneously or can be catalyzed by glutaminyl cyclase (GS). The pyroglutamate cyclization of the fusion protein can be spontaneous by encoding N-terminal glutamine or glutamate. In one embodiment, the method can further include incubating the fusion protein with glutaminyl cyclase. In some embodiments, the method includes introducing the sequence QFA into the N-terminus of the protein. The QFA sequence is added to the target protein and co-expressed with the GS enzyme to produce the N-terminal cyclized protein.

[0192] In one embodiment, the enzymatic cyclization of the N-terminal glutamine of the fusion protein can be performed in vitro or co-translationally in cells, as follows:

[0193] A fusion protein comprising a RING domain and with a tev cleavage site is expressed to expose glutamine at the N-terminus. The fusion protein is expressed on a polycistronic vector comprising a glutamine cyclase. The plasmid is co-transfected with a second plasmid encoding the tev protease. During expression, the tev protease cleaves the RING fusion protein, exposing the N-terminal glutamine, which is cyclized by the glutamine cyclase. The cyclized RING fusion protein with pyroglutamic acid at the N-terminus is then purified, for example, based on a C-terminal His tag. In such embodiments, the following fusion proteins are expressed during the production of the fusion protein of the present invention.

[0194] 1.T7P-RBS-ENLYVQQFA-RR-PY-6His-Stop-RBS-bQC(E45Q)-Stop-T7T

[0195] 2.T7P-RBS-MBP-TEVP(S219V)

[0196] Wherein T7P=T7 promoter, RBS=ribosome binding site, ENLYVQ (SEQ ID NO: 16) is the tev cleavage site, QFA is the recognition site of glutamine cyclase (QC), R=RING, PY=PRYSPRY domain, 6His=6 histidines, Stop=stop codon, T7T=T7 terminator, MBP=maltose binding protein, TEVP=TEV protease

[0197] Additional methods for modifying fusion proteins include replacing or modifying the N-terminal amino acid of the fusion protein with an amino acid sequence that inhibits the ability of Ube2W to ubiquitinate the fusion protein, such as replacing or modifying the N-terminal amino acid of the RING domain if the RING domain is located at the N-terminus. In some embodiments, only the N-terminal amino acid can be replaced with an amino acid sequence that inhibits the ubiquitination of the fusion protein by Ube2W.

[0198] In some embodiments, the amino acids at positions 1, 2, 3, 4 and 5 are modified or substituted to provide an amino acid sequence that inhibits ubiquitination of the fusion protein by Ube2W. Preferably, the method comprises modifying or replacing at least the amino acids at positions 1, 2 and 3, more preferably at least the amino acid at position 1. For example, in one embodiment, the method comprises replacing the -N-terminal amino acid of the RING domain at the N-terminus with a polyproline sequence, for example, the method may comprise replacing the amino acids at positions 1, 2, 3, 4 and 5 with a polyproline sequence or other sequence that can inhibit ubiquitination of the fusion protein by Ube2W. The amino acid segment may replace an equivalent amount of amino acids at the beginning of the fusion protein. In some embodiments, the method comprises adding an amino acid sequence that can block ubiquitination of the fusion protein by Ube2W to the N-terminus of the fusion protein, for example, if the fusion protein comprises a RING domain at the N-terminus, the method comprises adding an amino acid sequence to the N-terminus of the first RING domain.

[0199] The terms "comprising" or "comprises" may be replaced with the terms "consisting of", "consists of", "consisting essentially of" or "consists essentially of" and vice versa, wherever they appear in the text.

[0200] The contents of all publications cited herein in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

[0201] The present invention will be further understood by reference to the following examples.

[0202] Example

[0203] method

[0204] Plasmids

[0205] A complete list of plasmids used in this study including primary sequences of all constructs can be found at Fig.11 Found in . For lentiviral production, the construct was inserted into a modified version of pSMPP (Addgene No. 104970), in which the SFFV promoter and puromycin resistance sequence were replaced by the PGK1 promoter and the Zeocin resistance sequence (pPMEZ), respectively. For in vitro mRNA transcription, the construct was inserted into pGEMHE (Liman et al., 1992), which contains UTR and polyA sequences for optimal mRNA stability and translation. For protein purification, the construct was inserted into a derivative vector of the pOP and pET (Novagen) series of vectors.

[0206] Lentivirus production

[0207] The lentiviral plasmid construct (FuGENE 6, Promega) was co-transfected with HIV-1 GagPol expressor pcRV1 (gift from Dr. Stuart Neil) and pMD2G (Addgene plasmid number 12259) donated by Didier Trono. Fig.11 ) After 3 days, lentiviral particles were collected from the supernatant of HEK293T cells. The supernatant was filtered through 0.45 μm before storage at -80°C.

[0208] In vitro transcription of mRNA

[0209] The pGEMHE plasmid construct ( Fig.11 ) and synthesized 5'-capped mRNA using T7 polymerase (NEB HiScribe T7 ARCA kit) according to the manufacturer's instructions. mRNA concentration was quantified using a Qubit 4 fluorometer (ThermoFisher) and an RNA broad range assay kit (ThermoFisher; Q10211).

[0210] Protein expression and purification

[0211] A complete list of purified proteins used in this study can be found at ( Fig.12 ). Ube2W, Ube2N, and Ube2V2, as well as TRIM21 R, R-PS, RR-PS, T21R-vhhGFP4, and mEGFP were expressed in E. coli BL21 DE3. Ubiquitin and Ube1 were expressed in E. coli Rosetta 2DE3 cells as previously described (Kiss et al., 2021). Cells were grown at 37°C and 220 rpm until OD 600 After induction, the temperature was lowered to 18°C ​​and cultured overnight. TRIM21 and E2 enzymes were induced with 0.5 mM IPTG and 10 μM ZnCl2, and ubiquitin and Ube1 were induced with 0.2 mM IPTG. mEGFP was expressed in ZY auto-induction medium (Studier, 2005) at 37°C and 220 rpm. At OD 600When the pH was 0.7, the temperature was lowered to 18 ° C and expressed overnight. After centrifugation, the cells were resuspended in 50mM Tris pH 8.0, 150mM NaCl, 10μM ZnCl2, 1mM DTT, 20% Bugbuster (Novagen) and Complete protease inhibitor (Roche, Switzerland). For His-tagged proteins, 20mM imidazole was added to the buffer. Cleavage was performed by ultrasonic treatment. TRIM21-R-PS and -RR-PS were expressed with N-terminal GST-SUMO tags, and TRIM-R, Ube2W, Ube2V2 and Ube1 were expressed with N-terminal GST-tags, followed by TEV protease cleavage sites, and purified by glutathione agarose resin (GE Healthcare) balanced in 50mMTris pH 8.0, 150mM NaCl and 1mM DTT. Tags were cleaved (with SUMO or TEV protease, respectively) on beads overnight at 4°C. After cleavage with SUMO protease, there was no cleavage mark on TRIM21-R-PS and -RR-PS. After TEV cleavage, a GSH-trace was left at the N-terminus on TRIM21-R, a G-trace on Ube2N, a GSQEF-trace on Ube2V2, and a GSH-trace on Ube2W. For Ube1, no protease cleavage was performed, and the GST-Ube1 fusion protein was eluted using 50mM Tris pH 8.0, 150mM NaCl, 10mM reduced glutathione, and 1mM DTT. mEGFP was expressed with an N-terminal His-tag without a protease cleavage site, and Ube2N was expressed with an N-terminal His-tag followed by a TEV protease cleavage site. TRIM21 R-vhhGFP4 was expressed as a His-SUMO fusion protein to produce a native TRIM21 N-terminus after cleavage by SUMO protease during purification. The His-tagged protein was purified by Ni-NTA resin equilibrated in 50mM Tris pH 8.0, 150mM NaCl, 20mM imidazole, and 1mM DTT. The protein was eluted in 50mM Tris pH 8.0, 150mM NaCl, 1mM DTT, and 300mM imidazole. For Ube2N, the protein was dialyzed overnight in 50mM Tris pH 8.0, 150mM NaCl, 1mM DTT, and 20mM imidazole for TEV cleavage of the His-tag. Then, the His-tagged TEV protease was removed by Ni-NTA resin. For T21R-vhhGFP4, SUMO protease was used to cut on Ni-NTA resin overnight at 4°C. The equilibration buffer was used for elution the next day.Finally, size exclusion chromatography was performed on HiLoad 26 / 60 or 16 / 600 Superdex 75 preparative columns (GE Healthcare) for all proteins except GST-Ube1, for which HiLoad 26 / 60 or 16 / 600 Superdex 200 preparative columns (GE Healthcare) were used in 20 mM Tris pH 8.0, 150 mM NaCl, and 1 mM DTT. Ubiquitin purification was performed according to the protocol established by the Pickart laboratory (Pickart and Raasi, 2005). Ubiquitin was purified by sonication (lysis buffer: 50 mM Tris pH 7.4, 1 mg mL -1 Lysozyme (Sigma Aldrich, St. Louis, USA), 0.1 mg mL -1 After cell lysis with DNAse (Sigma-Aldrich, St. Louis, USA), perchloric acid at a total concentration of 0.5% was added to the stirred lysate at 4°C. The (milky) lysate was incubated on a stirrer at 4°C for an additional 30 minutes to complete precipitation. Next, the lysate was centrifuged (19,500 rpm) at 4°C for 30 minutes. The supernatant was dialyzed overnight (3,500 MWCO) in 3L of 50mM sodium acetate solution, pH 4.5. Ub was then purified by cation exchange chromatography using a NaCl gradient (0-1000mM NaCl in 50mM NaAc pH 4.5) using a 20mL SP column (GE Healthcare). Finally, size exclusion chromatography was performed in 20mM Tris pH 7.4 on a HiLoad 26 / 60Superdex75 preparative grade column (GE Healthcare). Escherichia coli BL21 DE3 cells were grown in a 4% culture medium supplemented with 15 Isotope-labeled proteins were expressed in M9 minimal medium containing NH4Cl (Sigma-Aldrich ISOTEC). Chaetomium thermophilum Naa50 containing a C-terminal His-tag was expressed in Escherichia coli Rosetta 2 cells grown at 37°C and 220 rpm in ZY auto-induction medium (Studier, 2005). 82-289 (Naa50ΔΔ). 600 When the CtNaa50 was 0.7, the temperature was lowered to 18°C ​​for expression overnight. 82-289The purification steps are as follows: cells are harvested, resuspended in buffer A500 (20 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole) supplemented with a protease inhibitor cocktail (SERVA Electrophoresis GmbH, Germany), and lysed with a microfluidizer (M1-10L, microfluidics). The lysate was clarified at 50,000 g, 4 ° C for 30 minutes and filtered through a 0.45 μm filter. The supernatant was applied to a 1 mL HisTrap FF column (GE Healthcare) for Ni-IMAC (immobilized metal affinity chromatography) purification. The column was eluted with buffer A500 and the protein was eluted with buffer A500 supplemented with 250 mM imidazole. CtNaa50 was subsequently purified by SEC (size exclusion chromatography) using a Superdex 75 26 / 60 gel filtration column (GE Healthcare) in buffer G500 (20 mM HEPES pH 7.5, 500 mM NaCl). 82-289 SUMO protease (MBP-Ulp1 (based on the R3 sequence (Lau et al., 2018))) was purified using an MBPTrap HP 5ml column and eluted with 50mM Tris pH 8, 150mM NaCl, 1mM DTT and 10mM maltose. Finally, the eluted fractions were separated on a HiLoad 26 / 60 Superdex 75pg SEC column (150mM NaCl, 50mM Tris pH 8 and 1mM DTT).

[0212] Cell culture

[0213] HEK293T (ATCC) and NIH3T3-CAV1-EGFP (Shvets et al., 2015) cells were cultured in DMEM medium (Gibco; 31966021) supplemented with 10% calf serum and penicillin-streptomycin. RPE-1 cells (ATCC) were cultured in DMEM / F-12 medium (Gibco; 10565018) supplemented with 10% calf serum and penicillin-streptomycin. All cells were grown in a humidified environment at 37°C and 5% CO2 and were checked regularly to ensure that they were free of mycoplasma. NIH3T3 cells were male. HEK293T and RPE-1 cells were female. For proteasome inhibition experiments, MG132 (Sigma; C2211) was used at a final concentration of 25 μM, and epoxomicin (Sigma; 324801) was used at 10 μM. After electroporation, cells were grown in medium supplemented with 10% calf serum but without antibiotics. Live cell real-time imaging was performed using an IncuCyte S3 live cell analysis system (Sartorius) placed in a 37°C, 5% CO2 humidified incubator. For real-time imaging of the IncuCyte, the cell culture medium was replaced with Fluorobrite (Gibco; A1896701) supplemented with 10% calf serum and GlutaMAX (Gibco; 35050061).

[0214] Cell lines

[0215] The cell lines used and produced in this article are detailed in (Table 1). RPE-1TRIM21 KO cells (Zeng et al., 2021), HEK293T TRIM21 KO cells (Zeng et al., 2019) and NIH3T3-CAV1-EGFP (Shvets et al., 2015) have been previously described. For stable expression of CAV1-mEGFP and CAV1-mEGFP-Halo, RPE-1 cells were transduced with lentiviral particles at a multiplicity of infection of about 0.1 transduction units per cell, and GFP-positive cell populations were then selected by flow cytometry. In order to stably express TRIM21-HA at endogenous levels, RPE-1TRIM21 KO cells were reconstructed with TRIM21-HA constructs under the regulation of the natural TRIM21 promoter, as previously described (Zeng et al., 2019).

[0216] Electroporation

[0217] use Electroporation was performed with the Transfection System (Thermo Fisher Scientific). The cells were washed with PBS and then electroporated at 1-8 × 10 7Resuspend in Buffer R at a concentration of 10 cells / mL. For each electroporation reaction, add 1-8x10 5 Mix 10 cells with 2 μl of the antibody (usually 0.5 mg / ml) or mRNA (usually 0.5 μM) or protein to be delivered. Pipette the mixture into 10 μl The cells were electroporated at 1400 V, 20 ms, 2 pulses in a pipette tip and transferred to medium without antibiotics.

[0218] Measurement of fluorescence in living cells

[0219] In order to quantify the GFP fluorescence in living cells, the IncuCyte live cell analysis system (Sartorius) was used to collect and analyze images. In the IncuCyte software, the integrated density (area and average intensity product) of GFP fluorescence was normalized according to the total cell area (phase). The data values ​​in each experiment were normalized relative to the internal control.

[0220] Antibody

[0221] Antibodies and concentrations used for traditional immunoblotting (IB), capillary-based immunoblotting (Jess), and electroporation (EP) are detailed in (Table 2). All antibodies used for electroporation were either purchased in azide-free form or passed through an Amicon Ultra-0.5 100 KDa centrifugal filter device (Millipore) to remove traces of azide and replace the buffer with PBS.

[0222] Adv5 neutralization assay

[0223] Adenovirus serotype 5 2.6-del CMV-eGFP (Adv5-GFP, Viraquest) was diluted to 1.1×10 9 TU / mL, and 16uL was incubated with the indicated concentrations of anti-hexon recombinant humanized IgG1 9C12 or 9C12H433A (Foss et al., 2016) or PBS at a 1:1 ratio. After incubation for 1 hour at room temperature, the complex was diluted with 250μL Fluorobrite medium and then used for Adv5 neutralization assay. For infection experiments, 4x10 6HEK293T TRIM21 KO cells were plated and resuspended in 2 mL Fluorobrite medium. 50 μL of each cell suspension was mixed with Adv5:9C12 or Adv5:9C12H433A complex (for immediate infection) or Fluorobrite (for delayed infection) in a 96-well plate at a 1:1 ratio. For delayed infection, electroporated cells were allowed to adhere to the plate for 2 hours, then the medium was replaced with 50 μL Fluorobrite medium and infected with 50 μL Ad5:9C12 complex. After 16 hours post-infection, the infection level was quantified using the IncuCyte system by measuring the ratio of GFP fluorescent area to total cell area. The infection level was plotted relative to Adv5-GFP infection in the absence of 9C12 antibody.

[0224] NFκB signaling assay

[0225] HEK293T TRIM21 KO cells were transfected with 2ug pGL4.32 NF-κB luciferase plasmid (Promega) in 200uL OptiMEM (Thermo Fisher) using 12uL of Viafect (Promega). After 24 hours, 4x10 6 The transfected cells were electroporated and then resuspended in 1 mL of DMEM. For infection experiments, Adv5:9C12 complexes were prepared as described above, except that Ad5-GFP was diluted to 1.1x10 10 , 9C12 was used at a concentration of 20ug / mL, and the complex was diluted to 150μL DMEM after incubation for 1 hour. 50μL of cell suspension after electroporation was mixed with Adv5:9C12 complex or PBS (control) in a 1:1 ratio, and then lysed with 100uLSteadyLite luciferase reporter reagent (PerkinElmer) after 4 hours. As an internal control, TNF-α was used at a concentration of 10ng / uL. PheraStar FS (BMG LabTech) was used to record luminescence intensity.

[0226] Immunoblotting

[0227] The samples were electrophoresed on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific) and then transferred to nitrocellulose membranes. The membranes were incubated in blocking buffer (PBS, 0.1% Tween 20, 5% milk) at room temperature for 1 hour before incubation with antibodies. Antibodies used for immunoblotting (IB) and their dilutions in blocking buffer are detailed in (Table 2). HRP-coupled antibodies were detected by enhanced chemiluminescence (Amersham, GE Healthcare) and X-ray film. IRDye-coupled antibodies were detected using the LI-COR Odyssey CLx imaging system.

[0228] Capillary immunoblotting

[0229] The RIPA buffer protein extracts were diluted 1:2 in 0.1x sample buffer (bio-techne; 042-195) and analyzed on the Jess Simple Western system using a 12-230 kDa separation module (bio-techne) according to the manufacturer's instructions. Antibodies and dilutions used for capillary immunoblotting (Jess) are detailed in (Table 2). Protein peak areas were quantified using Compass software (bio-techne) and normalized to the amount of internal reference protein loaded in each capillary.

[0230] In vitro ubiquitination assay

[0231] Ube2W-dependent TRIM21-monoubiquitination assays were performed in 50 mM Tris pH 7.4, 150 mM NaCl, 2.5 mM MgCl2, and 0.5 mM DTT. Reaction components included 2 mM ATP, 1 μM GST-Ube1, 80 μM ubiquitin, and the indicated concentrations of Ube2W and TRIM21. The reaction was terminated by adding LDS sample buffer containing 50 mM DTT at 4 °C. Next, the samples were boiled at 90 °C for 2 minutes. For reactions using 10 μM TRIM21, visualization was performed only by instant blue staining LDS-PAGE. The polyubiquitin chain extension assay was performed as described above, but 0.5 μM Ube2N / Ube2V2 was added instead of Ube2W. For the determination of antibody-induced monoubiquitination, similar conditions were used as for the monoubiquitination assay for LDS-PAGE analysis described above. However, the concentration of TRIM21 needed to be reduced to 100nM and the concentration of GST-Ube1 to 0.25μM. Anti-GFP antibody (9F9.F9) was added in an amount equimolar to TRIM21. The reaction was started by adding Ube2W (0, 50, 100, 200nM). The reaction was terminated by adding LDS sample buffer at 4°C. The samples were boiled at 90°C for 2 minutes and resolved by LDS-PAGE. TRIM21 was visualized using Western blot. The in vitro reconstruction of Trim-Away ubiquitination events was performed similarly to the antibody-induced monoubiquitination experiments described above. The E2 enzyme concentrations were 200nM Ube2W and 0.5μM Ube2N / Ube2V2, and 200nM His-mEGFP was used as the Trim-Away target.

[0232] Acetylation and monoubiquitination assays

[0233] N-terminal acetylation of TRIM21 is mediated by Chaetomium thermophilum N-acetyltransferase (NAT) Naa50ΔΔ. The acetylation reaction was carried out in 50mM Tris pH 7.4 and 150mM NaCl at 25°C for 4 hours. The reaction system contained 20μM TRIM21, 1mM acetyl-CoA and 1μM CtNaa50ΔΔ. After the acetylation reaction was completed, it was mixed with Ube2W-ubiquitination mixture in a 1:1 ratio, and the mixture contained 100mM Tris pH7.4, 300mM NaCl, 5mM MgCl2, 1mM DTT, 4mM ATP, 2μM GST-Ube1, 160μM ubiquitin and 2μM Ube2W. The Ube2W ubiquitinase reaction was performed at 37°C for 1 hour and then terminated by adding LDS sample buffer containing 50 mM DTT at 4°C, followed by boiling the samples at 90°C for 2 minutes. Visualization was performed by instant blue staining LDS-PAGE only.

[0234] NMR spectroscopy

[0235] Two-dimensional NMR measurements ( 15 N-HSQC) is equipped with 5mm 1 H- 13 C- 15 The data were processed with the program Topspin (Bruker BioSpin GmbH, Germany) and analyzed with the program CCPNanalysis v2 (Vranken et al., 2005). The sample buffer was replaced with 50 mM deuterated Tris pH 7.0, 150 mM NaCl and 1 mM deuterated DTT (Cambridge Isotopes, United Kingdom).

[0236] Chemical shift perturbations (CSPs) were calculated using formula (3):

[0237] (3)

[0238] where Δδ N,HN is CSP, Δδ( 1 H) and Δδ( 15N) is the chemical shift difference between the position of the proton or nitrogen signal in the absence and presence of titrant. Chemical shift assignments for TRIM21 refer to previous publications (Dickson et al., 2018).

[0239] Mass spectrometry

[0240] The protein gel pieces cut out were decolorized with 50% v / v acetonitrile: 50mM ammonium bicarbonate. After reduction with 10mM DTT and alkylation with 55mM iodoacetamide, the protein was digested overnight with 6ng / μL Asp-N (Promega, UK) at 37°C. Peptides were extracted in 2% v / v formic acid: 2% v / v acetonitrile and analyzed subsequently by nanoscale capillary LC-MS / MS, using Ultimate U3000 HPLC (Dionex (Thermo Scientific Dionex, San Jose, USA) of Thermo Scientific, San Jose, USA), with a flow rate of 300nL / min. Peptides were enriched on a C18 Acclaim PepMap100 5 μm, 100 μm×20 mm nanoViper (Dionex, Thermo Scientific, San Jose, USA) before separation on a C18 T3 1.8 μm, 75 μm×250 mm nanoEase column (Waters, Manchester, UK). Peptides were eluted with an acetonitrile gradient and the analytical column outlet was directly connected to a quadrupole-orbitrap mass spectrometer (Q-Exactive HFX, Thermo Scientific, USA) using a nanoflow electrospray ion source. For data-dependent analysis, the resolution of the full MS spectrum was set to 60,000, followed by 12 MS / MS analyses. MS spectra were collected in the m / z range of 300-1,800. The resulting LC-MS / MS spectra were searched against the protein database (UniProt KB) using the Mascot search engine program. The database search parameters were set as follows: precursor ion tolerance of 5 ppm, fragment ion tolerance of 0.1 Da. Multiple modifications were set in the search parameters: two enzyme cleavage misses, variable modifications of methionine oxidation, cysteine ​​carbamoyl methylation, pyroglutamic acid, and protein N-terminal acetylation. The proteomics software Scaffold 4 was used to view the fragment spectra.

[0241] Statistical analysis

[0242] Mean (average / mean), standard deviation (sd), standard error of the mean (sem), and statistical significance based on Student's t-test (two-tailed) and one-way or two-way ANOVA were calculated in Microsoft Excel or Graphpad Prism. Significance is indicated by the following symbols: ns (not significant, P>0.05), * (P≤0.05), ** (P≤0.01), *** (P≤0.001), **** (P≤0.0001).

[0243] Crystallography

[0244] TRIM21-RING:Ube2W V30K / D67K / C91K Crystals of the complex were grown in 2 nL drops at 17°C at a protein concentration of 10 mg / ml in a solution of 0.1 M Bicine pH 9.0, 5% PEG 6000, 0.1 M TCEP hydrochloride. Diffraction experiments were performed at the ID233 beamline of the European Synchrotron Radiation Facility using a Dectris PILATUS 6M detector with a wavelength set to Using XDS Processing The protein structure was analyzed by molecular replacement using Phaser software (Adams et al., 2010) with the TRIM21RING domain (5OLM (Dickson et al., 2018)) and residues 1-118 of Ube2W (2MT6 (Vittal et al., 2015)) as search models. Model building and real space refinement were performed in coot software (Emsley and Cowtan, 2004), and structure refinement was performed using REFMAC5 and phenix-refine (Afonine et al., 2012) software. The model and structure factors have been deposited in the PDB database with accession code 8A58.

[0245] result

[0246] To understand how TRIM21 recruits Ube2W, we solved the crystal structure of the RING domain (R) in complex with a dimerization mutant of Ube2W (Vittal et al., 2013b) in which the active site cysteine ​​is also replaced by lysine (Ube2W V30K / D67K / C91K ) replacement. Two copies of each Ube2W and RING were found in the asymmetric unit, and the two RINGs formed homodimers, consistent with previous descriptions (Dickson et al., 2018; Kiss et al., 2021; Kiss et al., 2019) ( Figure 1A). RING and Ube2W are connected to each other through the typical RING:E2 interface ( Figure 1 B). The activated state of Ube2W~Ub was simulated by superimposing the R:Ube2W structure with Ube2N~Ub in the previously determined Ub-R:Ube2N~Ub:Ube2V2 structure (Kiss et al., 2021). Figure 1 C). Overall, when activated by TRIM21, the arrangement of Ube2W-Ub is similar to that of Ube2N-Ub (Kiss et al., 2021; Kiss et al., 2019). In this model, the donor ubiquitin is in a closed conformation, stabilized by two RING protomers. Interestingly, this model also suggests that TRIM21E13 may engage ubiquitin K11 to stabilize the closed conformation ( Figure 1 D) E13 is part of a triionic motif that has been shown to drive the Ube2N-Ub interaction (Kiss et al., 2019). 15 N-labeled TRIM21 triionic mutants to Ube2W V30K / D67K / C91K NMR titrations were performed to test whether this motif is involved in the binding of Ube2W. Mutation of the triionic residues E12 and E13 to alanine did not result in a significant reduction in the observed chemical shift perturbations (CSPs). Figure 7 AC). Moreover, the tri-ion mutant had only a modest effect on TRIM21 monoubiquitination ( Figure 7 D). Both TRIM21 mutants E13A and E13R showed a slight decrease in activity, suggesting that residue E13 can indeed interact with ubiquitin K11, but this is not as critical as for its action on Ube2N~Ub (Kiss et al., 2019). When the RING in the R:Ube2W structure was compared to the structures of apo- (Dickson et al., 2018) and engaged Ube2N~Ub (Kiss et al., 2019), it was noted that the N- and C-terminal helices of the RING partially unfolded when bound to Ube2W ( Figure 7 E). While this may reflect differences in the crystallization process, it may also indicate that Ube2W has the potential to destabilize the 4-helix bundle, thereby creating a disordered N-terminus for modification. Even so, this is not enough to explain how Ube2W is able to modify the N-terminus of the RING to which it binds, as it is far from the E2 enzyme active site.

[0247] It is hypothesized that since Ube2W is usually a dimer (Vittal et al., 2013a), it may adopt a catalytic RING topology similar to the previously described Ube2N / Ube2V2 heterodimer (Kiss et al., 2021). In this arrangement, two RINGs can form a dimer as an enzyme to activate the donor ubiquitin on one Ube2W monomer, while the third RING acts as a substrate and is directed by the second Ube2W monomer to attack the N-terminus ( Figure 1 E). This hypothesis was tested using two TRIM21 constructs carrying either one (R) or two (RR) RING and PRYSPRY (PS) domains (R-PS and RR-PS). Both constructs were efficiently monoubiquitinated by Ube2W ( Figure 1 F). However, although dimerization of Ube2W is not thought to be important for activity (Vittal et al., 2013, Cell Biochem Biophys), when monomeric Ube2W is used V30K / D67K This activity disappears when Figure 1 F). This is consistent with the idea that Ube2W directs one RING domain toward substrates by dimerization ( Figure 1 E). Surprisingly, no differences were observed between R-PS and RR-PS, likely because relatively high TRIM21 concentrations were sufficient to drive dimerization of the RING of R-PS. It has been previously shown that TRIM21 is activated in cells through substrate-induced aggregation (Zeng et al., 2021), and indeed, at near-physiological enzyme concentrations, IgG Fc needs to be added to in vitro ubiquitination assays to induce TRIM21-mediated K63 chain formation by Ube2N / Ube2V2 (Kiss et al., 2021). Therefore, R-PS or RR-PS concentrations were subsequently reduced and Ube2W was titrated in the presence or absence of IgG. Under these conditions, monoubiquitination was only observed in RR-PS ( Figure 1 G and H). In addition, IgG greatly stimulated RR-PS monoubiquitination ( Figure 1 H and I). Importantly, we observed that endogenous TRIM21 also depends on dimeric Ube2W for substrate degradation in cells. During the Trim-Away experiment, Ube2W was electroporated into V30K / D67K As a positive control, wild-type Ube2N / Ube2V2 was compared with a mutant in which the residue responsible for deprotonation of K63 on the receptor ubiquitin was replaced by alanine (Ube2N N119A / Ube2V2) substitution (Kiss et al., 2021). This catalytically inactive mutant is unable to degrade CAV1-mEGFP ( Figure 1 J). These results confirm previous observations using siRNA that both Ube2W and Ube2N / Ube2V2 are required for TRIM21 activity (Fletcher et al., 2015b; McEwan et al., 2013). In summary, the cellular and in vitro data suggest that antibody binding promotes monoubiquitination of the TRIM21 RING by dimeric Ube2W through a trans-acting mechanism similar to Ube2N / Ube2V2 and that this is required for substrate degradation.

[0248] Although Ube2W is required for substrate degradation and TRIM21 can be monoubiquitinated by E2 enzymes in vitro, this does not prove that the two are interdependent. To investigate the necessity of TRIM21 N-terminal monoubiquitination, it was decided to block it biochemically by N-acetylation. N-acetylation is an irreversible modification that is catalyzed in cells by N-acetyltransferases (NATs) using the cofactor acetyl-CoA (Aksnes et al., 2019). NAT from Chaetomium thermophilum, a thermostable enzyme that can interact with human proteins (Weyer et al., 2017), was selected and incubated with RR-PS in the presence of acetyl-CoA. Successful N-terminal acetylation was confirmed by LC-MS / MS ( Figure 8 A). N-terminally acetylated RR-PS (Ac-RR-PS) was added together with Ube2W and ubiquitin to test whether monoubiquitination was inhibited ( Figure 2 A). In the absence of acetylation, all RR-PS were monoubiquitinated, whereas RR-PS incubated with NAT and acetyl-CoA remained mostly unubiquitinated ( Figure 2 B) The extent of monoubiquitination inhibition is proportional to the incubation time of NAT and acetyl-CoA ( Figure 8 B). Importantly, the formation of free K63-linked ubiquitin chains was not affected, indicating that the acetylated RING is still catalytically active ( Figure 8 C) These results both confirm that the TRIM21 RING is monoubiquitinated by Ube2W at its N-terminus and suggest that this ubiquitination can be inhibited by N-terminal acetylation.

[0249] We tested whether this process is required for substrate degradation by specifically blocking N-terminal ubiquitination of RING. We electroporated RR-PS or Ac-RR-PS together with anti-GFP antibody into cells expressing CAV1-mEGFP, and monitored the kinetics of substrate degradation by fluorescence detection ( Figure 2C). Consistent with previous Trim-Away results, electroporation of anti-GFP antibody drives substrate degradation via endogenous TRIM21 ( Figure 2 D). However, by introducing exogenous RR-PS, CAV1-mEGFP degradation was significantly accelerated. Importantly, ligase acetylation (Ac-RR-PS) had no effect on this, and degradation proceeded with the same kinetics ( Figure 2 D). Next, we tested whether N-terminal acetylation is required for the antiviral function of TRIM21. Cells were electroporated with RR-PS or Ac-RR-PS and then infected with adenovirus 5 (AdV5) encoding the gene for GFP in the presence of the anti-hexon antibody 9C12. Toxigenic infection was monitored by measuring GFP expression 24 h after infection ( Figure 2 E). Previous experiments have shown that when TRIM21 binds to antibody-coated viruses, it blocks infection by mediating proteasomal degradation of virions (Mallery et al., 2010). As expected, electroporation of RR-PS neutralized AdV5 in an antibody dose-dependent manner ( Figure 2 F). Ac-RR-PS was at least as active as RR-PS in neutralizing AdV5 infection and was more effective at intermediate antibody concentrations. In addition to mediating virion degradation, TRIM21 also activates innate immune signaling upon detection of antibody-coated virus (McEwan et al., 2013). Therefore, infection experiments were repeated in cells containing a luciferase gene driven by the NF-κB promoter. RR-PS and Ac-RR-PS were equally effective in stimulating NF-κB-driven transcription in response to antibody-coated Adv5 ( Figure 2 G). In both cases, this was dependent on direct engagement of the antibody, as the 9C12 mutant H433A, which specifically abolishes binding to PRYSPRY (McEwan et al., 2012), was unable to activate NF-κB. Taken together, these data suggest that N-terminal ubiquitination of RING ligases is not essential for the antiviral function of Trim-Away or TRIM21.

[0250] It was reasoned that if RING ligase autoubiquitination is not required for substrate degradation, it may be involved in mediating its own turnover. Therefore, acetylated RR-PS was electroporated into RPE-1 cells and protein levels were monitored after 1 hour ( Figure 3 A). Comparison of epoxomicin-treated and untreated cells revealed that unacetylated RR-PS is readily degraded by the proteasome ( Figure 3 B, first two lanes). In contrast, epoxomicin had little effect on Ac-RR-PS protein levels, suggesting that inhibition of N-terminal ubiquitination blocks proteasomal degradation ( Figure 3 B, last two lanes). These data suggest that, although ligase N-terminal ubiquitination is not required for substrate degradation, it can be used to modulate ligase levels. To test this, electroporation experiments were repeated with RR-PS or Ac-RR-PS, but after a delay of several hours, Trim-Away or AdV5 neutralization was measured ( Figure 3 C). For the Trim-Away experiment, this was achieved by co-electroporating the RNA encoding the antibody construct responsible for recruiting the RR-PS to the substrate (vhhGFP4-Fc). Thus, upon expression of vhhGFP4-Fc, Trim-Away was delayed for several hours. Under this protocol, there was no longer any substrate degradation in cells electroporated with the RR-PS ( Figure 3 D, with Figure 2 D). In contrast, Ac-RR-PS degraded CAV1-mEGFP as efficiently as Trim-Away immediately after ligase electroporation ( Figure 3 D). For the AdV5 neutralization experiment, cells were infected 2 hours after ligase introduction. In this case, neutralization by RR-PS was severely impaired, while Ac-RR-PS was significantly more effective in inhibiting infection ( Figure 3 E).

[0251] Since autoubiquitination of the ligase is not required for substrate degradation, we investigated whether this is instead driven by substrate ubiquitination. To test this, in vitro ubiquitination experiments were performed using RR-PS, anti-GFP antibodies, and recombinant mEGFP substrate ( Figure 4 A). Simultaneous ubiquitination of RR-PS, antibody heavy chain, and mEGFP was observed ( Figure 4 A and B). Monoubiquitination was observed when incubated with Ube2W alone, whereas anchored polyubiquitin chains were formed when Ube2N / V2 was co-added. Importantly, substrate ubiquitination occurred only in the presence of both RR-PS and antibody ( Fig. 9 ). This is consistent with the requirement of the antibody to recruit the TRIM21 ligase to its substrate. Next, we investigated whether substrate ubiquitination occurs independently of ligase ubiquitination or whether modification of the latter is required for stimulation of catalytic activity. To this end, we repeated the in vitro ubiquitination assay in the presence of both E2 enzymes and compared RR-PS with Ac-RR-PS. Ligase acetylation blocked autoubiquitination but did not interfere with polyubiquitination of the antibody heavy chain or the substrate ( Figure 4C). These data suggest that TRIM21 catalyzes polyubiquitination of antibody-coated substrates and that this process can occur independently of the ligase's own ubiquitination. However, the fact that TRIM21, antibody, and substrate can be polyubiquitinated simultaneously in vitro is consistent with in vivo Trim-Away data indicating that all three components are degraded simultaneously (Clift et al., 2017).

[0252] Next, we attempted to monitor substrate ubiquitination during Trim-away degradation in living cells. Figure 5 A). Two kinase substrates, ERK1 and IKKα, were selected and analyzed by western blot at different time points after antibody electroporation. For both substrates, high molecular weight bands or smearing consistent with polyubiquitination were observed 30 minutes after electroporation ( Figure 5 B and C). These high molecular weight bands decreased over the next few hours, along with a decrease in substrate protein levels. To obtain further evidence for substrate polyubiquitination, the IKKα Trim-Away experiment was repeated in the presence of the proteasome inhibitor MG132. Addition of MG132 had no effect on control cells, but in the presence of the electroporated antibody, a higher molecular weight ladder was clearly observed ( Figure 5 D). When performing a time course experiment, the results show that the IKKα ubiquitinated species is formed first and then depleted as the protein is degraded. Treatment with MG132 both blocks degradation and leads to a steady accumulation of the ubiquitinated species ( Fig.10 A). TRIM21 was also detected and a decrease in its protein level was observed, which was also similar to substrate depletion ( Figure 5 B and C). Higher molecular weight bands were observed for TRIM21, which may also indicate the occurrence of polyubiquitination; however, while these bands increased during ERK1 Trim-Away, they did not change in the IKKα experiment. To test whether antibody-dependent substrate ubiquitination was mediated by TRIM21, the experiments were repeated in TRIM21 knockout cells reconstituted with HA-tagged TRIM21 or empty vector control. Antibody-induced ERK1 laddering was observed in knockout cells reconstituted with TRIM21-HA but not empty vector, indicating polyubiquitination ( Figure 5 E) These data indicate that during Trim-Away in living cells, substrates are ubiquitinated in an antibody- and TRIM21-dependent manner.

[0253] It has been shown previously that Trim-Away can be achieved in the absence of antibody by directly fusing substrate-targeted nanobodies to domains from TRIM21 (Zeng et al., 2021). This approach was used to determine if there was something special about the ternary complex formed between TRIM21:antibody:substrate that is required for substrate ubiquitination. Two fusion constructs were tested, one fused to the TRIM21 RING, B-box, and coiled-coil (T21RBCC-) with an anti-GFP nanobody (vhhGFP4) and the other fused to the RING domain alone (T21R-) with an anti-GFP nanobody (vhhGFP4). These fusion constructs were transduced as RNA into cells expressing CAV1-mEGFP and ubiquitination and degradation were monitored. Effective Trim-Away ( Fig.10 C). Treatment with MG132 inhibits degradation and leads to the simultaneous accumulation of ubiquitinated substrates ( Fig.10 C). These results suggest that TRIM21-mediated substrate ubiquitination and degradation requires neither antibodies nor the ternary complex. Two RING inactivating mutations were introduced into the T21R-vhhGFP4 construct (T21R I18R / M72E -vhhGFP4) (Zeng et al., 2021), both substrate ubiquitination and degradation were completely abolished, indicating that these processes are driven by the catalytic activity of TRIM21 RING.

[0254] The above data indicate that activated TRIM21 can catalyze the simultaneous ubiquitination of itself, antibodies, and substrates. However, blocking N-terminal TRIM21 ubiquitination did not prevent substrate degradation. To exclude the possibility that ligase lysine autoubiquitination drives substrate degradation, a variant of T21R-vhhGFP4 was prepared in which all lysines were mutated to arginines ( Figure 6 A; T21R K0 -vhhGFP4 K0 Notably, removal of all lysines from T21R-vhhGFP4 had no effect on the rate or efficiency of substrate degradation ( Figure 6 B and C). Furthermore, simultaneous blocking of the N-terminus by acetylation had no effect. Taken together, these data suggest that TRIM21 is independent of canonical autoubiquitination for substrate degradation. Importantly, however, substrate turnover can be uncoupled from ligase turnover, as inhibition of N-terminal autoubiquitination reduced ligase depletion without altering substrate degradation ( Figure 6 C).

[0255] To test whether Trim-Away is driven by substrate lysine ubiquitination, a model substrate containing the dodecameric ALFAtag repeat sequence, which naturally contains no lysine residues ( Gotzke et al., 2019 ), was designed and fused to either wild-type or lysine-free anti-GFP nanobodies ( Figure 6 D). These substrates were expressed in wild-type or TRIM21 knockout (T21KO) RPE-1 cells and then electroporated with ALFAtag nanobody fused to IgG Fc (vhhALFA-Fc). ALFA-Fc is expected to bind to the ALFAtag substrates and recruit endogenous TRIM21 through Fc interaction, leading to TRIM21 aggregation, activation, and degradation ( Figure 6 E). Indeed, this is what was observed, with significant degradation observed in wild-type cells but not in T21KO cells ( Figure 6 F and G). Note that in the absence of TRIM21, addition of ALFA-Fc actually increases substrate levels, suggesting that the nanobody:tag complex is more stable. Importantly, degradation is independent of substrate lysine, as both wild-type and lysine-free substrates are degraded equally ( Figure 6 F and G). As expected from the Trim-Away assay, endogenous TRIM21 was also degraded along with each substrate ( Figure 6 H). Since the degraded form of the TRIM21: substrate complex may require lysine ubiquitination on only one side, the assay was modified to remove lysine from both the ligase and the substrate. To this end, the model substrate was supplemented with a model ligase comprising a TRIM21 RING fused to an anti-ALFAtag nanobody (T21R-vhhALFA; Figure 6 I). In this assay, the ALFAtag substrate is expected to recruit multiple T21R-vhhALFA ligases, leading to ligase aggregation, activation, and degradation ( Figure 6 I). As previously mentioned, removing all lysine from the substrate had no effect on substrate degradation ( Figure 6 J and K). However, strikingly, Trim-Away is equally effective when both the ligase and substrate are lysine-free ( Figure 6 J and K). Taken together, these data suggest that TRIM21-mediated degradation is not driven by ligase autoubiquitination, nor is substrate lysine ubiquitination. This finding may explain the high efficiency of Trim-Away in degrading different substrates (Clift et al., 2017).

[0256] The data also show that while TRIM21 activation results in ubiquitination of both the ligase and substrate, autoubiquitination plays a regulatory role and is not required for substrate degradation. Substrate binding stimulates the E2 enzyme Ube2W to autoubiquitinate the N-terminal RING, but when inhibited, such as by N-terminal acetylation, it does not prevent substrate ubiquitination or substrate degradation, nor does it affect TRIM21 activity. The inventors found that uncoupling the degradation of the ligase and substrate prevents ligase recycling and prolongs functional persistence in cells.

[0257] Thus, the data also demonstrate that by blocking the N-terminus of RING-containing protein constructs, inhibiting the autoubiquitination of the N-terminal RING, it is possible to increase the cellular half-life of RING-containing fusion proteins while substantially not affecting substrate degradation.

[0258] Although the present disclosure has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present disclosure.

[0259] Materials used:

[0260] Table 1:

[0261]

[0262]

[0263] Table 2:

[0264]

[0265]

[0266] References

[0267] Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, B.V., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221.

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Claims

1. A fusion protein comprising: - at least a first RING domain; and - an adaptor domain capable of positioning the RING domain and the substrate together, and The fusion protein is incapable of N-terminal autoubiquitination.

2. The fusion protein according to claim 1, wherein the fusion protein is: -N-terminally acetylated; -N-terminally methylated; or - comprising a chemical moiety coupled to the N-terminus of the fusion protein, wherein the chemical moiety inhibits ubiquitination of the fusion protein.

3. The fusion protein according to claim 1, wherein the N-terminus of the fusion protein comprises an N-acetyltransferase recognition site, preferably wherein the N-terminus of the fusion protein comprises the sequence DDDI or EEEI.

4. The fusion protein according to claim 1, wherein the N-terminus of the fusion protein is capable of N-terminal cyclization, preferably wherein the fusion protein is capable of N-pyroglutamate cyclization. The fusion protein according to claim 1 , wherein the fusion protein comprises glutamic acid, glutamine or pyroglutamic acid as the N-terminal residue.

6. The fusion protein according to claim 1, wherein at least the N-terminal amino acid of the fusion protein is substituted with an amino acid or an amino acid sequence, which inhibits the ability of the E2 enzyme Ube2W to ubiquitinate the fusion protein.

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein: - comprises a second RING domain, wherein the second RING domain is located between the first RING domain and the adapter domain; - does not contain a coiled-coil domain and / or a B-box domain; and / or - comprises a linker sequence, said linker sequence being located between said RING domain and said adaptor sequence.

8. The fusion protein according to any one of claims 1 to 7, wherein the RING domain is derived from a TRIM polypeptide. 9 . The fusion protein according to claim 8 , wherein the TRIM polypeptide is selected from the group consisting of TRIM5, TRIM7, TRIM19, TRIM21, TRIM25, TRIM28 and TRIM 32, preferably the TRIM polypeptide is TRIM21.

10. The fusion protein according to any one of claims 1 to 9, wherein the adaptor sequence is: - a protein targeting domain selected from a PRYSPRY domain, an antibody or an antibody fragment thereof, or an antibody mimetic, wherein the antibody fragment is preferably selected from the group consisting of: Fab, Fab', F(ab')2, scFab, Fv, scFV, dAB, a VL fragment thereof, a VH fragment thereof, and a V HH fragment; or a. - encoding a protein or a fragment thereof capable of localizing said RING domain to said substrate.

11. A nucleic acid construct encoding the fusion protein according to any one of claims 1 to 10.

12. A nucleic acid construct comprising a first nucleic acid sequence encoding a first RING domain and a second nucleic acid sequence encoding an adaptor domain, wherein the nucleic acid construct encodes a fusion protein that is incapable of autoubiquitination.

13. The nucleic acid construct according to claim 11 or 12, wherein the construct does not encode a coiled-coil domain; does not encode a B-box domain or does not encode a coiled-coil domain and a B-box domain.

14. The nucleic acid construct according to any one of claims 11 to 13, which is in the form of a vector, preferably wherein the vector is a viral delivery vector, more preferably an adeno-associated virus (AAV) vector.

15. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 10 or the nucleic acid according to any one of claims 11 to 14, and a pharmaceutically acceptable carrier and / or excipient.

16. The fusion protein according to any one of claims 1 to 10 or the nuclear construct according to any one of claims 11 to 14 for use as a medicament. 17 . A method for degrading a target protein in a cell, the method comprising introducing the fusion protein according to any one of claims 1 to 10 or the nucleic acid construct according to any one of claims 11 to 14 into the cell.

18. A method for increasing the cellular half-life of a fusion protein, wherein the fusion protein comprises a RING domain and an adaptor domain, wherein the adaptor domain is capable of positioning the RING domain and a substrate together, the method comprising modifying the fusion protein so that the fusion protein cannot undergo N-terminal autoubiquitination.

19. The method of claim 18, wherein modifying the fusion protein comprises: - incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA to N-terminally acetylate the fusion protein; - methylating the N-terminal amino acid of the fusion protein; - coupling a chemical moiety to the N-terminal amino acid of the fusion protein, wherein the chemical moiety reduces the ability of the E2 enzyme Ube2W to ubiquitinate the fusion domain; - introducing an N-acetyltransferase recognition site into the N-terminus of the fusion protein, preferably the sequence DDDI, and optionally incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA to N-terminally acetylate the fusion protein; - modifying the fusion protein so that the fusion protein can undergo N-terminal cyclization; - modifying the fusion protein so that the fusion protein is capable of N-terminal pyroglutamate cyclization; - introducing a glutamic acid or glutamine residue into the N-terminus of the fusion protein and optionally incubating the fusion protein with glutamine cyclase; or - replacing at least the N-terminal amino acid of the RING domain with an amino acid or an amino acid sequence, which inhibits the ability of the E2 enzyme Ube2W to ubiquitinate the fusion protein.

20. A method for producing a fusion protein according to any one of claims 1 to 10, the method comprising: (a) culturing a host cell comprising a first vector encoding a fusion protein under conditions allowing expression of the fusion protein, wherein the fusion protein comprises a RING domain and an adaptor domain; and (b) obtaining the expressed protein from the host cell, wherein the fusion protein is incapable of N-terminal autoubiquitination.

21. The method of claim 20, wherein the first vector further encodes glutamine cyclase and the expressed fusion protein comprises an N-terminal glutamic acid or an N-terminal glutamine, and the method comprises culturing the host cell under conditions that allow expression of the fusion protein and the glutamine cyclase.

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