Modified fusion proteins and nucleic acid constructs

Abstract

The present invention relates to modified fusion proteins and nucleic acid constructs suitable for use in intracellular proteolysis. The fusion protein includes a RING domain and an adapter domain capable of localizing the RING domain and a substrate. The fusion protein is not susceptible to N-terminal auto-ubiquitination and has an extended cellular half-life. The present invention also relates to compositions containing these fusion proteins and nucleic acids, as well as 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 use in intracellular proteolysis. The fusion proteins are not susceptible to N-terminal auto-ubiquitination and have an extended half-life in cells. The present invention also relates to compositions containing these fusion proteins and nucleic acids, and the use of the fusion proteins and nucleic acid constructs in therapy.

Background Art

[0002] Proteolysis occurs naturally within cells, preventing the generation of misfolded proteins and providing an endogenous mechanism that mediates cellular responses. The main pathway for proteolysis is via the ubiquitin-proteasome system (UPS). The ability to manipulate the UPS to redirect this system to achieve targeted proteolysis within cells holds great potential for applications in research, drug discovery, and therapeutics.

[0003] By selectively depleting target proteins, it becomes possible to study protein functions and dynamic protein interactions at the cellular level. Such selective depletion is particularly used in drug discovery, where small molecules known as "proteolysis-targeting chimeras" (PROTACs) can be used to redirect proteolysis and induce selective depletion of target proteins (Non-Patent Document 1). Similarly, techniques such as "Trim-Away™" utilize the E3 ubiquitin ligase known as TRIM21, a specific component of the UPS, to selectively deplete target proteins bound to antibodies (Non-Patent Documents 2, 3, 4, 5). Further strategies utilizing constructs containing the RING domain of E3 ubiquitin ligases are disclosed in Patent Document 1. These newly emerging tools and drug discovery platforms enable the study of protein interactions in the post-translational environment, avoiding many limitations associated with genetic manipulation that are time-consuming and costly and do not provide phenotypic insights.

[0004] Targeted proteolysis holds promise for use in therapeutic applications (Non-Patent Document 6), particularly in diseases associated with excessive protein production or abnormal protein aggregation. Using targeted proteolysis 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. Examples of TRIM proteins include those that suppress viral infection (TRIM5 (Non-Patent Document 7), TRIM21 (Non-Patent Document 8), TRIM22 (Non-Patent Document 9), TRIM25 (Non-Patent Document 10)), those that activate innate immunity (TRIM32 (Non-Patent Document 11), TRIM56 (Non-Patent Document 12), TRIM65 (Non-Patent Document 13), RIPLET (Non-Patent Document 14)), and those that suppress transcription (TRIM4 (Non-Patent Document 15), TRIM28 (Non-Patent Document 16)). In particular, the intracellular antibody receptor TRIM21 plays a role in mediating targeted proteolysis in Trim-Away™. Despite their importance, the ubiquitination mechanism of TRIM ligases remains unclear. TRIM ligases contain both a substrate-targeting domain and a catalytic domain within a single polyprotein. However, the mechanism by which TRIM catalyzes ubiquitination is not fully understood, particularly with regard to activation, ubiquitin priming, and chain elongation.

[0006] The most recent mechanisms of TRIM catalysts have been reported mainly by experiments on two antiviral proteins, TRIM5 and TRIM21. Both proteins are dimers containing a RING domain, a B-box domain, a coiled-coil domain, and a PRYSPRY domain. Each RING domain is located at the end of the opposite side of the elongated antiparallel coiled-coil (Non-Patent Document 17). While ubiquitination of the monomeric RING may be detected in vitro, dimerization is required for full cellular activity (Non-Patent Document 18, Non-Patent Document 3). TRIM21 also results in supramolecular cluster formation (Non-Patent Document 3), including on the surface of the viral capsid (Non-Patent Document 19), but is retained on its substrate by an intermediate antibody molecule (Non-Patent Document 8). The Fab of each antibody binds to the substrate, while the Fc binds to TRIM21 PRYSPRY (Non-Patent Document 20).

[0007] The TRIM ligase is degraded together with its substrate. This has been shown during HIV infection in the case of TRIM5 (Non-Patent Document 21) and with a wide range of substrates during Trim-Away in the case of TRIM21 (Non-Patent Document 2). Furthermore, since TRIM21 and its substrate are degraded with a consistent kinetics, it is suggested that they are processed together as a complex (Non-Patent Document 2). As evidence for the autodegradation of the TRIM ligase, the photoinduced cluster formation of the TRIM21 RING-cytochrome2 fusion was sufficient to cause the degradation of the ligase (Non-Patent Document 3). On the other hand, the autodegradation of TRIM5 can be easily induced by ectopic overexpression (Non-Patent Document 22). This results in the formation of large oligomers called "cytosomes", which are likely driven by the trimerization of the B-box (Non-Patent Document 23, Non-Patent Document 24).

[0008] It has been proposed that only the self-ubiquitination of ligase drives the recruitment of the proteasome, which may result in the degradation of the entire TRIM:substrate complex (Non-Patent Document 22, Non-Patent Document 25, Non-Patent Document 8, Non-Patent Document 26). Degradation of the entire TRIM:substrate complex may increase the intracellular turnover of proteins containing TRIM.

[0009] Therefore, there is a need for further fusion proteins and corresponding nucleic acid constructs that can be used to selectively degrade intracellular proteins having an improved cellular half-life. Such fusion proteins are considered to be useful particularly in both the fields of therapy and research.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Summary of the Invention

[0012] The present invention relates to a fusion protein that is suitable for proteolysis in cells and does not undergo N-terminal auto-ubiquitination, and a nucleic acid construct encoding such a protein. Specifically, it relates to a fusion protein comprising at least one RING domain and an adapter sequence that is suitable for proteolysis in cells. The inventors have surprisingly found that by modifying the N-terminus of the fusion protein, the degradation of the ligase and the degradation of the substrate are decoupled. The inventors have provided a fusion protein that can be used for proteolysis, in which the target substrate is degraded but the fusion protein is not degraded.

[0013] In a first aspect, the present invention provides a fusion protein comprising at least a first RING domain, an adapter domain capable of localizing the RING domain and the substrate, and wherein the fusion protein is incapable of N-terminal auto-ubiquitination.

[0014] The fusion protein containing this RING domain cannot undergo N-terminal auto-ubiquitination. The fusion protein cannot function 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, particularly Ube2W. The E2 enzyme Ube2W can still bind to the fusion protein. The fusion protein can use Ube2W to ubiquitinate the substrate of the fusion protein, but the N-terminal ubiquitination (e.g., auto-ubiquitination) of the fusion protein itself is inhibited. The inventors have found that when the ability of the RING-containing fusion protein to undergo N-terminal auto-ubiquitination is inhibited, the turnover of the fusion protein becomes slower, i.e., the cellular half-life is prolonged, and the fusion protein can be maintained intracellularly for a longer time without interfering with the degradation of the substrate. The fusion proteins are not degraded together with their target substrates. The modified fusion protein has an extended half-life intracellularly compared to the equivalent unmodified fusion protein, while still maintaining the ability to degrade substrate proteins intracellularly.

[0015] The second aspect of the present invention provides a nucleic acid construct encoding the fusion protein according to the first aspect.

[0016] The third aspect of the present invention provides a nucleic acid construct comprising a first nucleic acid sequence encoding the first RING domain and a second nucleic acid sequence encoding an adapter domain, which encodes a fusion protein that cannot undergo N-terminal auto-ubiquitination.

[0017] 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 additive.

[0018] The fifth aspect of the present invention provides the fusion protein according to the first aspect or the nucleic acid construct according to the second aspect for use as a medicament.

[0019] A sixth aspect of the present invention provides a method for degrading a target protein in a cell, the method comprising introducing into the cell a fusion protein according to the first aspect or a nucleic acid construct according to the second aspect.

[0020] A seventh aspect of the present invention provides a method for extending the half-life of a fusion protein comprising a RING domain and an adaptor domain, wherein the adaptor domain can localize the RING domain and a substrate, the method comprising modifying the fusion protein so that it cannot undergo N-terminal auto-ubiquitination.

[0021] An eighth aspect of the present invention provides a method for producing a fusion protein according to the first aspect, the method comprising: (a) culturing a host cell containing a first vector encoding a fusion protein comprising a RING domain and an adaptor domain under conditions that allow expression of the fusion protein; (b) obtaining the protein expressed from the host cell; wherein the fusion protein cannot undergo N-terminal auto-ubiquitination.

[0022] Further aspects and embodiments of the present invention are described below.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0024] The inventors have found that when the N-terminal auto-ubiquitination of the E2 enzyme Ube2W of a fusion protein containing at least one RING domain and an adaptor domain is inhibited, the half-life of the fusion protein in cells is extended while its cellular activity is still maintained.

[0025] Therefore, the present invention provides at least a first RING domain, An adapter sequence capable of localizing the RING domain and the substrate, A fusion protein comprising that is not N-terminally self-ubiquitinatable is provided.

[0026] The fusion proteins of the present invention have E3 ubiquitin ligase activity, but they cannot be N-terminally self-ubiquitinylated. In some embodiments, the fusion protein cannot be ubiquitinated. The fusion protein can bind to the E2 enzyme Ube2W and use it to ubiquitinate a substrate, but is modified so that it itself is not ubiquitinated (self-ubiquitinated). "Not capable of N-terminal self-ubiquitination", "not susceptible to N-terminal self-ubiquitination", "inhibited from N-terminal self-ubiquitination", or "not capable of being N-terminal self-ubiquitinated" etc. mean that the fusion protein itself cannot be self-ubiquitinated, but can ubiquitinate other proteins that are present. In other words, the present invention provides a fusion protein containing a RING that cannot N-terminally ubiquitinate itself, but is still catalytically active and can N-terminally ubiquitinate other proteins.

[0027] The RING needs to have an activity that mediates the degradation of the target substrate, but the active RING itself will be degraded unless its self-ubiquitination 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 self-ubiquitination of the RING-containing fusion protein without affecting its ability to mediate the degradation of the substrate. When the N-terminus of the fusion protein is modified to inhibit the self-ubiquitination of the fusion protein, the fusion protein will remain in the cell for a longer period of time when delivered, thereby resulting in a sufficient period of survival to degrade multiple copies of the substrate. The fusion proteins are no longer degraded together with their targets, thereby resulting in a more efficient and longer-lasting depletion of the protein.

[0028] Even in the absence of the target substrate, the RING may have some residual activity and may be a target for other ligases. Therefore, blocking the N-terminus makes the fusion protein containing the RING more persistent in the cell.

[0029] The N-terminus of the fusion protein is modified such that auto-ubiquitination of the fusion protein is inhibited. Modifying the N-terminus of the fusion protein prevents N-terminal ubiquitination of the fusion protein by an E2 enzyme, such as Ube2W. This is achieved by preventing the reactive N-terminus of the fusion protein from being covalently modified with ubiquitin by an E2 enzyme, such as Ube2W. The fusion protein can still bind to Ube2W. The E2 enzyme, particularly Ube2W, can still bind to the E2-binding site of the RING domain. However, the bound Ube2W does not cause ubiquitin conjugation to the N-terminus of the fusion protein.

[0030] The E2-binding site of the RING domain retains its ability to bind to its E2 enzyme and preferably retains its ability to bind to Ube2W.

[0031] In one embodiment, the first RING domain is at the N-terminus of the fusion protein and the adapter domain is located at the C-terminus of the RING domain. Alternative embodiments may include an adapter domain at the N-terminus of the fusion protein.

[0032] In one embodiment, the fusion protein is N-terminally acetylated, i.e., the fusion protein contains an acetyl group at its N-terminal residue. Blocking the N-terminus of the fusion protein with an acetyl group prevents the N-terminus from being ubiquitinated and prevents the fusion protein from being degraded, for example, during Trim-Away.

[0033] In some embodiments, the N-terminus of the fusion protein is blocked with 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 the Ube2W ubiquitination of the fusion protein. Chemical moieties that can be conjugated to the N-terminus include, in addition to acetyl, other amine-reactive moieties such as methyl. Thus, other modifications include cases where the fusion protein is N-terminally methylated, i.e., where the fusion protein contains a methyl group at its N-terminal residue.

[0034] In some embodiments, the N-terminus of the fusion protein contains an N-acetyltransferase recognition site. The N-acetyltransferase recognition site is a short amino acid sequence recognized by the N-acetyltransferase enzyme. Preferably, this recognition site contains the sequence DDDI (SEQ ID NO: 14) or EEEI (SEQ ID NO: 15), more preferably DDDI. The presence of these sites enables acetylation of the fusion protein, so that the fusion protein comes to contain an acetyl group at its N-terminus.

[0035] In some embodiments, the N-terminus of the fusion protein can undergo N-terminal cyclization, preferably, the fusion protein can undergo N-pyroglutamic acid cyclization. To promote N-terminal cyclization of the fusion protein, the fusion protein can contain glutamic acid or glutamine as the N-terminal residue. In some embodiments, the N-terminal glutamine is part of the sequence GFA at the N-terminus of the fusion protein.

[0036] When the fusion protein undergoes N-terminal cyclization, the resulting fusion protein will contain N-terminal pyroglutamine as the N-terminal residue. Thus, in some embodiments, the fusion protein comprises an N-terminal pyroglutamic acid residue. As used herein, pyroglutamic acid broadly refers to an amino acid derivative in which the free amino group of glutamic acid or glutamine cyclizes 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 protects the fusion protein from N-terminal auto-ubiquitination via the E2 enzyme Ube2W.

[0037] In some embodiments, the N-terminal amino acid of the fusion protein is substituted or modified with an amino acid or amino acid sequence that inhibits the ability of an E2 enzyme, such as Ube2W, to ubiquitinate the fusion protein. In some embodiments, the N-terminal amino acid may be substituted with an amino acid sequence that inhibits Ube2w ubiquitination of the fusion protein, preferably E2 enzyme ubiquitination. In some embodiments, the amino acids at positions 1, 2, 3, 4, and 5 are modified or substituted to provide a sequence that inhibits Ube2W ubiquitination of the fusion protein. Preferably, at least the amino acids at positions 1, 2, and 3, more preferably at least the amino acid at position 1, are substituted or modified. For example, in one embodiment, the N-terminal amino acid may be substituted with a polyproline sequence, i.e., the amino acids at positions 1, 2, 3, 4, and 5 may be substituted with a polyproline sequence or another sequence capable of blocking Ube2W ubiquitination of the fusion protein, preferably E2 enzyme ubiquitination. At the start of the fusion protein, a stretch of amino acids may be replaced by an equivalent number of amino acids. In some embodiments, the N-terminus of the fusion protein is modified by adding to the N-terminus a sequence that blocks Ube2W ubiquitination of the fusion protein, preferably E2 enzyme ubiquitination, for example, by adding it to the N-terminus of the first RING domain or adapter domain. Thus, in some embodiments, the present invention provides a fusion protein comprising a RING domain and an adapter domain that cannot be ubiquitinated.

[0038] The fusion proteins of some embodiments of the present invention are 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, and may be represented as.

[0039] Here, RING = ring domain, AD = adapter domain, Ac = acetyl group, NATRS = N-acetyltransferase recognition site, E = glutamic acid, Q = glutamine, PCA = pyroglutamic acid, and a linker sequence may optionally be present between the domains. The fusion protein is shown with the adapter domain located at the N-terminus of the RING domain(s), but the adapter domain and the RING domain(s) can be in any order as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal auto-ubiquitination, for example, preventing Ube2W from ubiquitinating the fusion protein.

[0040] The RING domain of the fusion protein can be derived from any suitable polypeptide. RING domains are known in the art and are described in Freemont PS et al (1991) and function as E3 ligases (Meroni G and Roux G, 2005).

[0041] 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 directs ubiquitin-conjugating enzymes to substrates. Members of the RING (Really Interesting New Gene) domain family typically have a consensus sequence of 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 Joazeiro C, 2009). The RING E3 ligase domain is found in various proteins. Other RING domains include the RING domain derived from the protein X-linked mammalian inhibitor of apoptosis (XIAP) and the RING domain of DER3 / Hrd1. Thus, the use of RING domains derived from other protein families in fusion proteins is also encompassed. The present invention is particularly applicable to RING domains that are capable of auto-ubiquitination, i.e., that may have auto-ubiquitination activity.

[0042] Preferably, the RING domain of the fusion protein is derived from a TRIM polypeptide. The TRIM family contains a number of RING E3 ligases (Marin, I. et al, 2012). In a preferred embodiment, the RING domain is derived from the TRIM21 polypeptide, preferably human TRIM21. The sequence of human TRIM21 is shown in SEQ ID NO: 1 (Uniprot: P19474). MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLL KNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKH RDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHA EFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSA LELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWL ILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCR DSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSF YNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY (SEQ ID NO: 1)

[0043] The RING domain of human TRIM21 includes at least amino acids 3 to 81 of the human TRIM21 sequence shown in SEQ ID NO: 1, preferably amino acids 1 to 85 of the human TRIM21 amino acid sequence shown in SEQ ID NO: 1. The RING domain containing amino acids 1 to 85 of human TRIM21 has the sequence: MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQ (SEQ ID NO: 2) and includes.

[0044] Accordingly, in one embodiment of the present invention, the RING domain includes amino acids 3 to 81 of SEQ ID NO: 2, preferably amino acid residues 1 to 81 of SEQ ID NO: 2, or a variant thereof. In one embodiment, the RING domain includes the sequence of SEQ ID NO: 2 or a variant thereof, and preferably, the RING domain of the fusion protein consists of the sequence of SEQ ID NO: 2 or a variant thereof.

[0045] Amino acids 3 to 81 of human TRIM21 have the sequence: SAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQ (SEQ ID NO: 3) and includes.

[0046] Amino acids 1 to 81 of human TRIM21 have the sequence: MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQ (SEQ ID NO: 4) comprises

[0047] In some embodiments, the RING domain comprises amino acid residues 2 to 81 of human TRIM21. In some embodiments, the RING domain consists of amino acid residues 2 to 81 of human TRIM21.

[0048] Amino acids 2 to 81 of human TRIM21 have the sequence: ASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQ (SEQ ID NO: 5) comprises

[0049] Preferably, the variant sequence has at least 60% identity to the reference sequence at the amino acid level using the default parameters of the BLAST computer program (Atschul et al., 1990, provided by the HGMP (Human Genome Mapping Project)). More preferably, the variant sequence of SEQ ID NO: 2 can have at least 65%, 70%, 75%, 80%, 85%, 90%, even more preferably 95% (even more preferably at least 99%) identity to the sequence of SEQ ID NO: 2 at the amino acid level.

[0050] "Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences that is determined by comparing the sequences. In the art, identity sometimes also means the degree of sequence relatedness (homology) between polypeptide or polynucleotide sequences, which is determined by the match between the strings of such sequences in some cases. There are numerous methods for measuring identity between two polypeptide sequences or two polynucleotide sequences, but the methods commonly used to determine identity are embodied in computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., 1984), BLASTP, BLASTN, and FASTA (Atschul et al., 1990).

[0051] The N-terminal methionine of the expressed fusion protein can be cleaved from the expressed protein either co-translationally or post-translationally, for example, by methionine aminopeptidase. Thus, when referring to the N-terminal amino acid of the modified fusion protein, this includes the N-terminal residue of the fusion protein after excision of the methionine residue of the expressed fusion protein and other co-translational or post-translational processing, such as residue 2 of SEQ ID NO:2 in the modified fusion protein. Thus, when the RING domain is at the N-terminus of the fusion protein, in some embodiments, it is the arginine residue of the RING domain that will be modified, for example, it will be acetylated or modified in such a way that the ability of Ube2W to ubiquitinate the fusion protein is inhibited by other means.

[0052] In some embodiments, RING domains from TRIM polypeptides other than TRIM21 can be used, for example, RING domains from TRIM5, TRIM7, TRIM19, TRIM25, TRIM28, and / or TRIM32, preferably the RING domain from TRIM5 can be used.

[0053] The fusion protein comprises at least one RING domain, i.e., one, two, three, or more RING domains, preferably, the fusion comprises two or three RING domains, more preferably two RING domains.

[0054] In some embodiments, the fusion protein comprises a second RING domain, where the second RING domain is 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 part of a catalytic RING topology that enables proteolysis of the target protein.

[0055] A fusion protein comprising two RING domains can be a more efficient degrader of the target protein than the corresponding fusion protein comprising only one RING domain, but the dual RING fusion protein is constitutively active even in the absence of substrate (e.g., under steady state conditions). That is, such fusion proteins are faster and more efficient, but their half-lives are shorter. Thus, for example, inhibiting N-terminal auto-ubiquitination by blocking the N-terminus of a fusion protein comprising the RING-RING format is particularly beneficial for the RING-RING fusion protein format. Such RING-RING fusion proteins that cannot undergo N-terminal auto-ubiquitination have a longer cellular half-life and improved function compared to single RING domain fusion proteins that can undergo constitutive N-terminal ubiquitination, and also have a longer half-life compared to the corresponding RING-RING fusion proteins having auto-ubiquitination activity.

[0056] When the fusion contains two RING domains, the adapter domain is preferably at the C-terminus of the first RING domain and the second RING domain. Separate domains of the fusion protein can be provided in the order of RING domain - RING domain - adapter domain, from the N-terminus to the C-terminus. In such an embodiment, the amino acid sequence of the first RING domain is linked to the N-terminus of the second RING domain, and the adapter domain is linked to the C-terminal domain of the second RING domain. Such fusion proteins containing at least two RING domains are described in Patent Document 1, where the adapter protein is described as a protein targeting domain, the content of which is incorporated herein by reference.

[0057] When the fusion protein contains two RING domains, the RING domains have sequences capable of dimerizing with each other to form a RING dimer. Preferably, the RING domains contain the same sequence. In one embodiment, both the first RING domain and the second RING domain contain the sequence of SEQ ID NO: 2. When the RING domains contain 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 contains the sequence of SEQ ID NO: 2, and the second RING domain contains a variant sequence of SEQ ID NO: 2, or vice versa. The variant sequence can have at least 65%, 70%, 75%, 80%, 85%, 90%, preferably 95% (more preferably at least 99%) identity to the sequence of SEQ ID NO: 2.

[0058] The fusion protein contains an adaptor domain. The adaptor domain helps localize the fusion protein near the target substrate (i.e., the target protein to be degraded). The adaptor domain is a polypeptide sequence that can place the fusion protein at an appended position to the target substrate. The adaptor domain may directly associate with the target protein, or may bind to an antibody that binds to the target protein or an antibody that binds to that antibody. The adaptor protein may be located at the C-terminus or N-terminus of the construct, and preferably, the adaptor protein is located at the C-terminus of the fusion protein.

[0059] In one embodiment, the adaptor domain is referred to as a protein targeting domain. The protein targeting domain directs the fusion protein to the target protein to be degraded (substrate), also referred to as the target protein of interest. The protein targeting domain binds to the target protein, or an antibody or a fragment or antibody mimetic that binds to the target protein, and may also be referred to as a "protein binding domain". The protein targeting domain may either directly bind to the target protein to form a fusion protein - target protein complex, or bind to an antibody, an antibody fragment, 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.

[0060] 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 located at the C-terminus or N-terminus of the RING domain (e.g., RING-PRYSPRY, RING-RING-PRYSPRY, PRYSPRY-RING-RING, or PRYSPRY-RING).

[0061] The N-terminus of the fusion protein is modified to prevent self-ubiquitination 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 glutamate (E) residue, glutamine (Q) residue, or pyroglutamic acid (PCA) residue, and such constructs are AC-RING-RING-PRYSPRY; NATRS-RING-RING-PRYSPRY; Q-RING-RING-PRYSPRY; E-RING-RING-PRYSPRY; PCA-RING-RING-PRYSPRY; AC-RING-PRYSPRY; NATRS-RING-PRYSPRY; Q-RING-PRYSPRY; E-RING-PRYSPRY; PCA-RING-PRYSPRY represented as.

[0062] The fusion protein is shown with the adaptor domain located at the N-terminus of the RING domain(s), but the adaptor domain and the RING domain(s) can be in any order as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal auto-ubiquitination, such that, for example, Ube2W cannot ubiquitinate the fusion protein.

[0063] The PRYSPRY domain can be derived from a TRIM polypeptide, preferably TRIM21, more preferably human TRIM21. The PRYSPRY domain is composed of the PRY and SPRY regions at positions 286 to 337 and 339 to 465 of the human TRIM21 amino acid sequence shown in SEQ ID NO: 1.

[0064] Amino acids 286 to 337 of human TRIM21 are AVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHF (SEQ ID NO: 7) are.

[0065] Amino acids 339 to 465 of human TRIM21 are SGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPL (SEQ ID NO: 8) are.

[0066] Preferably, the PRYSPRY domain has the sequence: AVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCR DSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPL (SEQ ID NO: 9) comprises

[0067] 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 to 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 the HGMP (Human Genome Mapping Project). More preferably, the variant sequence of SEQ ID NO: 9 can have at least 65%, 70%, 75%, 80%, 85%, 90%, preferably 95% (even more preferably at least 99%) identity to the sequence of SEQ ID NO: 9 at the amino acid level.

[0068] The PRYSPRY domain of the fusion protein binds to the Fc of an antibody or an antibody fragment thereof, such as the Fc region of human IgG1. The fusion protein binds to an antibody that binds to the target protein.

[0069] Since Fc is a dimer, it can be bound by two PRYSPRY domains. The PRYSPRY domain of the first fusion protein binds to one monomer of Fc, while the PRYSPRY domain of the second fusion protein binds to the second monomer of Fc. As a result, the two fusion proteins are co-localized and the RING dimers of each fusion protein are in proximity, so that one RING dimer of one fusion protein is available to mediate the ubiquitination of the other RING dimer.

[0070] In a further 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, their VL fragments, their VH fragments, and sdAbs (i.e., nanobodies) such as their VHH fragments. Preferably, it is scFV or VHH.

[0071] 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 TRIM21, and the VHH binds to the target protein. Preferably, the VHH is at the C-terminus of the RING domain. Preferably, the fusion protein does not contain a coiled-coil domain and / or a B-box domain derived from TRIM located between the VVH domain and the RING domain, and more preferably, the fusion protein does not contain any coiled-coil domain sequence or B-box domain sequence at all. The N-terminus of the fusion protein is modified so as to prevent self-ubiquitination of the fusion protein. For example, as discussed above, the N-terminus of the first RING is N-acetylated (Ac), the fusion protein contains an N-acetyltransferase recognition site (NATRS), or the fusion protein contains an N-terminal glutamate (E) residue, glutamine (Q) residue, or pyroglutamic acid (PCA) residue, and such constructs are AC-RING-VHH; NATRS-RING-VHH; Q-RING-VHH; E-RING-VHH; PCA-RING-VHH represented as

[0072] The fusion protein is shown with the VHH located at the N-terminus of the RING domain, but the VHH and RING domains can be in any order as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal auto-ubiquitination, such that, for example, Ube2W cannot bind to the fusion protein. If the fusion protein is in the VHH-RING order, the N-terminus of the VHH can be N-acetylated.

[0073] In a preferred embodiment, the fusion protein comprises a first RING domain, a second RING domain, and a VHH domain, wherein the RING domains are derived from a TRIM polypeptide, preferably TRIM21, and wherein the VHH binds to a target protein. Preferably, the VHH is at the C-terminus of the first RING domain and the second RING domain. Preferably, the fusion protein does not contain a coiled-coil domain and / or a B-box domain derived from TRIM located between the VVH domain and the second RING domain, and more preferably, the fusion protein does not contain any coiled-coil domain sequence or B-box domain sequence at all. The N-terminus of the fusion protein is modified to prevent auto-ubiquitination of the fusion protein. For example, as discussed above, the N-terminus of the first RING is N-acetylated (Ac), the fusion protein contains an N-acetyltransferase recognition site (NATRS), or the fusion protein contains an N-terminal glutamic acid (E) residue, glutamine (Q) residue, or pyroglutamic acid (PCA) residue, and such constructs can be AC-RING-RING-VHH; NATRS-RING-RING-VHH; Q-RING-RING-VHH; E-RING-RING-VHH; PCA-RING-RING-VHH represented as.

[0074] The fusion protein is shown with the VHH located at the N-terminus of multiple RING domains. However, as long as the N-terminus of the fusion protein contains a modification that inhibits N-terminal auto-ubiquitination and prevents, for example, Ube2W from binding to the fusion protein, the VHH and the multiple RING domains can be in any order. When the fusion protein has the order VHH-RING-RING, the N-terminus of the VHH can be N-acetylated.

[0075] An antibody, antibody fragment, or antibody mimetic of the fusion protein, such as a VHH, specifically binds to a target protein. The fusion protein directly binds to the target protein at the target sequence of the target protein to be degraded. Since many proteins are oligomers (or at least dimers) or part of a protein complex, the antibody domain of the first fusion protein can bind to one of the monomers of the oligomer or protein complex, while the antibody domain of the second fusion protein can bind to the second monomer of the oligomer or protein complex.

[0076] 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. The pathogenic form of the target protein may contain a repeat domain or be a multimeric form of the protein.

[0077] The target protein can be an intracellular protein selected from the group consisting of huntingtin and tau. When the intracellular protein is huntingtin, in one embodiment, the protein targeting domain of the fusion protein binds to the polyglutamic acid sequence of huntingtin.

[0078] In one embodiment, the adapter domain encodes a protein or a fragment thereof that can place the RING domain on a substrate.

[0079] Preferably, the fusion protein does not contain either the B-box domain or the coiled-coil domain of TRIM21 located between the first RING domain and the adapter domain. In one embodiment where the fusion contains two RING domains, the fusion protein may not contain either a B-box domain or a coiled-coil domain derived from any protein 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 contain a B-box domain such as the B-box domain derived from TRIM21, and preferably does not contain a B-box domain derived from any protein. In one embodiment, the fusion does not contain a coiled-coil domain derived from TRIM21, and preferably does not contain a coiled-coil domain derived from any protein.

[0080] The B-box domain of human TRIM21 contains amino acids 91 to 128 of the human TRIM21 amino acid sequence shown in SEQ ID NO: 1. The coiled-coil domain of human TRIM21 contains amino acids 128 to 238 of the human TRIM21 amino acid sequence shown in SEQ ID NO: 1.

[0081] The B-box domain may contain the sequence: RCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVPL (SEQ ID NO: 10) and may include.

[0082] Thus, in one embodiment, the fusion protein does not contain the sequence of SEQ ID NO: 10 or its variants.

[0083] The coiled-coil domain may contain the sequence: EEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHS (SEQ ID NO: 11) and may include.

[0084] Thus, in one embodiment, the fusion protein does not include the sequence of SEQ ID NO: 11 or its variants.

[0085] Preferably, the fusion protein does not include either the sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or their functional variants. Preferably, the variant sequence has at least 60% identity to 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 SEQ ID NO: 11 can have at least 65%, 70%, 75%, 80%, 85%, 90%, preferably 95% (even more preferably at least 99%) identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11 at the amino acid level.

[0086] In embodiments having two RING domains, by not including either a coiled-coil domain or a B-box domain between the second RING domain and the adapter domain, it helps to enable the RING dimer of the fusion protein to be close to the RING dimer of the second fusion protein that is co-localized on the target protein (or an antibody that binds to the target protein).

[0087] However, in some embodiments, the fusion construct may include a coiled-coil domain, a B-box domain, or both a coiled-coil domain and a B-box domain. If the coiled-coil domain and / or B-box is present in a fusion protein that includes two RING domains, they should be located at a sufficient distance from the adapter domain and the RING domain so that the RING dimer of the first fusion protein and the RING dimer of the second fusion protein that is co-localized on the target protein (or an antibody that binds to the target protein) can still be close, for example, when both are bound to the same Fc.

[0088] A linker sequence may be provided between the RING domain and the adapter domain and between each RING domain present in the fusion protein.

[0089] The linker sequence may be derived from the sequence of the TRIM polypeptide, where the linker sequence does not encode the coiled-coil domain and / or the B-box domain of the TRIM polypeptide.

[0090] In other embodiments, standard linker sequences known in the art can also be used. For example, the amino acid sequence of polyglycine or polyserine can be used, or a linker sequence containing a combination of glycine residues and serine residues, such as a linker having the sequence GSGGGGS (SEQ ID NO: 12), can also be used. The length of the linker can be of various sizes.

[0091] However, in embodiments containing two RING domains, the linker sequence between the two RING domains should be of a length sufficient to confer flexibility to the fusion protein and allow dimerization of the two RING domains present. In one embodiment, the linker sequence between the RING domains is between 1 amino acid and 50 amino acids in length, preferably between 1 amino acid and 35 amino acids, between 1 amino acid and 30 amino acids, between 1 amino acid and 25 amino acids, between 1 amino acid and 20 amino acids, between 1 amino acid and 15 amino acids, or between 1 amino acid and 10 amino acids in length. More preferably, the linker is between 1 amino acid and 6 amino acids in length, for example, 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids in length. In some embodiments, there may be no linker between the first RING domain and the second RING domain.

[0092] 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 the RING dimer of the first fusion protein to be in proximity to the RING dimer of the second fusion protein when co-localized on the target protein (or an antibody that binds to the target protein). The linker should be of sufficient length to allow the formation of the catalytic RING topology with the RING domain of the second protein.

[0093] In one embodiment, the linker sequence between the adapter domain and the RING domain is between 5 and 50 amino acids in length, preferably between 5 and 40 amino acids, between 5 and 30 amino acids, between 5 and 25 amino acids, between 10 and 25 amino acids, between 15 and 25 amino acids, between 15 and 20 amino acids, or between 10 and 20 amino acids. More preferably, the linker is between 10 and 20 amino acids in length.

[0094] In one embodiment, the linker sequence may be derived from the sequence of a TRIM polypeptide, where the linker sequence does not encode the coiled-coil domain and / or the B-box domain of the 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, this sequence consists of the sequence GTQGERGLKKMLRTC (SEQ ID NO: 13).

[0095] Accordingly, one embodiment of the present invention includes a 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 between the RING domain and the adapter domain, wherein the RING domain is derived from a TRIM polypeptide, preferably TRIM21, and preferably, the fusion protein does not include a coiled-coil domain or a B-box domain, and the fusion protein cannot be N-terminally auto-ubiquitinated. Preferably, the first RING domain is N-acetylated (Ac), and the fusion protein includes an N-acetyltransferase recognition site (NATRS) at its N-terminus, or the fusion protein includes an N-terminal glutamic acid (E) residue, glutamine (Q) residue, or pyroglutamic acid (PCA) residue.

[0096] Another embodiment of the present invention includes a fusion protein comprising one RING domain, an adapter domain located 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 TRIM21, and preferably, the fusion protein does not include a coiled-coil domain or a B-box domain, and the fusion protein cannot be N-terminally auto-ubiquitinated. Preferably, the N-terminus of the fusion protein is modified to prevent auto-ubiquitination of the fusion protein. Preferably, the RING domain is N-acetylated (Ac), and the fusion protein includes an N-acetyltransferase recognition site (NATRS) at its N-terminus, or the fusion protein includes an N-terminal glutamic acid (E) residue, glutamine (Q) residue, or pyroglutamic acid (PCA) residue.

[0097] "Fusion protein" and "fusion polypeptide" refer to a polypeptide having two or more moieties linked to each other by a covalent bond, each moiety having specific properties which may be the same or different. Such properties may be biological properties such as activity in vitro or in vivo. Such properties may also be simple chemical or physical properties such as binding to a target antigen, catalysis of a reaction, etc. The two moieties may be directly linked by a single peptide bond or may be linked via a peptide linker containing one or more amino acid residues. Generally, the two moieties and the linker are in frame with each other.

[0098] The term "fusion protein" as used herein generally means one or more proteins linked to each other by chemical means including hydrogen bonds or salt bridges, or by peptide bonds by protein synthesis, or both. Typically, a fusion protein will be prepared by standard DNA recombination techniques in the art and may be referred to herein as a recombinant fusion protein.

[0099] The present invention also provides a nucleic acid construct encoding the fusion protein of the present invention. The nucleic acid construct may comprise 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 comprise a third nucleic acid sequence encoding a second RING domain located between the first RING domain and the adapter domain. The nucleic acid can encode a fusion protein that cannot self-ubiquitinate and includes an N-terminus that inhibits the ubiquitination of Ube2W of the fusion protein. The nucleic acid construct encodes a fusion protein that inhibits the ubiquitination of Ube2W of the fusion protein, but the fusion protein encoded by the nucleic acid construct can bind to Ube2W.

[0100] In some embodiments, the nucleic acid 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 the sequence DDDI (SEQ ID NO: 14) or EEEI (SEQ ID NO: 15) at the N-terminus.

[0101] In some embodiments, the nucleic acid construct encodes a fusion protein comprising a glutamine or glutamate residue as the N-terminal residue. In some embodiments, the nucleic acid construct may further comprise a sequence encoding glutaminyl cyclase.

[0102] In some embodiments, the nucleic acid construct encodes a fusion protein having an amino acid sequence that inhibits Ube2W ubiquitination of the fusion protein at the N-terminus. In addition to the RING domain and the adapter domain, an amino acid sequence may be present. Alternatively, the amino acid sequence may replace the N-terminal sequence of the RING domain or the adapter domain (depending on which domain is located at the N-terminus). For example, if the nucleic acid construct encodes a fusion protein sequence having a RING domain at the N-terminus, the nucleic acid construct encodes the RING domain and the N-terminal residue of the RING domain is replaced with an amino acid sequence that inhibits Ube2W ubiquitination of the fusion protein, for example, the N-terminal residue of the RING domain may be replaced with a polyproline sequence. In another embodiment, if the nucleic acid construct encodes a fusion protein sequence having a RING domain at the N-terminus, the nucleic acid construct comprises an additional nucleic acid sequence encoding an amino acid sequence that inhibits Ube2W ubiquitination of the fusion protein, where the amino acid sequence that inhibits Ube2W ubiquitination of the fusion protein is located at the N-terminus of the RING domain and is replaced with a certain sequence.

[0103] In some embodiments, the nucleic acid construct does not encode a coiled-coil domain, does not encode a B-box domain, or does not encode either the coiled-coil domain or the B-box domain.

[0104] These nucleic acid constructs may be provided in the form of a vector, such as an expression vector, among others, chromosomal vectors, episomal vectors, and vectors derived from viruses, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, baculoviruses, papovaviruses such as SV40, vaccinia virus, adenovirus, lentivirus, fowlpox virus, pseudorabies virus, and retroviruses, and vectors derived from combinations thereof, such as plasmids and vectors derived from genetic elements of bacteriophages such as cosmids and phagemids. Generally, any vector suitable for maintaining, increasing, or expressing nucleic acids to express polypeptides in a host can be used for expression in this regard. The vector may contain a plurality of, for example, two or more nucleic acid constructs as defined above. Preferably, the vector is a viral delivery vector, preferably an adeno-associated virus (AAV) vector or a lentivirus vector.

[0105] The nucleic acid constructs of the present invention preferably include a promoter or other regulatory sequences that control the expression of the nucleic acid. The promoter or other regulatory sequences can be operably linked to the nucleic acid sequence encoding the domain of the fusion protein. The promoters and other regulatory sequences that control the expression of the nucleic acid are specified and are known in the art. Those skilled in the art will note that it may not be necessary to utilize the entire promoter or other regulatory sequences. Only the minimal essential regulatory elements may be required, and in fact, such elements can be used to construct chimeric sequences or other promoters.

[0106] The term "nucleic acid construct" generally refers to nucleic acids of any length that can be DNA, cDNA, or RNA such as mRNA obtained by cloning or generated by chemical synthesis. The DNA can be single-stranded or double-stranded. The single-stranded DNA can be a coding sense strand, or a non-coding or antisense strand. For therapeutic use, the nucleic acid construct is preferably in a form capable of being expressed in the subject to be treated.

[0107] The present invention also provides a host cell containing such a nucleic acid construct. The fusion protein can be expressed in various cells.

[0108] The present invention also provides a method for preparing the fusion protein of the present invention, which includes culturing or maintaining a host cell containing the above nucleic acid construct or vector under conditions such that the host cell produces the fusion protein, and optionally further includes isolating the fusion protein.

[0109] In one aspect of the present invention, a method for producing the fusion protein of the present invention is provided. This method (a) culturing a host cell containing a first vector encoding a fusion protein containing a RING domain and an adapter domain under conditions enabling the expression of the fusion protein; (b) obtaining the protein expressed from the host cell; and wherein the fusion protein cannot be N-terminally auto-ubiquitinated.

[0110] In some embodiments, the first vector also an N-acetyltransferase recognition site at the N-terminus of the fusion protein, preferably having the sequence DDDI (SEQ ID NO: 14) or EEEI (SEQ ID NO: 15); a glutamic acid residue or a glutamine residue at the N-terminus of the fusion protein, or The sequence QFA at the N-terminus of the fusion protein, An amino acid sequence at the N-terminus of a fusion protein that inhibits ubiquitination of the fusion protein by Ube2W, such as a polyproline sequence, encodes it.

[0111] In some embodiments, the first vector also encodes glutaminyl cyclase, the expressed fusion protein contains an N-terminal glutamate residue or an N-terminal glutamine residue, and the method includes culturing a host cell under conditions that allow expression of the fusion protein and glutaminyl cyclase.

[0112] For example, in one embodiment, the first vector also expresses a tev cleavage site along with the fusion protein, and the second vector expresses tev protease. The fusion protein can be expressed on a multicistronic vector that also expresses glutaminyl 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, which is cyclized by glutaminyl cyclase. Then, the cyclized RING fusion protein having pyroglutamic acid at the N-terminus is purified, for example, based on a C-terminal His tag. In such an embodiment, during the production of the fusion protein of the present invention, the following fusion proteins are expressed.

[0113] 1. T7P-RBS-ENLYVQQFA-R-R-PY-6His-Stop-RBS-bQC(E45Q)-Stop-T7T 2. T7P-RBS-MBP-TEVP(S219V)

[0114] Here, T7P = T7 promoter, RBS = ribosome binding site, ENLYVQ (SEQ ID NO: 16) is the tev site, QFA is the site for glutaminyl cyclase (QC), R = RING, PY = PRYSPRY domain, 6His = 6 histidines, Stop = stop codon, T7T = T7 terminator, MBP = maltose binding protein, TEVP = TEV protease.

[0115] In some embodiments, the method includes modifying the N-terminus of the expressed fusion protein. The modification of the N-terminus of the fusion protein can be performed in various ways.

[0116] 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 includes incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA. NAT can add an acetyl group to the N-terminus of the fusion protein.

[0117] By blocking the N-terminus of the fusion protein with an acetyl group, the N-terminus is prevented from being ubiquitinated and the fusion protein is prevented from being degraded, for example, during Trim-Away.

[0118] In some embodiments, the first vector encodes a fusion protein having a glutamate residue or a glutamine residue at the N-terminus of the expressed fusion protein, and the method further includes incubating the fusion protein with glutaminyl cyclase. In some embodiments, the vector encodes a fusion protein having the sequence QFA as the N-terminal sequence. Glutaminyl cyclase cyclizes the free amino group of glutamate or glutamine to form a lactam, resulting in pyroglutamic acid as the N-terminal residue of the fusion protein. By blocking the N-terminus of the fusion protein with a pyroglutamic acid residue, the N-terminus is prevented from being ubiquitinated and the fusion protein is prevented from being degraded, for example, during Trim-Away.

[0119] In some embodiments, the fusion protein can be expressed with an N-terminal methionine. Such residues can be cleaved post-translationally from the expressed protein, for example, by methionine aminopeptidase. Thus, when referring to the N-terminal amino acid of the fusion protein to be modified, this includes the N-terminal residue of the fusion protein after excision of the methionine residue of the expressed fusion protein. For example, residue 2 of SEQ ID NO:2 is the N-terminal residue of the fusion protein to be modified. Thus, when the RING domain is at the N-terminus of the fusion protein, in some embodiments, the arginine residue of the RING domain will be modified. For example, it can be acetylated or modified in such a way that autoubiquitination via Ube2W is inhibited or blocked by other means, or it can be replaced with an amino acid that inhibits the ability of Ube2W to ubiquitinate the fusion protein.

[0120] In some embodiments, the method comprises methylating the N-terminal amino acid of the expressed fusion protein or 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.

[0121] Thus, in some embodiments, the method provides a fusion protein that cannot be ubiquitinated.

[0122] Also provided is a pharmaceutical composition comprising the fusion protein or nucleic acid construct of the present invention. The pharmaceutical composition may comprise various pharmaceutically acceptable carriers and / or additives. Suitable pharmaceutically acceptable carriers and / or additives are 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 administration, intramuscular administration, oral administration, or intraperitoneal administration. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a solution, gel, powder, tablet, capsule, or foam.

[0123] The fusion protein and nucleic acid construct of the present invention can be used as a medicine for treatment. In one embodiment, the present invention also provides the treatment of neurological disorders such as Alzheimer's disease or Huntington's disease. In other embodiments, the present invention provides the treatment of infectious diseases such as viral infections such as HIV. In a further embodiment, the present invention provides the treatment of trinucleotide repeat disorders, particularly trinucleotide repeat disorders in which the trinucleotide repeat is present within the coding sequence of a gene. Trinucleotide repeat disorders that can be treated using the fusion protein or nucleic acid construct of the present invention include Huntington's disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxia.

[0124] The treatment of neurological disorders, infectious diseases, or trinucleotide repeat disorders includes administering to a subject the fusion protein, nucleic acid, or pharmaceutical composition of the present invention.

[0125] In one embodiment, the treatment comprises administering a fusion protein comprising a first RING domain and an adapter domain, the fusion protein being unable to self-ubiquitinate at the N-terminus. The adapter domain is located at the C-terminus of the first RING domain and the second RING domain. Preferably, the fusion protein to be administered does not include a coiled-coil domain or a B-box domain. In some embodiments, the fusion protein includes a second RING domain located between the first RING domain and the adapter domain.

[0126] In one embodiment of the present invention, the treatment comprises 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, the nucleic acid encoding a fusion protein that is unable to self-ubiquitinate at the N-terminus. The nucleic acid construct encodes a fusion protein in which the protein targeting domain is located at the C-terminus of the RING domain. Preferably, the nucleic acid construct to be administered does not include a sequence encoding a B-box domain or a sequence encoding a coiled-coil domain. In some embodiments, the nucleic acid construct includes a third nucleic acid sequence encoding a second RING domain located between the first RING domain and the adapter domain.

[0127] When the disorder to be treated is a neurological disorder 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 that specifically binds to tau, a fragment of the antibody, or an antibody mimetic. When the disorder to be treated is Huntington's disease, the adapter domain may encode a sequence that targets huntingtin. In one embodiment, the adapter domain may encode an antibody that specifically binds to the polyglutamic acid sequence of huntingtin, a fragment of the antibody, or an antibody mimetic.

[0128] The nucleic acid construct according to the present invention can also be administered by a delivery vector. Examples of the delivery vector include viral delivery vectors such as adenovirus delivery vectors, retrovirus delivery vectors, or lentivirus delivery vectors known in the art. Other non-viral delivery vectors include lipid delivery vectors including liposome delivery vectors known in the art.

[0129] The treatment includes both prevention (prevention before occurrence) and therapeutic treatment. The terms "treat", "treating", or "treatment" (or equivalent terms) mean that the severity of the condition of an individual is reduced, or at least partially improved or remitted, and / or at least some reduction, alleviation, or decrease of at least one clinical symptom is achieved, and / or the progression of the condition is suppressed or delayed, and / or the prevention or delay of the onset of a disease or illness is seen.

[0130] The terms "patient", "individual", or "subject" include human subjects and other mammalian subjects that receive either preventive treatment or therapeutic treatment with the fusion protein or nucleic acid construct described herein. Mammalian subjects include primates, such as non-human primates. Mammalian subjects also include, but are not limited to, experimental animals commonly used in research, such as rabbits and rodents such as rats and mice.

[0131] The fusion protein and nucleic acid construct of the present invention can also be used, for example, as a research tool for studying the degradation of proteins in cells or in samples.

[0132] Therefore, in one embodiment of the present invention, a method for degrading intracellular proteins is provided, which includes administering the fusion protein or nucleic acid of the present invention. The cells may be in vitro cells.

[0133] A further embodiment of the present invention provides a method for degrading a protein in a sample, which includes introducing the fusion protein or nucleic acid construct of the present invention into the sample.

[0134] In one embodiment, a method for degrading a protein in a cell or a sample includes administering a fusion protein that includes a first RING domain and an adaptor domain and cannot be N-terminally auto-ubiquitinated. The adaptor domain is preferably located at the C-terminus of the RING domain. Preferably, the administered fusion protein does not include a coiled-coil domain or a B-box domain. In some embodiments, the fusion protein includes a second RING domain located between the first RING domain and the adaptor domain.

[0135] In one embodiment of the present invention, a method for degrading a protein in a cell or a sample includes administering a nucleic acid construct that includes a first nucleic acid sequence encoding a first RING domain and a second nucleic acid sequence encoding an adaptor domain, and the nucleic acid encodes a fusion protein that cannot be N-terminally auto-ubiquitinated. Preferably, the administered nucleic acid construct does not include a sequence encoding a B-box domain or a sequence encoding a coiled-coil domain. In some embodiments, the nucleic acid construct includes a third nucleic acid sequence encoding a second RING domain located between the first RING domain and the adaptor domain. The nucleic acid construct may have the other features described above.

[0136] An antibody, an antibody fragment, or an antibody mimetic that targets the target protein, or a nucleic acid encoding the antibody, the antibody fragment, or the antibody mimetic can also be administered to the cell or the sample. The "target protein" is the protein to be degraded. The antibody, the antibody fragment, or the antibody mimetic can specifically bind to the target protein.

[0137] The above method is particularly useful for degrading proteins in cells that do not endogenously express TRIM21. The above method is particularly useful for degrading intracellular proteins. However, in some embodiments, when targeting a pathogen such as a virus, for example, the antibody will bind to the target protein extracellularly. The antibody-target will translocate into the cell, where the fusion protein will bind to the antibody-target and degrade the protein.

[0138] The fusion protein or nucleic acid can be introduced into cells by transfection, for example, by injection including microinjection or by electroporation, or by transduction, for example, by the use of a viral delivery vector such as an AAV vector. Other delivery techniques suitable for introducing the fusion protein and nucleic acid constructs into cells are known in the art.

[0139] The provided fusion protein has an extended cellular half-life compared to the corresponding fusion protein that can undergo self-ubiquitination.

[0140] In one aspect of the invention, there is provided a method of extending the cellular half-life of a fusion protein comprising a RING domain and an adapter sequence, wherein the adapter sequence can localize the RING domain and a substrate, the method comprising modifying the fusion protein so that it does not undergo N-terminal self-ubiquitination.

[0141] "The cellular half-life is extended" means that the cellular half-life of the modified fusion protein is extended compared to the corresponding fusion protein without the modification.

[0142] Modifying the fusion to be unable to undergo N-terminal auto-ubiquitination involves modifying the N-terminus of the fusion protein as compared to an unmodified fusion protein capable of N-terminal auto-ubiquitination. By modifying the N-terminus, a fusion protein that cannot be ubiquitinated by Ube2W is obtained. This is achieved by preventing the reactive N-terminus of the fusion protein from being covalently modified with ubiquitin by Ube2W. The N-terminus of the fusion protein can be modified in various ways.

[0143] In one embodiment, the method includes incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA to N-terminally acetylate the fusion protein. By blocking the N-terminus of the fusion protein with an acetyl group, the N-terminus is prevented from being ubiquitinated and the protein is prevented from being degraded, for example, during Trim-Away.

[0144] The fusion protein can also be modified by introducing an N-acetyltransferase recognition site at the N-terminus of the fusion protein. Examples of N-acetyltransferase recognition sites include sequences such as DDDI and EEEI. In some embodiments, the method may further include incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA to N-terminally acetylate the fusion protein. The presence of an N-acetyltransferase recognition site makes the fusion protein a substrate for cytoplasmic N-acetyltransferase. Cytoplasmic N-acetyltransferase can add an acetyl group to the N-terminus of the fusion protein.

[0145] Other techniques include methylating the N-terminal amino acid of the fusion protein or coupling a chemical moiety that inhibits the ability of the E2 enzyme, particularly Ube2W, to ubiquitinate the fusion protein to the N-terminal amino acid of the fusion protein.

[0146] The fusion protein may be modified such that the fusion protein can undergo N-terminal cyclization. Preferably, the method includes modifying the fusion protein such that the fusion protein can undergo N-terminal pyroglutamic acid cyclization.

[0147] To provide a fusion protein capable of undergoing N-terminal pyroglutamic acid cyclization, the method may include introducing a glutamic acid residue or a glutamine residue at the N-terminus of the fusion protein. Preferably, the method includes encoding an N-terminal glutamic acid residue in the fusion protein.

[0148] Proteins starting 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, in a fusion protein having an N-terminal RING domain, the presence of pyroglutamic acid at the N-terminus of the fusion protein is thought to protect the RING domain from N-terminal auto-ubiquitination via Ube2W.

[0149] This reaction may occur spontaneously or may be catalyzed by a glutaminyl cyclase (GS) enzyme. The pyroglutamic acid cyclization of the fusion protein may potentially occur spontaneously by encoding an N-terminal glutamine or glutamic acid. In one embodiment, the method may further include incubating the fusion protein with glutaminyl cyclase. In some embodiments, the method includes introducing the sequence QFA at the N-terminus of the protein. By adding this QFA sequence to the target protein and co-expressing it with the GS enzyme, it becomes possible to produce a protein with a cyclized N-terminus.

[0150] In one embodiment, the enzyme-catalyzed cyclization of the N-terminal glutamine of the fusion can be carried out in vitro or simultaneously with translation intracellularly as follows:

[0151] A fusion protein containing a RING domain is expressed with a TEV cleavage site leaving an exposed glutamine at the N-terminus. The fusion protein is expressed on a multicistronic vector containing glutaminyl cyclase. This plasmid is co-transfected with a second plasmid encoding TEV protease. During expression, the TEV protease cleaves the RING fusion protein to expose the N-terminal glutamine, which is cyclized by glutaminyl 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.

[0152] 1. T7P-RBS-ENLYVQQFA-R-R-PY-6His-Stop-RBS-bQC(E45Q)-Stop-T7T 2. T7P-RBS-MBP-TEVP(S219V)

[0153] Here, T7P = T7 promoter, RBS = ribosome binding site, ENLYVQ (SEQ ID NO: 16) is the TEV site, QFA is the site for glutaminyl cyclase (QC), R = RING, PY = PRYSPRY domain, 6His = 6 histidines, Stop = stop codon, T7T = T7 terminator, MBP = maltose binding protein, TEVP = TEV protease.

[0154] A further method of modifying the fusion protein involves substituting or modifying the N-terminal amino acid of the fusion protein, for example, the N-terminal amino acid of the RING domain if located at the N-terminus, with an amino acid sequence that inhibits the ability of Ube2W to ubiquitinate the fusion protein. In some embodiments, only the N-terminal amino acid may be substituted with an amino acid sequence that results in inhibition of Ube2w ubiquitination of the fusion protein.

[0155] 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 includes modifying or substituting the amino acids at at least positions 1, 2, and 3, more preferably the amino acids at at least position 1. For example, in one embodiment, the method includes substituting the N-terminal amino acids of the RING domain located at the N-terminus with a polyproline sequence. For example, the method may include substituting the amino acids at positions 1, 2, 3, 4, and 5 with a polyproline sequence or other sequence capable of inhibiting Ube2W ubiquitination of the fusion protein. At the start of the fusion protein, a stretch of amino acids may be replaced by an equal number of amino acids. In some embodiments, the method includes adding an amino acid sequence capable of blocking Ube2W ubiquitination of the fusion protein to the N-terminus of the fusion protein. Here, for example, if the fusion protein includes a RING domain at the N-terminus, the method includes adding an amino acid sequence to the N-terminus of the first RING domain.

[0156] The terms "comprising" or "comprises", wherever they appear in this specification, can be replaced by the terms "consisting of", "consists of", "consisting essentially of", or "consists essentially of", and vice versa.

[0157] The content of all publications cited herein is hereby incorporated by reference in its entirety to more fully describe the state of the art to which the present invention pertains.

[0158] The present invention will be further understood by reference to the following examples.

Examples

[0159] Method Plasmid A complete list of the plasmids used in this study can be found in Figure 11, including the primary sequences of all constructs. To generate lentiviruses, constructs were inserted into a modified version of pSMPP (Addgene number 104970) (pPMEZ) in which the SFFV promoter and puromycin resistance sequence were replaced with the PGK1 promoter and zeocin resistance sequence, respectively. To transcribe mRNA in vitro, constructs were inserted into pGEMHE (Liman et al., 1992), which contains UTR and polyA sequences for optimal mRNA stability and translation. To purify proteins, constructs were inserted into derivatives of vectors from the pOP and pET (Novagen) series.

[0160] Generation of Lentiviruses Three days after co-transfection (FuGENE 6, Promega) of the lentiviral plasmid construct (Figure 11) with the HIV-1 GagPol expression construct pcRV1 (gift from Dr. Stuart Neil) and pMD2G (gift from Didier Trono) (Addgene plasmid number 12259), lentiviral particles were collected from the HEK293T cell supernatant. The supernatant was filtered through 0.45 μm and stored at -80 °C.

[0161] In Vitro Transcription of mRNA The pGEMHE plasmid construct (Figure 11) was linearized and 5’-capped mRNA was synthesized using T7 polymerase (NEB’s HiScribe T7 ARCA kit) according to the manufacturer’s instructions. mRNA concentration was quantified using a Qubit 4 fluorometer (ThermoFisher) and the RNA Broad Range assay kit (ThermoFisher, Q10211).

[0162] Protein Expression and Purification The full list of purified proteins used in this study can be found in (Figure 12). Ube2W, Ube2N, and Ube2V2, as well as TRIM21 R, R-PS, R-R-PS, T21R-vhhGFP4, and mEGFP, were expressed in Escherichia coli BL21 DE3. Ubiquitin and Ube1 were expressed in Escherichia coli Rosetta 2 DE3 cells described previously (Kiss et al., 2021). Cells were grown at 37 °C and 220 rpm until the OD 600 reached approximately 0.7. After induction, the temperature was lowered to 18 °C overnight. For TRIM21 and E2, induction was performed using 0.5 mM IPTG and 10 μM ZnCl2, and for ubiquitin and Ube1, induction was performed using 0.2 mM IPTG. mEGFP was expressed in ZY autoinduction medium (Studier, 2005) at 37 °C and 220 rpm. OD 600When it reached 0.7, the temperature was decreased to 18 °C overnight for expression. After centrifugation, the cells were resuspended in 50 mM Tris (pH 8.0), 150 mM NaCl, 10 μM ZnCl2, 1 mM DTT, 20% Bugbuster (Novagen), and Complete protease inhibitor (Roche, Switzerland). For His-tagged proteins, 20 mM imidazole was added to the buffer. Lysis was performed by sonication. TRIM21-R-PS and TRIM21-R-R-PS were expressed with an N-terminal GST-SUMO tag, and TRIM-R, Ube2W, Ube2V2, and Ube1 were expressed with an N-terminal GST tag followed by a TEV protease cleavage site and purified via glutathione sepharose resin (GE Healthcare) equilibrated in 50 mM Tris (pH 8.0), 150 mM NaCl, and 1 mM DTT. The tags were cleaved overnight at 4 °C on the beads (using SUMO protease or TEV protease respectively). No trace of cleavage occurred in TRIM21-R-PS and TRIM21-R-R-PS with SUMO protease cleavage. With TEV cleavage, a trace of N-terminal GSH occurred in TRIM21-R, a trace of N-terminal G occurred in Ube2N, a trace of N-terminal GSQEF occurred in Ube2V2, and a trace of N-terminal GSH occurred in Ube2W. For Ube1, protease cleavage was not performed, and the GST-Ube1 fusion protein was eluted using 50 mM Tris (pH 8.0), 150 mM NaCl, 10 mM reduced glutathione, and 1 mM DTT. mEGFP was expressed without a protease cleavage site together with an N-terminal His tag, and Ube2N was expressed with an N-terminal His tag followed by a TEV protease cleavage site. By expressing TRIM21 R-vhhGFP4 as a His-SUMO fusion protein, the native N-terminus of TRIM21 was generated after SUMO protease cleavage during purification. His-tagged proteins were purified via Ni-NTA resin equilibrated in 50 mM Tris (pH 8.0), 150 mM NaCl, 20 mM imidazole, and 1 mM DTT.The protein was eluted in 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM DTT, and 300 mM imidazole. For Ube2N, His-tag TEV cleavage was carried out overnight by dialyzing the sample against 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM DTT, and 20 mM imidazole. Subsequently, His-tagged TEV protease was removed by Ni-NTA resin. For T21R-vhhGFP4, SUMO protease cleavage was carried out overnight at 4 °C on Ni-NTA resin. The next day, elution was performed using equilibration buffer. Finally, size-exclusion chromatography of all proteins was carried out either on a HiLoad 26 / 60 or 16 / 600 Superdex 75 prep grade column (GE Healthcare) in 20 mM Tris (pH 8.0), 150 mM NaCl, and 1 mM DTT, except for GST-Ube1, for which a HiLoad 26 / 60 or 16 / 600 Superdex 200 prep grade column (GE Healthcare) was used. Ubiquitin purification was carried out according to the protocol established by the Pickart lab (Pickart and Raasi, 2005). After cell lysis by sonication (lysis buffer: 50 mM Tris (pH 7.4), 1 mg·mL. -1 of lysozyme (Sigma Aldrich, St. Louis, USA), 0.1 mg·mL -1DNase (Sigma Aldrich, St. Louis, USA)) was added to the stirred lysate at 4 °C with a total concentration of 0.5% perchloric acid. The (milky white) lysate was further incubated in a stirrer at 4 °C for 30 minutes to complete precipitation. Next, the lysate was centrifuged at 4 °C for 30 minutes (19,500 rpm). The supernatant was dialyzed overnight (3500 MWCO) against 3 L of 50 mM sodium acetate (pH 4.5). Subsequently, Ub was purified using a NaCl gradient (0 mM - 1000 mM NaCl in 50 mM NaAc (pH 4.5)) via cation exchange chromatography using a 20 mL SP column (GE Healthcare). Finally, size exclusion chromatography was performed on a HiLoad 26 / 60 Superdex 75 prep grade column (GE Healthcare) in 20 mM Tris (pH 7.4). 15 Isotopically labeled proteins were expressed using Escherichia coli BL21 DE3 cells in M9 minimal medium supplemented with NH4Cl (Sigma-Aldrich ISOTEC). Chaetomium thermophilum Naa50 containing a C-terminal His tag 82~289 (Naa50ΔΔ) was expressed using E. coli Rosetta 2 cells in ZY autoinduction medium (Studier, 2005) grown at 37 °C and 220 rpm. OD 600 When it reached 0.7, the temperature was lowered to 18 °C overnight for expression. CtNaa50 82~289was purified as follows: Cells were harvested, resuspended in buffer A500 (20 mM HEPES (pH 7.5), 500 mM NaCl, 20 mM imidazole) supplemented with protease inhibitor mix (SERVA Electrophoresis GmbH, Germany), and lysed using a microfluidizer (M1-10L, Microfluidics). The lysate was clarified at 50,000 g for 30 min at 4 °C and filtered through a 0.45-μm membrane. The supernatant was applied to a 1-mL HisTrap FF column (GE Healthcare) for Ni-IMAC (immobilized metal affinity chromatography) purification. The column was washed with buffer A500 and the protein was eluted with buffer A500 supplemented with 250 mM imidazole. Subsequently, CtNaa50 82~289 was 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). SUMO protease (MBP-Ulp1 (based on the R3 sequence (Lau et al., 2018))) was purified using an MBPTrap HP 5-ml column and eluted with 50 mM Tris (pH 8), 150 mM NaCl, 1 mM DTT, and 10 mM maltose. Finally, the eluted fractions were separated on a HiLoad 26 / 60 Superdex 75pg SEC column (150 mM NaCl, 50 mM Tris (pH 8), and 1 mM DTT).

[0163] Cell culture HEK293T (ATCC) cells and NIH3T3-CAV1-EGFP (Shvets et al., 2015) cells were cultured in DMEM medium (Gibco, 31966021) supplemented with 10% fetal bovine serum and penicillin-streptomycin. RPE-1 cells (ATCC) were cultured in DMEM / F-12 medium (Gibco, 10565018) supplemented with 10% fetal bovine serum and penicillin-streptomycin. All cells were grown at 37 °C in a 5% CO2 humidified atmosphere and were regularly checked for the absence of mycoplasma. The sex of NIH3T3 cells is male. The sex of HEK293T cells and RPE-1 cells is female. For the 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, the cells were grown in medium supplemented with 10% fetal bovine serum without antibiotics. Live imaging was performed using an IncuCyte S3 live cell analysis system (Sartorius) housed in a 37 °C, 5% CO2 humidified incubator. For live imaging with the IncuCyte, the cell culture medium was replaced with Fluorobrite (Gibco, A1896701) supplemented with 10% fetal bovine serum and GlutaMAX (Gibco, 35050061).

[0164] Cell line The cell lines used and generated in this manuscript are detailed in (Table 1). RPE-1 TRIM21 KO cells (Non-Patent Document 3), HEK293T TRIM21 KO cells (Zeng et al., 2019), and NIH3T3-CAV1-EGFP (Shvets et al., 2015) have been described previously. To stably express CAV1-mEGFP and CAV1-mEGFP-Halo, lentiviral particles were introduced into RPE-1 cells at a multiplicity of infection of approximately 0.1 transduction units per cell, and the GFP-positive population was selected by flow cytometry. To stably express TRIM21-HA at endogenous levels, RPE-1 TRIM21 KO cells were reconstituted under the control of the native TRIM21 promoter using the TRIM21-HA construct as previously described (Zeng et al., 2019).

[0165] Electroporation Electroporation was performed using the Neon™ transfection system (Thermo Fisher). Cells were washed with PBS and resuspended in Buffer R at a concentration of 1×10 7 cells ~ 8×10 7 cells per ml. For each electroporation reaction, 10.5 μl of a volume of 1×10 5 cells ~ 8×10 5 cells was mixed with 2 μl of antibody (typically 0.5 mg / ml) or mRNA (typically 0.5 μM) or protein to be delivered. The mixture was taken up in a 10 μl Neon™ pipette tip and electroporated at 1400 V, 20 ms, 2 pulses, and transferred to antibiotic-free medium.

[0166] Measurement of fluorescence in live cells To quantify GFP fluorescence in live cells, images were acquired and analyzed using an IncuCyte live cell analysis system (Sartorius). Within the IncuCyte software, the integrated density (product of area and average intensity) for GFP fluorescence was normalized for each image against the total cell area (phase). Values were normalized against the internal control within each experiment.

[0167] antibody The antibodies and concentrations used for conventional immunoblotting (IB), capillary-based immunoblotting (Jess), and electroporation (EP) are detailed in (Table 2). All antibodies used for electroporation were purchased in azide-free form or passed through an Amicon Ultra-0.5 100KDa centrifugal filter device (Millipore) to remove trace amounts of azide and the buffer was replaced with PBS.

[0168] Adv5 neutralization assay Adenovirus serotype 5 2.6-del CMV-eGFP (Adv5-GFP, Viraquest) was diluted to 1.1×10 9 T.U. / mL in PBS and 16 μL was incubated 1:1 with anti-hexon recombinant humanized IgG1 9C12 or 9C12H433A (Foss et al., 2016) at the indicated concentrations or with PBS. After incubation for 1 hour at room temperature, the complex was diluted with 250 μL of Fluorobrite medium and used for the Adv5 neutralization assay. To infect, 4×10 6Individual HEK293T TRIM21 KO cells were electroporated with PBS or R-R-PS±N-acetylation and resuspended in 2 mL of Fluorobrite medium. 50 μL of each cell suspension was combined 1:1 with the Adv5:9C12 complex or the Adv5:9C12H433A complex (for immediate infection) or Fluorobrite (for delayed infection) in a 96-well plate. For delayed infection, the electroporated cells were allowed to adhere to the plate for 2 hours, after which the medium was replaced with 50 μL of Fluorobrite and the cells were infected with 50 μL of the Ad5:9C12 complex. Infection levels were quantified by measuring the GFP fluorescence area relative to the total cell area 16 hours after infection using the IncuCyte system. Infection levels were plotted against Adv5-GFP infection in the absence of the 9C12 antibody.

[0169] NFκB signaling assay HEK293T TRIM21 KO cells were transfected with 2 μg of the pGL4.32 NF-κB luciferase plasmid (Promega) using 12 μL of Viafect (Promega) in 200 μL of OptiMEM (Thermo Fisher). After 24 hours, 6 Individual transfected cells were electroporated with PBS or R-R-PS±N-acetylation and resuspended in 1 mL of DMEM medium. To infect, the Adv5:9C12 complex was prepared as described above, except that Ad5-GFP was diluted to 1.1×10 10 , 9C12 was used at 20 μg / mL, and the complex was incubated for 1 hour and then diluted in 150 μL of DMEM. 50 μL of the electroporated cell suspension was mixed 1:1 with the Adv5:9C12 complex or PBS (control) and then lysed in 100 μL of SteadyLite Plus luciferase reporter (PerkinElmer) 4 hours later. As an internal control, TNF-α was used at 10 ng / μL. Luminescence was recorded on a PheraStar FS (BMG LabTech).

[0170] Immunoblotting The sample was run on a NuPAGE 4%–12% Bis-Tris gel (ThermoFisher) and transferred onto a nitrocellulose membrane. The membrane was incubated in blocking buffer (PBS, 0.1% Tween 20, 5% milk) for 1 h at room temperature and then incubated with antibodies. The antibodies and dilutions (in blocking buffer) used for immunoblotting (IB) are detailed in (Table 2). Antibodies conjugated with HRP were detected by enhanced chemiluminescence (Amersham, GE Healthcare) and X-ray film. Antibodies conjugated with IRDye were detected using a LI-COR Odyssey CLx imaging system.

[0171] Capillary-based immunoblotting RIPA buffer protein extracts were diluted 1:2 with 0.1× sample buffer (bio-techne, 042-195) and electrophoresed in a Jess Simple Western system using a 12 kDa–230 kDa separation module (bio-techne) according to the manufacturer's instructions. The antibodies and dilutions used for capillary-based immunoblotting (Jess) are detailed in (Table 2). Protein peak areas were quantified using Compass software (bio-techne) and normalized to the internal protein loading control within each capillary.

[0172] In vitro ubiquitination assay The Ube2W-dependent TRIM21 monoubiquitination assay was performed in 50 mM Tris (pH 7.4), 150 mM NaCl, 2.5 mM MgCl2, and 0.5 mM DTT. The reaction components were 2 mM ATP, 1 μM GST-Ube1, 80 μM ubiquitin, and the indicated concentrations of Ube2W and TRIM21, respectively. The reaction was stopped by adding LDS sample buffer containing 50 mM DTT at 4 °C. The samples were then boiled at 90 °C for 2 min. For reactions using 10 μM TRIM21, visualization was performed by instant blue-stained LDS-PAGE only. The polyubiquitin chain elongation assay was performed as above, but 0.5 μM Ube2N / Ube2V2 was added instead of Ube2W. For antibody-induced monoubiquitination, the same conditions as described above for monoubiquitination analyzed by LDS-PAGE were used. However, the concentration of TRIM21 was reduced to 100 nM and the concentration of GST-Ube1 was reduced to 0.25 μM. Anti-GFP antibody (9F9.F9) was added at a 1 molar equivalent to TRIM21. The reaction was initiated by the addition of Ube2W (0 nM, 50 nM, 100 nM, 200 nM). The reaction was stopped by adding LDS sample buffer at 4 °C. The samples were boiled at 90 °C for 2 min and separated by LDS-PAGE. TRIM21 was visualized using Western blot. The in vitro reconstitution of Trim-Away ubiquitination events was performed as in the above antibody-induced monoubiquitination experiment. The E2 concentrations were 200 nM Ube2W and 0.5 μM Ube2N / Ube2V2, and His-mEGFP was used at 200 nM as the Trim-Away target.

[0173] Acetylation and Monoubiquitination Assays The N-terminal acetylation of TRIM21 was mediated by Chaetomium thermophilum N-acetyltransferase (NAT) Naa50ΔΔ. The acetylation reaction was carried out at 25 °C for 4 h in 50 mM Tris (pH 7.4) and 150 mM NaCl. The reaction contained 20 μM of TRIM21, 1 mM of acetyl-CoA, and 1 μM of CtNaa50ΔΔ. After the acetylation reaction was completed, it was mixed 1:1 with a Ube2W ubiquitination mix containing 100 mM Tris (pH 7.4), 300 mM NaCl, 5 mM MgCl2, and 1 mM DTT, 4 mM ATP, 2 μM GST-Ube1, 160 μM ubiquitin, and 2 μM Ube2W. The Ube2W ubiquitination reaction was carried out at 37 °C for 1 h and stopped by adding LDS sample buffer containing 50 mM DTT at 4 °C, followed by boiling the sample at 90 °C for 2 min. Visualization was performed only by instant blue staining LDS-PAGE.

[0174] NMR spectroscopy Two-dimensional NMR measurements ( 15 N-HSQC) were performed at 25 °C on a Bruker Avance I 600 MHz spectrometer equipped with a 5 mm 1 H- 13 C- 15 N cryogenic probe. The data were processed with the program Topspin (Bruker BioSpin GmbH, Germany) and analyzed with the program CCPN analysis v2 (Vranken et al., 2005). The sample was buffer-exchanged into 50 mM deuterated Tris (pH 7.0), 150 mM NaCl, and 1 mM deuterated DTT (Cambridge Isotopes, UK).

[0175] Chemical shift perturbation (CSP) was calculated using Equation (3): (3) Δδ N,HN = √(Δδ( 1 H) 2 +(Δδ( 15 N) 2 × 0.14) (where Δδ N,HN is the CSP and Δδ(1 H) and Δδ( 15 N) was calculated using the difference in chemical shift between the positions of the proton signal or nitrogen signal in the absence and presence of the titrant). The assignment of TRIM21 was used from a previous publication (Non-Patent Document 18).

[0176] Mass spectrometry The excised protein gel pieces were decolorized with 50% (volume / volume) acetonitrile: 50 mM ammonium bicarbonate. After reduction with 10 mM DTT and alkylation with 55 mM iodoacetamide, the protein was digested overnight at 37 °C with Asp-N (Promega, UK) at 6 ng·μL -1 and the peptides were extracted in 2% (volume / volume) formic acid: 2% (volume / volume) acetonitrile and subsequently at 300 nL·min -1Analysis was performed by nano-scale capillary LC-MS / MS using an Ultimate U3000 HPLC (Thermo Scientific Dionex, Sunnyvale, USA) set to a flow rate of. Peptides were trapped on a C18 Acclaim PepMap100 (5 μm, 100 μm × 20 mm) (nanoViper) (Thermo Scientific Dionex, Sunnyvale, USA) and then separated on a C18 T3 (1.8 μm, 75 μm × 250 mm) (nanoEase) column (Waters, Manchester, UK). Peptides were eluted by a gradient of acetonitrile, and the outlet of the analytical column was directly connected to a quadrupole Orbitrap mass spectrometer (Q-Exactive HFX, Thermo Scientific, USA) using a nanoflow electrospray ionization source. In data-dependent analysis, a resolution of 60,000 was used for the full MS spectrum, followed by 12 MS / MS runs. MS spectra were collected over the m / z range of 300 - 1800. The obtained LC-MS / MS spectra were searched against a protein database (UniProt KB) using the Mascot search engine program. Database search parameters were restricted to a precursor ion tolerance of 5 ppm and a fragment ion tolerance of 0.1 Da. Multiple modifications were set in the search parameters: variable modifications for two missed enzyme cleavages, methionine oxidation, cysteine carbamidomethylation, pyroglutamic acid, and protein N-terminal acetylation. Fragmented spectra were visualized using the proteomics software Scaffold 4.

[0177] Statistical analysis Mean (average), standard deviation (s.d.), standard error of the mean (s.e.m), and statistical significance based on Student's t-test (two-tailed) and one-way ANOVA or two-way ANOVA were calculated in Microsoft Excel or GraphPad Prism. Significance was defined as ns (not significant, P > 0.05), * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001), ****It is represented by the display of (P≦0.0001).

[0178] Crystallography TRIM21-RING:Ube2W V30K / D67K / C91K Crystals of the complex were grown at 17 °C at 10 mg / ml in 2 nl droplets in 0.1 M bis-tris (pH 9.0), 5% PEG 6000, 0.1 M TCEP hydrochloride. Diffraction experiments were performed at beamline ID23 of the European Synchrotron Radiation Facility using a Dectris PILATUS 6M detector at a wavelength of 0.984004 Å. Diffraction data at 2.25 Å were processed using XDS. The structure was solved by molecular replacement using Phaser (Adams et al., 2010) with the TRIM21 RING domain (5OLM (Non-Patent Document 18)) and residues 1 to 118 of Ube2W (2MT6 (Vittal et al., 2015)) as search models. Model building and real-space refinement were carried out in coot (Emsley and Cowtan, 2004) and refinement was carried out using REFMAC5 and phenix-refine (Afonine et al., 2012). The model and structure factors are deposited in the PDB with accession code 8A58.

[0179] Results To understand the mechanism by which TRIM21 recruits Ube2W, a Ube2W dimer mutant in which the cysteine at the active site was also replaced with lysine (Vittal et al., 2013b) (Ube2W V30K / D67K / C91K) and the crystal structure of the RING domain (R) that forms a complex with it was elucidated. In the asymmetric unit, there are two copies each of Ube2W and RING, where the two RINGs form a homodimer as previously described (Non-Patent Document 18, Kiss et al., 2021, Kiss et al., 2019) (Figure 1A). RING and Ube2W bind to each other via the classical RING:E2 interface (Figure 1B). By superimposing the R:Ube2W structure and Ube2N~Ub from the previously determined Ub-R:Ube2N~Ub:Ube2V2 structure (Kiss et al., 2021), the activation of Ube2W~Ub was modeled (Figure 1C). 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 adopts a closed conformation and is stabilized by both RING protomers. Interestingly, this model also suggests the possibility that the E13 of TRIM21 binds to K11 of ubiquitin to stabilize the closed conformation (Figure 1D). E13 is part of the tri-ionic motif that has been confirmed to drive Ube2N~Ub interactions (Kiss et al., 2019). We have Ube2W V30K / D67K / C91K against 15By performing NMR titrations of the N - tagged TRIM21 tri - ionic mutants, it was verified whether this motif is involved in Ube2W binding. Even when the tri - ionic residues E12 and E13 were mutated to alanine, no obvious decrease in the observed chemical shift perturbation (CSP) was brought about (Figures 7A - 7C). Furthermore, the tri - ionic mutants had only a slight effect on the monoubiquitination of TRIM21 (Figure 7D). Since both E13A and E13R of the TRIM21 mutants showed only a slight decrease in activity, residue E13 may indeed interact with K11 of ubiquitin, suggesting that this is not as important as Ube2N~Ub (Kiss et al., 2019). Comparing the R:Ube2W structure in the apo - bound form (Non - Patent Document 18) and the Ube2N~Ub - bound structure (Kiss et al., 2019) of the RING, it was found that the N - terminal helix and C - terminal helix of the RING were partially unfolded when Ube2W was bound (Figure 7E). This may reflect differences in crystallization, but it is thought to suggest that Ube2W destabilizes the four - helix bundle, thereby potentially generating a disordered N - terminus for modification. Nevertheless, since Ube2W is located far from the E2 active site, it is insufficient to explain how Ube2W can modify the N - terminus of the bound RING.

[0180] Since Ube2W is normally a dimer (Vittal et al., 2013a), it was hypothesized that it could utilize a catalytic RING topology similar to that previously described for the Ube2N / Ube2V2 heterodimer (Kiss et al., 2021). In such an arrangement, the two RINGs form a dimer and function as an enzyme, activating donor ubiquitin on one Ube2W monomer, while the third RING functions as a substrate and is oriented by the second Ube2W monomer to allow for attack on the N-terminus (Figure 1E). We tested this hypothesis using two TRIM21 constructs (R-PS and R-R-PS) that have either one (R) or two (R-R) RINGs and the PRYSPRY (PS) domain. Both constructs were efficiently monoubiquitinated by Ube2W (Figure 1F). However, although Ube2W dimerization was thought to be unimportant for activity (Vittal et al., 2013, Cell Biochem Biophys), monomeric Ube2W V30K / D67KWhen used, this activity disappeared (Figure 1F). This is consistent with the use of Ube2W dimerization to orient one of the RING domains as a substrate (Figure 1E). Surprisingly, no difference was observed between R-PS and R-R-PS, probably because the relatively high TRIM21 concentration was sufficient to drive RING dimerization of R-PS. Previously, we showed that TRIM21 is activated by substrate-induced cluster formation in cells (Non-Patent Document 3), and that, on the contrary, addition of IgG Fc to in vitro ubiquitination experiments is necessary to induce TRIM21-mediated K63 chain formation by Ube2N / Ube2V2 under concentrations close to physiological enzyme concentrations (Kiss et al., 2021). Therefore, we decreased the concentration of R-PS or R-R-PS and titrated Ube2W both in the presence and absence of IgG. Under these conditions, monoubiquitination was observed only in the case of R-R-PS (Figures 1G and 1H). Furthermore, R-R-PS monoubiquitination was greatly stimulated by the addition of IgG (Figures 1H and 1I). Importantly, it was also observed that endogenous TRIM21 similarly depends on dimeric Ube2W for the degradation of substrates in cells. When Ube2W V30K / D67K was electroporated, unlike wild-type Ube2W, the degradation of the intracellular substrate CAV1-mEGFP induced by the anti-GFP antibody was inhibited. As a positive control, wild-type Ube2N / Ube2V2 was mutated to replace the residue responsible for deprotonation of K63 on acceptor ubiquitin with alanine (Ube2N N119Awas also compared with (Ube2V2)(Kiss et al., 2021). Catalytically inactive mutants were unable to degrade CAV1-mEGFP (Figure 1J). These results confirm previous observations using siRNA (Fletcher et al., 2015b, McEwan et al., 2013) that both Ube2W and Ube2N / Ube2V2 are required for TRIM21 activity. Collectively, the cellular and in vitro data suggest that antibody binding promotes TRIM21 RING monoubiquitination by dimeric Ube2W via a similar trans mechanism as Ube2N / Ube2V2 and that this is required for substrate degradation.

[0181] Ube2W is required for substrate degradation, and TRIM21 can be mono-ubiquitinated by E2 in vitro, but this does not prove that one requires the other. To investigate the necessity for N-terminal mono-ubiquitination of TRIM21, the inventors decided to biochemically block this via N-acetylation. N-acetylation is an irreversible modification catalyzed by N-acetyltransferase (NAT) using the cofactor acetyl-CoA intracellularly (Aksnes et al., 2019). The inventors selected a NAT from Chaetomium thermophilum, a heat-stable enzyme that can interact with human proteins (Weyer et al., 2017), and incubated this with R-R-PS in the presence of acetyl-CoA. Successful N-terminal acetylation was confirmed by LC-MS / MS (Figure 8A). N-terminally acetylated R-R-PS (Ac-R-R-PS) was added together with Ube2W and ubiquitin to verify whether mono-ubiquitination was inhibited (Figure 2A). In the absence of acetylation, all of the R-R-PS was mono-ubiquitinated, but most of the R-R-PS incubated with NAT and acetyl-CoA remained non-ubiquitinated (Figure 2B). The degree of mono-ubiquitination inhibition was proportional to the incubation time of NAT and acetyl-CoA (Figure 8B). Importantly, since the formation of free K63-linked ubiquitin chains was not impaired, it was demonstrated that the acetylated RING remained catalytically active (Figure 8C). These results confirm that the TRIM21 RING is mono-ubiquitinated by Ube2W at its N-terminus and that this can be inhibited by N-terminal acetylation.

[0182] Using a method that specifically blocks the N-terminal ubiquitination of RING, it was verified whether this is necessary for substrate degradation. Either R-R-PS or Ac-R-R-PS was electroporated into cells expressing CAV1-mEGFP together with an anti-GFP antibody, and the kinetics of substrate degradation was monitored by fluorescence detection (Figure 2C). Consistent with previous Trim-Away experiments, electroporation of the anti-GFP antibody drove substrate degradation via endogenous TRIM21 (Figure 2D). However, CAV1-mEGFP degradation was significantly accelerated by the delivery of exogenous R-R-PS. Importantly, acetylation of the ligase (Ac-R-R-PS) had no effect, and degradation proceeded with the same kinetics (Figure 2D). Next, it was verified whether N-terminal acetylation is required for the antiviral function of TRIM21. After electroporating either R-R-PS or Ac-R-R-PS into cells, they were infected with adenovirus 5 (AdV5) encoding the gene for GFP in the presence of the anti-hexon antibody 9C12. Proliferative infection was monitored by measuring GFP expression 24 hours after the challenge (Figure 2E). Previous experiments have shown that when TRIM21 binds to an antibody-coated virus, it blocks infection by mediating proteasomal degradation of the virion (Non-Patent Document 8). As expected, electroporation of R-R-PS neutralized AdV5 in an antibody dose-dependent manner (Figure 2F). Ac-R-R-PS had at least equivalent activity to R-R-PS in the neutralization of AdV5 infection and was more effective at intermediate antibody concentrations. In addition to mediating the degradation of virions, TRIM21 also activates innate immune signaling when it senses an antibody-coated virus (McEwan et al., 2013). Therefore, the infection experiment was repeated in cells containing the NF-κB promoter-driven luciferase gene. R-R-PS and Ac-R-R-PS were equally effective in stimulating NF-κB-driven transcription in response to antibody-coated Adv5 (Figure 2G). In both cases, this was dependent on direct antibody binding.This is because the 9C12 mutant H433A (Non-Patent Document 19) that specifically cleaves the binding to PRYSPRY was unable to activate NF-κB. In summary, the data indicate that N-terminal ubiquitination of the RING ligase is not necessarily required for both the Trim-Away or anti-viral function of TRIM21.

[0183] When autoubiquitination of the RING ligase is not required for substrate degradation, we inferred that this might be involved in mediating self-turnover. Therefore, acetylated R-R-PS was electroporated into RPE-1 cells, and protein levels were monitored 1 hour later (Figure 3A). Comparing epoxomicin-treated cells with untreated cells, it was revealed that non-acetylated R-R-PS was readily degraded by the proteasome (Figure 3B, the first two lanes). In contrast, epoxomicin had little effect on the Ac-R-R-PS protein level, indicating that inhibition of N-terminal ubiquitination blocks proteasomal degradation (Figure 3B, the last two lanes). This data suggests that N-terminal ubiquitination of the ligase is not required for substrate degradation but can be used to regulate ligase levels. To verify this, electroporation experiments were repeated using R-R-PS or Ac-R-R-PS, but neutralization of either Trim-Away or AdV5 was measured several hours later (Figure 3C). In the Trim-Away experiment, this was achieved by co-electroporating RNA encoding an antibody construct (vhhGFP4-Fc) that plays a role in recruiting R-R-PS to the substrate. Therefore, Trim-Away is delayed for several hours while vhhGFP4-Fc is expressed. In this experimental format, no substrate degradation was observed in cells electroporated with R-R-PS (Figure 3D, compare with Figure 2D). In contrast, Ac-R-R-PS degraded CAV1-mEGFP with approximately the same efficiency as when Trim-Away proceeds immediately after electroporation of the ligase (Figure 3D). In the AdV5 neutralization experiment, cells were infected 2 hours after ligase delivery. In this case, neutralization by R-R-PS neutralization was significantly attenuated, where Ac-R-R-PS inhibited infection much more efficiently (Figure 3E).

[0184] Since the self-ubiquitination of ligase is not required for substrate degradation, we investigated whether it could be driven by substrate ubiquitination instead. To verify this, we performed an in vitro ubiquitination experiment using R-R-PS, anti-GFP antibody, and recombinant mEGFP substrate (Figure 4A). Co-ubiquitination of R-R-PS, the antibody heavy chain, and mEGFP was observed (Figure 4A and Figure 4B). Monoubiquitination was observed when incubated with Ube2W alone, whereas polyubiquitin chains were obtained by this tethering in the condition containing Ube2N / V2 as well. Importantly, substrate ubiquitination occurred only when both R-R-PS and the antibody were present (Figure 9). This is consistent with the requirement of the antibody for recruiting the TRIM21 ligase to its substrate. Next, we investigated whether substrate ubiquitination occurs independently of ligase ubiquitination or whether the latter modification is necessary to stimulate catalytic activity. To do this, we repeated our in vitro ubiquitination assay in the presence of both E2s and compared R-R-PS with Ac-R-R-PS. Acetylation of the ligase blocked self-ubiquitination but did not prevent polyubiquitination of either the antibody heavy chain or the substrate (Figure 4C). This data indicates that TRIM21 catalyzes the polyubiquitination of antibody-coated substrates and that this can occur independently of ligase self-ubiquitination. Nevertheless, the fact that TRIM21, the antibody, and the substrate can be co-polyubiquitinated in vitro is consistent with the intracellular Trim-Away data indicating that all three components are degraded simultaneously (Non-Patent Document 2).

[0185] Next, we attempted to monitor substrate ubiquitination during Trim-away degradation in live cells (Figure 5A). We selected two kinase substrates, ERK1 and IKKα, and blotted for protein levels at various time points after antibody electroporation. For both substrates, high molecular weight bands or smearing consistent with polyubiquitination were observed 30 minutes after electroporation (Figures 5B and 5C). These high molecular weight bands decreased over the next several hours coincident with a decrease in substrate protein levels. To obtain further evidence for substrate polyubiquitination, we repeated the IKKα Trim-Away experiment in the presence of the proteasome inhibitor MG132. Addition of MG132 had no effect on control cells, but in the presence of electroporated antibody, higher molecular weight ladder formation was clearly observed (Figure 5D). When this was performed as a time-course experiment, it revealed that IKKα ubiquitinated species were first formed and then depleted coincident with proteolysis. Treatment with MG132 blocked both degradations and resulted in stable accumulation of ubiquitinated species (Figure 10A). Also, when we examined TRIM21, a decrease in protein levels was again observed in parallel with substrate depletion (Figures 5B and 5C). For TRIM21, higher molecular weight bands that may also indicate polyubiquitination were observed, but these increased during ERK1 Trim-Away, whereas no change was seen for IKKα. To verify whether antibody-dependent substrate ubiquitination is mediated by TRIM21, we repeated our experiments in TRIM21 knockout cells reconstituted with HA-tagged TRIM21 or an empty vector control. Antibody-induced ERK1 ladder formation, an indicator of polyubiquitination, was observed in knockout cells reconstituted with TRIM21-HA but not in the empty vector (Figure 5E). These data indicate that substrates are ubiquitinated in a manner dependent on antibody and TRIM21 during Trim-Away in live cells.

[0186] Previously, the inventors have demonstrated that Trim-Away can be performed even in the absence of antibodies by directly fusing a substrate-targeting nanobody to the domain from TRIM21 (Non-Patent Document 3). The inventors used this approach to determine whether there is anything special about the ternary complex formed between TRIM21:antibody:substrate required for substrate ubiquitination. Two fusion constructs were tested in which either the TRIM21 RING, B-box, and coiled-coil (T21RBCC-) or only the RING domain (T21R-) were fused to an anti-GFP nanobody (vhhGFP4). The fusion constructs were introduced as RNA into cells expressing CAV1-mEGFP, and both ubiquitination and degradation were monitored. Efficient Trim-Away was observed using either fusion construct (Figure 10C). Treatment with MG132 inhibited degradation and simultaneously resulted in the accumulation of ubiquitinated substrate (Figure 10C). These results indicate that neither antibodies nor ternary complexes are required for TRIM21-mediated substrate ubiquitination and degradation. Introduction of two RING-inactivating mutations into the T21R-vhhGFP4 construct (T21R I18R / M72E -vhhGFP4) (Non-Patent Document 3) completely abolished both substrate ubiquitination and degradation, suggesting that these processes are driven by the catalytic activity of the TRIM21 RING.

[0187] The above data indicate that activated TRIM21 can itself catalyze the simultaneous ubiquitination of antibodies and substrates. However, substrate degradation is not impaired even when N-terminal TRIM21 ubiquitination is blocked. To rule out the possibility that autoubiquitination of the ligase lysine drives substrate degradation, a mutant of T21R-vhhGFP4 with all lysines mutated to arginines was generated (Figure 6A, T21R K0 -vhhGFP4 K0)。Importantly, removal of all lysines from T21R-vhhGFP4 had no effect on either the rate or efficiency of substrate degradation (Figures 6B and 6C). Furthermore, simultaneous blockade of the N-terminus via acetylation had no effect. Collectively, these data indicate that substrate degradation by TRIM21 does not depend on classical auto-ubiquitination. However, importantly, inhibition of N-terminal auto-ubiquitination reduces ligase depletion without altering substrate degradation, thus decoupling substrate turnover from ligase turnover (Figure 6C).

[0188] To test whether Trim-Away is driven by ubiquitination of substrate lysines, we designed model substrates fused to wild-type or lysine-free anti-GFP nanobodies containing a dodecameric ALFAtag repeat that does not originally contain lysine residues (Gotzke et al., 2019) (Figure 6D). After expressing these substrates in either wild-type or TRIM21 knockout (T21KO) RPE-1 cells, we electroporated them with an ALFAtag nanobody fused to IgG Fc (vhhALFA-Fc). ALFA-Fc was expected to bind to the ALFAtag substrate, recruit endogenous TRIM21 via Fc interactions, and cause clustering, activation, and degradation of TRIM21 (Figure 6E). This was indeed observed, with significant degradation seen in wild-type cells but not in T21KO cells (Figures 6F and 6G). Notably, addition of ALFA-Fc increased the substrate level in the absence of TRIM21, suggesting that the nanobody:tag complex is more stable. Importantly, degradation was not dependent on substrate lysines as both wild-type and lysine-free substrates were equally well degraded (Figures 6F and 6G). As expected for Trim-Away experiments, endogenous TRIM21 was also degraded along with each substrate (Figure 6H). Since it is formally possible that degradation of the TRIM21:substrate complex requires only one target for lysine ubiquitination, we modified our assay to simultaneously remove lysines from both the ligase and the substrate. To do this, we complemented our model substrates with a model ligase containing the TRIM21 RING fused to an anti-ALFAtag nanobody (T21R-vhhALFA, Figure 6I). In this assay, the ALFAtag substrate was predicted to recruit multiple T21R-vhhALFA ligases and cause clustering, activation, and degradation of the ligase (Figure 6I). As before, removing all lysines from the substrate had no effect on substrate degradation (Figures 6J and 6K).However, notably, Trim-Away was equally efficient even when there was no lysine in both the ligase and the substrate (Figures 6J and 6K). In summary, our data show that ligase auto-ubiquitination does not drive TRIM21-mediated degradation, nor does it drive substrate lysine ubiquitination. This finding may explain the efficiency of Trim-Away in degrading diverse substrates (Non-Patent Document 2).

[0189] This data also shows that activation of TRIM21 results in ubiquitination of both the ligase and the substrate, while auto-ubiquitination is regulatory and not required for substrate degradation. Substrate binding stimulates N-terminal RING auto-ubiquitination by E2 Ube2W, but if inhibited, for example, by N-terminal acetylation, this does not prevent substrate ubiquitination or substrate degradation and has no effect on TRIM21 activity. We have found that by decoupling ligase degradation from substrate degradation, ligase recycling is impaired and its functional persistence in cells is extended.

[0190] Therefore, the data also demonstrate that it is possible to provide a fusion protein containing RING with an extended cellular half-life without substantially affecting substrate degradation by blocking the N-terminus of the RING-containing protein construct and inhibiting N-terminal RING auto-ubiquitination.

[0191] Although this disclosure has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this detailed description pertains only to the preferred embodiments and does not limit the scope of the disclosure.

[0192] Materials used:

[0193]

Table 1

[0194]

Table 2

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Claims

1. At least a first RING domain, and An adaptor domain capable of localizing the RING domain and a substrate, A fusion protein comprising the above, which cannot be N-terminally self-ubiquitinated.

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

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

4. The N-terminus of the fusion protein can undergo N-terminal cyclization, preferably, the fusion protein can undergo N-pyroglutamic acid cyclization, the fusion protein according to claim 1.

5. The fusion protein contains glutamic acid, glutamine, or pyroglutamic acid as an N-terminal residue, the fusion protein according to claim 1.

6. At least the N-terminal amino acids of the fusion protein are substituted with amino acids or amino acid sequences that inhibit the ability of the E2 enzyme Ube2W to ubiquitinate the fusion protein, the fusion protein according to claim 1.

7. The fusion protein Contains a second RING domain, wherein the second RING domain is between the first RING domain and the adaptor domain, Does not contain a coiled-coil domain and / or a B-box domain, and / or Contains a linker sequence between the RING domain and the adaptor sequence, the fusion protein according to any one of claims 1 to 6.

8. The RING domain is derived from a TRIM polypeptide, the fusion protein according to any one of claims 1 to 7.

9. The TRIM polypeptide is selected from the group consisting of TRIM5, TRIM7, TRIM19, TRIM21, TRIM25, TRIM28, and TRIM32, preferably TRIM21, the fusion protein according to claim 8.

10. 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 Fab, Fab', F(ab')2, scFab, Fv, scFV, dAb, their VL fragments, their VH fragments, and their V ＨＨ selected from the group consisting of fragments, or The fusion protein according to any one of claims 1 to 9, which encodes a protein or a fragment thereof capable of placing the RING domain on the 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 adapter domain, which encodes a fusion protein that cannot self-ubiquitinate.

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 either the coiled-coil domain or the B-box domain.

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

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 additive.

16. 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, used as a medicament.

17. A method for degrading a target protein in a cell, comprising introducing into the cell 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.

18. A method for extending the half-life of a fusion protein comprising a RING domain and an adapter domain, wherein the adapter domain can localize the RING domain and a substrate, the method comprising modifying the fusion protein so that it cannot undergo N-terminal self-ubiquitination.

19. The modification of the fusion protein is incubating the fusion protein with N-acetyltransferase (NAT) and acetyl CoA to acetylate the N-terminus of the fusion protein, methylating the N-terminal amino acid of the fusion protein, Coupling a chemical moiety that reduces the ability of the E2 enzyme Ube2W to ubiquitinate the fusion domain to the N-terminal amino acid of the fusion protein, Introducing an N-acetyltransferase recognition site, preferably the sequence DDDI, to the N-terminus of the fusion protein, and optionally incubating the fusion protein with N-acetyltransferase (NAT) and acetyl-CoA to N-terminally acetylate the fusion protein, Modifying the fusion protein such that the fusion protein can undergo N-terminal cyclization, Modifying the fusion protein such that the fusion protein can undergo N-terminal pyroglutamic acid cyclization, Introducing a glutamic acid residue or a glutamine residue to the N-terminus of the fusion protein and optionally incubating the fusion protein with glutaminyl cyclase, or Substituting at least the N-terminal amino acid of the RING domain with an amino acid or amino acid sequence that inhibits the ability of the E2 enzyme Ube2W to ubiquitinate the fusion protein, The method according to claim 18, comprising

20. A method for producing the fusion protein according to any one of claims 1 to 10, comprising (a) culturing a host cell containing a first vector encoding a fusion protein comprising a RING domain and an adapter domain under conditions that allow expression of the fusion protein, (b) obtaining the protein expressed from the host cell, The method, wherein the fusion protein is incapable of N-terminal auto-ubiquitination.

21. The method according to claim 20, wherein the first vector also encodes glutaminyl cyclase, and the expressed fusion protein contains 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 glutaminyl cyclase.

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