[0001] Proteins that traverse the secretory pathway undergo quality control in the endoplasmic reticulum (ER). Proteins that are present in excess, misfolded, or fail to assemble into native complexes are degraded by endoplasmic reticulum-associated degradation (ERAD).

[0002] ERAD depends on a number of proteins in the ubiquitin-proteasome pathway, including the Ubiquitin Conjugating Enzyme (E2) UBE2G2 (Ube2G2/E2G2), which acts with several ubiquitin ligases (E3s) in ERAD. UBE2G2 accepts ubiquitin from ubiquitin-activating enzyme (E1) and together with E3s catalyzes its covalent attachment to other proteins. Ancient ubiquitous protein 1 (AUP1) is an accessory protein required for degradation of some substrates and is believed to be critical for a wide range of substrates. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 UBE2G2 via its UBE2G2 binding region (G2BR ^AUP1 ).

[0003] ERAD is associated with various disorders due to loss of protein function as a result of the activity of the ERAD machinery. Conversely, acute or chronic ER stress (a form of cellular stress), becomes manifest when ERAD fails to keep up with misfolded protein production. Cellular pathogens can also coopt the ERAD pathway to increase their pathogenicity. ERAD- associated disorders include cystic fibrosis, cancer, disorders of cholesterol and/or triglyceride metabolism, Parkinson's disease, prion diseases of humans or animals, and viral infections, e.g., human cytomegalovirus (CMV) or human immunodeficiency virus (HIV).

[0004] Excessive ER degradation of mutant proteins results in insufficient cell surface expression of the mature protein. In the instance of the protein being a mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein, the insufficient surface expression causes cystic fibrosis.

[0005] On the other hand, failure to adequately degrade ER proteins can cause ER stress or exacerbate pre-existing ER stress. Cells respond to ER stress with a compensatory unfolded protein response (UPR), a widely conserved cellular stress response, but if this fails to compensate for the ER stress, the cells die as a consequence of a severe pro-apoptotic UPR that is activated. Cancer cells and cells undergoing metastasis are under constitutive ER stress. Thus, there is the potential to intensify ER stress to result in cancer cell death by inhibiting ERAD, by preventing UBE2G2 from accessing cellular ERAD E3s. This in turn has the potential to both prevent tumor growth and to prevent both the initiation of metastasis and growth of metastatic lesions. U.S. Patent No.8,420,776 demonstrated that expression of the gp78 G2BR (G2BR ^gp78 ) in myeloma cell lines did indeed elicit a UPR in the cells by binding and presumably sequestering UBE2G2 and resulted in apoptotic cell death.

[0006] There is a need in the art for compositions and methods capable of modulating ERAD activity. SUMMARY

[0007] Disclosed herein are AUP1 G2BR domain polypeptides, fusion proteins, conjugates, polynucleotides, methods of use, and methods of producing the compositions.

[0008] An isolated polypeptide comprises the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1), wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ , and X ₂₄ are each independently selected from any amino acid, or an amino acid sequence having at least 85% identity to residues 5-24 of SEQ ID NO:1.

[0009] A fusion protein or conjugate comprises a first moiety and a second moiety, wherein the first moiety consists of a G2BR domain consisting of the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1), wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ , and X ₂₄ are each independently selected from any amino acid, or an amino acid sequence having at least 85% identity to residues 5-24 of SEQ ID NO:1.

[0010] An isolated polynucleotide encodes the polypeptide or fusion protein.

Recombinant vectors and host cells are also disclosed.

[0011] A method of detecting the ubiquitin conjugating enzyme E2G2 (UBE2G2) in a sample comprises: contacting a sample with the polypeptide, fusion protein, or conjugate; and determining binding of the polypeptide fusion protein or conjugate to the UBE2G2 in the sample to detect the presence of UBE2G2 in the sample.

[0012] A method of modulating expression of a protein in a cell, comprises delivering the polypeptide, fusion protein, or conjugate to a eukaryotic cell expressing a protein under conditions such that expression of the protein is modulated.

[0013] A method of modulating degradation of a protein in a cell, comprises delivering the polypeptide, fusion protein, or conjugate to a eukaryotic cell comprising a protein under conditions such that degradation of the protein is modulated.

[0014] A method of reducing degradation of an over-expressed protein in a cell, comprises delivering the polypeptide, fusion protein, or conjugate to a eukaryotic cell expressing a protein under conditions such that degradation of the protein is reduced.

[0015] A method of inducing cell death comprises delivering the polypeptide, fusion protein, or conjugate to a eukaryotic cell under conditions such that degradation of endoplasmic reticulum proteins is inhibited and cell death is induced.

[0016] A composition comprises the polypeptide, the fusion protein or conjugate, the polynucleotide, or the vector for treatment of an endoplasmic reticulum-associated degradation (ERAD)-associated disorder.

[0017] A method of treating an ERAD-associated disorder in a patient comprises administering the polypeptide, fusion protein, conjugate, polynucleotide, or vector to a patient having an ERAD-associated disorder.

[0018] These and other advantages, as well as additional inventive features, will be apparent from the following Drawings, Detailed Description, Examples, and Claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following is a brief description of the drawings wherein like elements are numbered alike and which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

[0020] FIG.1 is a representation of a portion of the co-crystal structure of UBE2G2 and an AUP1 fragment (residues 376-410; only aa 379– 404 are visible in the structure), showing the strong salt bridge and hydrogen bond interaction between UBE2G2 and the AUP1 G2BR from two 180°-rotated sides.

[0021] FIG.2 is a schematic representation showing the residues of AUP1 involved in interactions with residues of UBE2G2; and also provides a comparison of the residues of the gp78 G2BR binding region interacting with the same residues of UBE2G2

[0022] FIG.3 is an image of gel results showing the presence or absence of binding between a given GST-AUP1 (aa 292-410 of SEQ ID NO:14) fusion polypeptide and recombinant, bacterially-expressed UBE2G2. The AUP1 G2BR (aa 378-404) sequences corresponding to each of the peptide abbreviations are as follows: RQKRR (WT) (SEQ ID NO:2); AAKRR (SEQ ID NO:3); RQKAA (SEQ ID NO:4); AAKAA(SEQ ID NO:7); RQERR(SEQ ID NO:8); AAAAA(SEQ ID NO:9); AAEAA(SEQ ID NO:10); EEKRR(SEQ ID NO:11); EEERR(SEQ ID NO:12); EEEEE (SEQ ID NO:13). The abbreviation GST stands for glutathione S-transferase. The lane labeled “INPUT 5%” in this figure and in FIG.4 shows an amount of UBE2G2 equivalent to 5% of that used to bind to each of the GST fusion polypeptides.

[0023] FIG.4 is an image of gel results showing binding of wild-type AUP1 G2BR (WT; SEQ ID NO:2), the R398A mutant (SEQ ID NO:5), the R400A mutant (SEQ ID NO:6), and the R398A/R400A mutant (SEQ ID NO:4) to UBE2G2.

[0024] FIG.5 is an image of a Western blot of a gel (used interchangeably with immunoblot or Western blot) showing degradation of NHK is inhibited in HT1080 cells lacking AUP1 (AUP1 k.o. cells) as compared to wild type HT1080 cells. EV: control plasmid; CHX:

cycloheximide; AUP1 ^WT : plasmid expressing FLAG-tagged wild type full-length AUP1;

AUP1 ^DG2BR : plasmid expressing a FLAG-tagged AUP1 mutant with the G2BR disrupted by deletion of 12 G2BR amino acids (aa 386-397) from full-length AUP1; NHK-HA: NHK tagged with the HA (hemagglutinin) tag; Hsp60: gel loading control, a protein not subject to ERAD. In all cellular experiments in this application material resolved on gels, prior to immunoblotting for specific proteins, represents detergent (Triton X-100 and deoxycholate) soluble cell lysates. CHX is used throughout this application to inhibit new protein synthesis thereby allowing for assessment of degradation of pre-existing protein.

[0025] FIG.6 is an image of a Western blot of a gel comparing NHK degradation in AUP1 k.o. cells transfected with a plasmid expressing FLAG-tagged wild-type AUP1, FLAG- tagged AUP1 R398A/R400A mutant, or the FLAG-tagged AUP1 G2BR deletion mutant. CHX: cycloheximide; AUP1 ^WT : plasmid expressing FLAG-tagged wild type AUP1; AUP1 ^RR-AA : plasmid expressing FLAG-tagged AUP1 R398A/R400A mutant; AUP1 ^DG2BR : plasmid expressing FLAG- tagged AUP1 mutant with G2BR deletion; NHK-HA: NHK tagged with the HA (hemagglutinin) tag; Hsp60: gel loading control, a protein not subject to ERAD.

[0026] FIG.7 is an image of a Western blot of a gel showing degradation of HA- tagged CD3-d in cells treated with cycloheximide. Cells are overexpressing G2BR ^gp78 (SEQ ID NO:15; 574-600 of full-length gp78), or G2BR ^{AUP1 WT} (SEQ ID NO:2; 378-404 of full-length AUP1) or G2BR ^SCR (SCR consists of the amino acids of the gp78 G2BR placed in a

‘scrambled’/random order), each fused to GFP (used interchangeably with green fluorescent protein, enhanced green fluorescent protein, and EGFP) and expressed from transfected plasmids. The fusion of the SCR sequence to GFP serves as a negative control and is expected to be no different than expressing GFP alone.

[0027] FIG.8 is an image of a Western blot of a gel showing the degradation of HA- tagged CD3-d in cells overexpressing G2BR ^SCR , G2BR ^{AUP1 WT} , G2BR ^{AUP1 R398A} , or G2BR ^{AUP1 R400A} in the presence of cycloheximide. The GFP-G2BR panel shows level of expression of each fusion protein. Hsp60 is a gel loading control protein not subject to ERAD.

[0028] FIG.9A is an image of a Western blot of a gel showing levels of the cell stress marker phospho-eIF2a from HT1080 cells expressing a GFP fusion of gp78 G2BR, AUP1 G2BR, or the scrambled gp78 G2BR sequence (SCR), which serves as a negative control.

[0029] FIG.9B is an image of a Western blot of a gel showing levels of the cell stress marker Binding of Immunoglobulin Protein (BiP) from HT1080 cells expressing a GFP fusion of gp78 G2BR, AUP1 G2BR, or a scrambled sequence (SCR).

[0030] FIG.9C is an image of a Western blot of a gel showing levels of the cell stress marker CCAAT/enhancer-binding protein homologous protein (CHOP) in HT1080 cells expressing a GFP fusion of gp78 G2BR, AUP1 G2BR, or a scrambled sequence (SCR).

[0031] FIG.10 shows survival curves from an experiment comparing athymic nude mice injected with HT1080 human fibrosarcoma cells expressing GFP-G2BR ^SCR or GFP- G2BR ^AUP1 .

[0032] FIG.11 presents gel images comparing UBE2G2 binding to GST-G2BR ^AUP1 or GST- G2BR ^gp78 . The lane labeled“5% INPUT” shows an amount of UBE2G2 equivalent to 5% of that used to bind to each of the GST fusion polypeptides. DETAILED DESCRIPTION

[0033] Compositions and methods for modulating endoplasmic reticulum-associated degradation are disclosed herein. In particular, the inventors have characterized through structural analysis, the sequence of ancient ubiquitous protein 1 (AUP1) that binds specifically to the ubiquitin conjugating enzyme UBE2G2 (one of ~40 E2s) with high affinity. Additionally, the inventors have identified mutations in the sequence that further increase or decrease the affinity of the interaction. The UBE2G2 binding domain of AUP1 is interchangeably referred to herein as the“UBE2G2 binding domain,”“UBE2G2 binding region,”“G2BR,”“AUP1 G2BR,” or“G2BR ^AUP1 .” The G2BR ^AUP1 has an affinity for UBE2G2 that is approximately 16 times greater than G2BR ^gp78 (interchangeably referred to as gp78 G2BR) making it substantially more effective in blocking ERAD. Further, mutations to G2BR ^AUP1 described herein unexpectedly increase binding of the G2BR ^AUP1 to UBE2G2.

[0034] AUP1 G2BR polypeptides, fusion proteins, and conjugates are disclosed herein. Polynucleotides encoding the AUP1 G2BR polypeptides are also disclosed, as are expression vectors and host cells for expression of the AUP1 G2BR polypeptides. Methods of using the AUP1 G2BR polypeptides, fusion proteins, conjugates, and polynucleotides in modulating protein degradation in the ER are also disclosed.

[0035] UBE2G2 is involved in endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded proteins; unassembled components of multi-protein complexes; and proteins that are expressed in excess whose degradation from the ER is critical to cellular homeostasis. AUP1 is an accessory protein required for degradation of some substrates and is believed to be critical for a wide range of substrates. The membrane-localized AUP1 includes an acyl transferase domain suggested to be involved in lipid droplet formation, and AUP1 has similarity to the ERAD RING finger ubiquitin ligase gp78 in having both a CUE domain and a UBE2G2 binding region (G2BR). The G2BR of AUP1 displays some similarity to that of gp78, disclosed in U.S. Patent No. 8,420,776, but binds UBE2G2 with significantly higher affinity.

[0036] The inventors have determined that expression of an isolated AUP1 G2BR will sequester UBE2G2 from ERAD complexes and inhibit processes requiring UBE2G2. This can be used as a strategy to reduce ER degradation of desired proteins, such as mutant CFTR or IgG in antibody-producing cells; or to inhibit processes such as the initiation of sterol synthesis; and to sensitize cells to toxic effects of therapeutics.

[0037] Failure to degrade ER proteins can also cause ER stress. Cells respond to ER stress with a compensatory unfolded protein response (UPR), a widely conserved cellular stress response, but if this fails to compensate for the ER stress, the cells die from a pro-apoptotic UPR. Cancer cells are under constitutive ER stress, this is also the case for cells undergoing metastasis. Thus, there is the potential to intensify this stress to result in cancer cell death by expression of an isolated G2BR that will sequester UBE2G2 from the ER components that require UBE2G2. This could also prevent both the successful initiation of metastasis as well as growth of metastatic lesions.

[0038] Thus, the disclosed AUP1 G2BR polypeptides and polynucleotides are useful as modulating agents in regulating a variety of cellular processes that involve or are influenced by ERAD. Accordingly, this disclosure provides isolated AUP1 G2BR polypeptides and isolated polynucleotides encoding AUP1 G2BR polypeptides, as well as methods of modulating protein degradation in the ER and/or treating ERAD-associated diseases and disorders.

[0039] The inventors have characterized the UBE2G2-binding region (G2BR) of AUP1 by obtaining a crystal structure of a fragment of AUP1 co-crystalized with UBE2G2. Based on the crystal structure, the inventors mutated several residues in the AUP1 G2BR involved in the interaction with UBE2G2 and identified mutants binding UBE2G2 with even higher apparent affinity than the wild-type (WT) AUP1 G2BR. Advantageously, expression of the isolated WT or mutant AUP1 G2BR in cells inhibits degradation of some substrates, likely by sequestration of UBE2G2 from its native complexes.

Polypeptides, Fusion Proteins, and Conjugates

[0040] In an aspect, the disclosure provides an isolated polypeptide comprising a G2BR domain consisting of the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1) wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ , and X ₂₄ are each independently selected from any amino acid; preferably X ₅ is R, A, E, K, or H, X ₆ is Q, A, H, E, K, or R, X ₁₃ is K, A, E, R, or H, X ₂₁ is G, V, L, I, F, D, R, A, or E, X ₂₃ is G, V, L, I, F, D, R, A, or E, and X ₂₄ is F or Y; more preferably X ₅ is R, A, or E, X ₆ is Q, A, or E, X ₁₃ is K, A, or E, X ₂₁ is R, A, or E, X ₂₃ is R, A, or E, and X ₂₄ is F; even more preferably X ₂₁ is A, X ₂₃ is A, and X ₂₄ is F. The G2BR domain amino acid sequence can be the sequence of residues 5 to 24 of SEQ ID NO:2

(RQESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:3

(AAESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:4

(RQESLQERKQALYEYAARAF); the sequence of residues 5 to 24 of SEQ ID NO:5

(RQESLQERKQALYEYAARRF); or the sequence of residues 5 to 24 of SEQ ID NO:6

(RQESLQERKQALYEYARRAF). The isolated G2BR polypeptide can have a binding affinity for UBE2G2 that is at least as high as that of the sequence of residues 5 to 24 of SEQ ID NO:2. In some embodiments, the G2BR domain amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1) does not have the sequence of residues 5 to 24 of SEQ ID NO:2.

[0041] The isolated G2BR polypeptide can be selected from a polypeptide consisting of SSWARQESLQERKQALYEYARRRFTER (SEQ ID NO:2); a polypeptide having at least 80% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide having at least 85% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide having at least 90% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide having at least 90% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide consisting of

SSWAAAESLQERKQALYEYARRRFTER (SEQ ID NO:3); a polypeptide consisting of SSWARQESLQERKQALYEYAARAFTER (SEQ ID NO:4); a polypeptide consisting of

SSWARQESLQERKQALYEYAARRFTER (SEQ ID NO:5); and a polypeptide consisting of SSWARQESLQERKQALYEYARRAFTER (SEQ ID NO:6).

[0042] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a molecule formed from the linking, in a defined order, of at least two amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0043] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics means chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0044] The term "isolated" or“purified,” used interchangeably herein, refers to a nucleic acid, a polypeptide, or other biological moiety that is removed from components with which it is naturally associated. The term "isolated" can refer to a polypeptide that is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type. The term "isolated" with respect to a polynucleotide can refer to a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome. Purity and homogeneity are typically determined using analytical chemistry techniques, for example polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In some embodiments, the term "purified" means that the nucleic acid or protein is at least 85% pure, specifically at least 90% pure, more specifically at least 95% pure, or yet more specifically at least 99% pure.

[0045] "Homology" refers to the percent identity between polynucleotide or polypeptide molecules. In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two DNA, or two polypeptide sequences, are "substantially homologous" to each other when the sequences exhibit at least about 50%, specifically at least about 75%, more specifically at least about 80%-85%, at least about 90%, and most specifically at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.

[0046] The G2BR domain polypeptide can be covalently bound to a label, a solid support, a lipid monolayer, or a heterologous polypeptide sequence.

[0047] The label can be a radiolabel (e. g., ¹²⁵ I, ³⁵ S, ³² P, ³³ P, ³ H), a fluorescent label, a chemiluminescent label, an enzymic label, or an immunogenic label.

[0048] The term“solid support” refers to a material or group of materials having a rigid or semi-rigid surface or surfaces. Examples of materials include plastics (e.g., polycarbonate), complex carbohydrates (e.g., agarose and sepharose), acrylic resins (e.g., polyacrylamide and latex beads), nitrocellulose, glass, silicon wafers, and positively charged nylon. In some aspects, at least one surface of the solid support can be substantially flat, although in some aspects it may be desirable to physically separate regions for different molecules with, for example, wells, raised regions, pins, etched trenches, or the like. In certain aspects, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. The solid support can be in the form of a nanoparticle.

[0049] As used herein,“heterologous” means that the sequence or cell originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention, or that the sequence is designed de novo without reference to any natural sequence. "Heterologous sequences" are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same or an analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. A“heterologous polypeptide” as used herein refers to a polypeptide which is not naturally included in the polypeptide sequence of the AUP1 G2BR polypeptide. A“heterologous cell” for expression of a polypeptide or nucleic acid refers to a cell that does not normally express that polypeptide or nucleic acid.

[0050] The heterologous polypeptide sequence can be any suitable polypeptide sequence known in the art, or a portion of such polypeptide as may be useful herein. The heterologous polypeptide can be, for example, a cell-penetrating peptide (CPP) that can transport the G2BR domain into a cell, a cell targeting peptide (CTP) which has high affinity and specificity to a specific cell or tissue target, a sequence to determine cellular localization and expression, to permit proper folding of the chimeric polypeptide in an expression system, and/or to facilitate isolation of the chimeric polypeptide. The heterologous polypeptide can be linked directly or via a linker to any portion of the G2BR domain polypeptide, for example to the amino terminal end or the carboxy terminal end of the G2BR domain sequence. For example, the heterologous polypeptide can be the first 45 amino acids of rat somatostatin, the FLAG® tag, a 6x histidine (his) tag, MYC tag, a fluorescent protein tag, V5 tag, HA (hemagglutinin)-tag, and/or glutathione S-transferase (GST). Determination of a suitable location for the heterologous polypeptide in the chimeric polypeptide relative to the amino end or the carboxy end of the G2BR domain sequence to obtain a particular functional aspect of the heterologous polypeptide on the chimeric polypeptide can be made by one of skill in the art.

[0051] A G2BR conjugate is also disclosed. The G2BR conjugate comprises an isolated G2BR polypeptide as a first moiety and a second moiety. The second moiety can be a heterologous polypeptide sequence, a label, a cross-linking moiety, or a moiety to stabilize the peptide alpha-helical structure. Moieties to stabilize alpha-helical structure include helix caps, non- natural amino acid substitutions, side-chain constraints, hydrogen bond surrogates (HBS), and hydrocarbon stapling moieties. A conjugate can be synthesized by any suitable method.

[0052] In HBS-modified peptides, a single N-terminal i and i+4 hydrogen bond is substituted with a covalent linkage. Introduction of the covalent bond creates a 13-membered ring, which mimics the single turn of an a-helix. HBS peptides have been shown to successfully modulate protein-biomolecule interactions in cell-free and cell culture assays (Mahon AB, et al. Drug Discov. Today Technol.2012 Spring; 9(1): e57–e62.)

[0053] One exemplary hydrocarbon stapling moiety is an olefin tether, to produce a “stapled” peptide (see Klein M, Exp Op. Drug Dis 2017, 12(11):1117-1125). A "stapled" peptide designates a peptide which comprises a chemical linkage (in addition to the amino acid chain) between two residues. The technique for olefin-based hydrocarbon stapling to constrain a helical peptide involves substitution of peptides at positions to span one side of the helix. The positions of the, alpha-di-substituted non-natural amino acids may include i,i + 3, i,i + 4, i,i + 7, or i,i + 11 [7]. The i,i + 3 and i,i + 4 staples span one turn of the helix, the i,i + 7 staple spans two helical turns, and the i,i + 11 staple spans three turns of the helix. The constrained helices of a stapled peptide have been shown to be cell permeable. A skilled artisan can obtain stapled peptides by using techniques which are available in the art, for example as described by Verdine and Hilinski, Methods in Enzymology, 2012 [12]. In a stapled G2BR domain polypeptide, the“staples” would involve two positions on the helix of the G2BR domain sequence that do not contact UBE2G2.

[0054] Also disclosed is a fusion protein comprising a first moiety and a second moiety. The first moiety consists of a G2BR domain consisting of the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1) wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ , and X ₂₄ are each defined as above. The G2BR domain amino acid sequence can be the sequence of residues 5 to 24 of SEQ ID NO:2 (RQESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:3 (AAESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:4 (RQESLQERKQALYEYAARAF); the sequence of residues 5 to 24 of SEQ ID NO:5 (RQESLQERKQALYEYAARRF); or the sequence of residues 5 to 24 of SEQ ID NO:6 (RQESLQERKQALYEYARRAF). In some embodiments, the G2BR domain amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1) does not have the sequence of residues 5 to 24 of SEQ ID NO:2. The G2BR domain amino acid sequence can be selected from a polypeptide consisting of SSWARQESLQERKQALYEYARRRFTER (SEQ ID NO:2); a polypeptide having at least 80% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide having at least 85% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide having at least 90% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide having at least 90% sequence identity to the amino acid sequence of residues 5 to 24 of SEQ ID NO:1; a polypeptide consisting of SSWAAAESLQERKQALYEYARRRFTER (SEQ ID NO:3); a polypeptide consisting of SSWARQESLQERKQALYEYAARAFTER (SEQ ID NO:4); a polypeptide consisting of SSWARQESLQERKQALYEYAARRFTER (SEQ ID NO:5); and a polypeptide consisting of SSWARQESLQERKQALYEYARRAFTER (SEQ ID NO:6). The second moiety comprises a heterologous polypeptide sequence.

[0055] With regard to the description above of fusion proteins or conjugates, the designation of first, second, and further moieties is made for clarity reasons to distinguish between the G2BR domain moiety, and moieties exhibiting other functions. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of a fusion protein.

[0056] As used herein, a G2BR“fusion protein,” or equivalently a“chimeric protein,” refers to a molecule, which does not occur in nature, in which all or a portion of a G2BR domain polypeptide sequence is part of the linear chimeric polypeptide sequence. The G2BR fusion protein comprises a G2BR domain polypeptide as a first moiety operatively linked to a heterologous polypeptide sequence as a second moiety. Within the fusion protein, the term“operatively linked” is intended to indicate that the G2BR domain polypeptide and the heterologous polypeptide sequence are fused in-frame to each other. The non-G2BR polypeptide can be fused to the N- terminus or C-terminus of the G2BR domain polypeptide. The chimeric polypeptide can be made by any method known in the art. For example, the chimeric polypeptide can be made by a recombinant expression system or can be synthesized. Fusion proteins can include multiple copies of a G2BR domain, which can have the same or different amino acid sequence.

[0057] The construction of a fusion protein often involves using linkers between functional moieties to be fused. Different kinds of linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers, can be used. Linkers can be used for example to increase stability or improve folding of fusion proteins, to increase expression, improve biological activity, enable targeting or alter pharmacokinetics of fusion proteins.

[0058] For example, in one embodiment, the fusion protein is a GST-G2BR fusion protein in which the G2BR sequence is fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant G2BR. In another embodiment, the fusion protein is a G2BR protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of G2BR can be increased through use of a heterologous signal sequence. In another embodiment, the fusion protein is a G2BR protein fused to a fluorescent protein (e.g., green fluorescent protein, or GFP). In a further embodiment, G2BR proteins can be fused to a peptide tag which can facilitate purification of the fusion protein, as well as detection and/or immunoprecipitation using antibodies directed to the peptide tag. In another embodiment, the fusion protein is a G2BR domain polypeptide fused to a cell-penetrating peptide (CPP) in order to enhance delivery of the fusion protein into cells.

[0059] Pharmaceutical potency of peptides is often restricted by their poor stability in vivo and/or by their low uptake in cells. Cell-penetrating peptides (CPPs) are short peptides (typically <30 amino acids long) that are able to penetrate biological membranes and drive the internalization of a bioactive cargo in cells. CPPs can improve both ex- vivo and in vivo delivery and efficiency of peptides, without activating the innate immune response or inducing toxic side effects. The first examples of CPPs were based on natural peptides derived from protein fragments, such as the Tat peptide (positions 48-60, derived from the transcription protein of HIV-l) and penetratin (derived from the amphiphilic Drosophila Antennapedia homeodomain), also referred to as Antennapedia. Subsequently, rationally designed CPPs have been developed to obtain greater efficiency of intracellular delivery (See Kalafatovic & Giralt, Molecules 2017 22:1929). The repertoire of available CPPs currently includes linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. Exemplary CPPs suitable for fusion or conjugation with a G2BR polypeptide disclosed herein include those in Table 1 below.

Table 1. Examples of linear CPP sequences

[0060] See for example Kalafatovic & Giralt, Molecules 201722:1929 for further CPP examples. Preferred CPP sequences include Tat (GRKKRRQRRRPQ, SEQ ID NO: 16),

Antennapedia (RQIKIWFQNRRMKWKK, SEQ ID NO: 17), Polyarginine (R9, SEQ ID NO: 20), and NM1 (KVRVRVRVpPTRVRV*RVK, where capital letters denote L-amino acids, non-capital letters denote D-amino acids, and * denotes peptide backbone N-methylation). Fusion proteins and conjugates of an AUP1 G2BR domain and a CPP sequence can be made, for example, by peptide synthesis or can be purchased from a number of commercial sources specializing in such synthesis, e.g., Creative Peptides (Shirley, NY) or DISCOVERY Peptides, Cambridge Research Biochemicals (Billingham, England, United Kingdom).

[0061] The G2BR polypeptides, fusion proteins, or conjugates of the invention can be used as immunogens to produce anti-G2BR antibodies in a subject, to detect G2BR target molecules (e.g., UBE2G2), and in screening assays to identify molecules which inhibit or enhance the interaction of G2BR with a G2BR target molecule (e.g., UBE2G2).

[0062] The G2BR polypeptides, fusion proteins, or conjugates can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The G2BR polypeptides, fusion proteins, or conjugates can be used to affect the bioavailability of a G2BR target molecule (e.g., UBE2G2). Use of G2BR polypeptides, fusion proteins, or conjugates may be useful therapeutically for the treatment of disorders caused by or associated with, for example, aberrant ERAD and/or aberrant protein stability (e.g., ERAD-associated disorders). ERAD-associated disorders include cystic fibrosis, cancer (including metastases), disorders of cholesterol and/or triglyceride metabolism, Parkinson's disease, prion diseases of humans or animals, and viral infections, e.g., human cytomegalovirus (CMV) or human immunodeficiency virus (HIV).

Polynucleotides, Recombinant Expression Vectors, and Host Cells

[0063] Also disclosed is an isolated polynucleotide. The isolated polynucleotide encodes an isolated AUP1 G2BR polypeptide or a fusion protein disclosed herein.

[0064] In an embodiment, the polynucleotide encodes the polypeptide comprising a G2BR domain consisting of the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1) wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ and X ₂₄ are as defined above. The encoded G2BR domain amino acid sequence can be the sequence of residues 5 to 24 of SEQ ID NO:2 (RQESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:3 (AAESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:4

(RQESLQERKQALYEYAARAF); the sequence of residues 5 to 24 of SEQ ID NO:5

(RQESLQERKQALYEYAARRF); or the sequence of residues 5 to 24 of SEQ ID NO:6

(RQESLQERKQALYEYARRAF). In some embodiments, the encoded G2BR domain amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1) does not have the sequence of residues 5 to 24 of SEQ ID NO:2.

[0065] The polynucleotide can encode the polypeptide comprising the sequence of any one of SEQ ID NOs:1-14, or a polypeptide having at least 80% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with the sequence of any one of SEQ ID NOs:1- 14. In preferred embodiments the sequence is any one of SEQ ID Nos: 3-13.

[0066] The polynucleotide can also encode a fusion protein comprising a G2BR domain and a heterologous amino acid sequence. The G2BR domain can consist of the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1) wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ and X ₂₄ are each defined as disclosed above.

[0067] Also disclosed is a recombinant vector comprising the isolated polynucleotide. In an embodiment, the recombinant vector permits inducible expression of the polypeptide or fusion protein encoded by the polynucleotide. Vectors permitting inducible expression are known. In certain embodiments, expression is induced by doxycycline or cycloheximide.

[0068] A host cell transfected with the recombinant vector or the polynucleotide is also disclosed.

[0069] The term“nucleic acid,”“polynucleotide,” or“oligonucleotide” includes DNA molecules and RNA molecules. A polynucleotide may be single-stranded or double-stranded. Polynucleotides can contain known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O- methyl ribonucleotides, peptide-nucleic acids (PNAs). A polynucleotide can be obtained by a suitable method known in the art, including isolation from natural sources, chemical synthesis, or enzymatic synthesis. Nucleotides may be referred to by their commonly accepted single-letter codes.

[0070] The term“operably linked” refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. A nucleic acid is“operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

[0071] The term "recombinant" can be used to describe a nucleic acid molecule and refers to a polynucleotide of genomic, RNA, DNA, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide can refer to a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, by a method known in the art. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0072] The term "vector" means a nucleic acid sequence to express a target gene in a host cell. Examples include a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Examples of viral vectors include a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. For example, the vector may be an expression vector including a membrane targeting or secretion signaling sequence or a leader sequence, in addition to an expression control element such as promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer. By“promoter” is meant minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included. If a promoter is inducible, there are sequences present that mediate regulation of expression so that the associated sequence is transcribed only when an inducer (e.g., cycloheximide) is available to the cell or tissue. The vector may be manufactured in various ways known in the art depending on the purpose. An expression vector may include a selection marker for selecting a host cell containing the vector. Further, a replicable expression vector may include an origin of replication. The term "recombinant vector" or “expression vector” means a vector operably linked to a heterologous nucleotide sequence for the purpose of expression, production, and isolation of the heterologous nucleotide sequence. The heterologous nucleotide sequence can be a nucleotide sequence encoding all or part of a G2BR polypeptide or a G2BR chimeric polypeptide disclosed herein. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., G2BR proteins, mutant forms of G2BR proteins, fusion proteins, and the like).

[0073] Accordingly, also disclosed is a method of producing a G2BR polypeptide or fusion protein disclosed herein. The method comprises culturing the host cell under conditions to produce the polypeptide.

[0074] The recombinant expression vectors of the invention can be designed for expression of G2BR proteins in prokaryotic or eukaryotic cells. For example, G2BR proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel (1990) Methods Enzymol.185:3-7. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0075] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0076] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69:301-315) and pET family vectors (MilliporeSigma), e.g. pET 11d (Studier et al. (1990) Methods Enzymol.185:60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[0077] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S. (1990) Methods Enzymol.185:119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0078] In another embodiment, the G2BR expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J.6:229-234), pMFa (Kuijan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp., San Diego, Calif.).

[0079] Alternatively, G2BR proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the Pac series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Surnmers (1989) Virology 170:31-39).

[0080] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0081] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinker et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473- 5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.4,873,316 and European Application Publication No.264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the .alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0082] A host cell can be any prokaryotic or eukaryotic cell. For example, a G2BR protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO), HEK293T, or COS cells). Other suitable host cells are known to those skilled in the art.

[0083] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms“transformation” and“transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[0084] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a G2BR protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0085] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a G2BR protein. Accordingly, the invention further provides methods for producing a G2BR protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a

recombinant expression vector encoding a G2BR protein has been introduced) in a suitable medium such that a G2BR protein is produced. In another embodiment, the method further comprises isolating a G2BR protein from the medium or the host cell.

Pharmaceutical Compositions

[0086] The G2BR polypeptides, fusion proteins, conjugates, and polynucleotides (also referred to herein as“active compounds”) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the G2BR polypeptide, fusion protein, conjugate, or polynucleotide and a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0087] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid;

buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0088] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum

monostearate and gelatin.

[0089] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a G2BR protein, conjugate, or fusion protein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0090] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0091] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0092] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fluidics acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0093] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0094] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0095] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0096] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.

Moreover, treatment of a subject with a therapeutically effective amount of a G2BR polypeptide, conjugate, or fusion protein can include a single treatment or can include a series of treatments. It will also be appreciated that the effective dosage of a G2BR polypeptide, conjugate, or fusion protein used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.

Uses and Methods of the Invention

[0097] In another aspect, methods of using the polypeptides, fusion proteins, conjugates, and polynucleotides disclosed herein are disclosed. [0098] The polypeptides, fusion proteins, conjugates, and nucleic acid molecules described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, and monitoring clinical trials); and c) methods of treatment (e.g., therapeutic and prophylactic). As described herein, a G2BR domain protein has one or more of the following activities: (i) interaction with a target molecule such as UBE2G2; (ii) modulation of UBE2G2 activity; (iii) modulation of ERAD; (iv) modulation of intra- or inter-cellular signaling and/or gene transcription (e.g.; either directly or indirectly); (v) modulation of protein stability (e.g., mutant CFTR, assembled or unassembled immunoglobulin molecules, CD3-d and/or TCR-a stability); and/or (vi) modulating tumor growth, invasiveness and/or metastasis.

[0099] The isolated nucleic acid molecules can be used, for example, to express a G2BR domain polypeptide or fusion protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect AUP1 G2BR mRNA (e.g., in a biological sample) or a genetic alteration in a G2BR, to modulate G2BR activity, and to modulate ERAD, as described further below.

[00100] The G2BR proteins can be used to detect UBE2G2 (e.g., in a biological sample), to modulate ERAD, and to treat disorders characterized by insufficient or excessive ERAD or insufficient or excessive stability of a particular protein, for example, ERAD-associated disorders. In addition, the G2BR proteins can be used to screen for naturally occurring G2BR target molecules, to screen for drugs or compounds which modulate G2BR activity, as well as to treat disorders characterized by insufficient or excessive ERAD or insufficient or excessive stability of a particular protein (e.g., an ERAD-associated disorder). Further, the G2BR proteins can be used to detect and isolate UBE2G2 proteins, regulate the bioavailability of UBE2G2 proteins, and modulate UBE2G2 activity

Detection assays

[00101] A method of detecting the ubiquitin conjugating enzyme E2G2 (UBE2G2) in a sample is disclosed. The method can comprise contacting a sample with a polypeptide, fusion protein, or conjugate disclosed herein; and determining binding of the polypeptide, fusion protein, or conjugate to the UBE2G2 in the sample to detect the presence of UBE2G2 in the sample. A “sample” or“biological sample” can be a cell, tissue, and/or biological fluid sample. The sample can be isolated from a subject by conventional means. A sample also includes a sample of cells or tissue grown in cell culture. Any cell type or tissue in which G2BR is expressed may be utilized in the assays described herein.

[00102] Portions or fragments of the DNA and polypeptide sequences identified herein can be used in numerous ways as polynucleotide and polypeptide reagents. For example, these sequences can be used to detect UBE2G2 protein in a biological sample.

[00103] An exemplary method for detecting the presence or absence of UBE2G2 protein in a biological sample comprises contacting a sample with a G2BR polypeptide, conjugate, or fusion protein; and determining binding of the G2BR polypeptide, conjugate, or fusion protein to the UBE2G2 in the sample to detect the presence of UBE2G2 in the sample. The contacting is performed under conditions such that binding occurs between UBE2G2 and the G2BR polypeptide, conjugate, or fusion protein. The method can further comprise obtaining a biological sample from a test subject or cell culture. Detecting the binding interaction between UBE2G2 and the G2BR polypeptide, conjugate, or fusion protein can comprise measuring an electrical property, measuring a change in an ion concentration, measuring a change in protein conformation, measuring binding of the test compound to the polypeptide, measuring a change in phosphorylation level, measuring a change in transcription level, measuring a change in second messenger level, measuring a change in neurotransmitter level, measuring a change in a spectroscopic characteristic, measuring a change in a hydrodynamic (e.g., shape) property, measuring a change in a chromatographic property, or measuring a change in solubility. The G2BR polypeptide, fusion protein, or conjugate can be detectably labeled, such as with a fluorescent label.

[00104] The terms "detectably labeled", "detectable label", or“label” refer to a material capable of producing a signal indicative of the presence of the label by any appropriate method illustratively including spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and/or immunochemical. Examples of detectable labels include a fluorescent moiety, a

chemiluminescent moiety, a bioluminescent moiety, a magnetic particle, an enzyme, a substrate, a radioisotope, and a chromophore.

[00105] In another aspect, the present invention provides a method for detecting the presence of G2BR activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of G2BR activity such that the presence of G2BR activity is detected in the biological sample.

[00106] A preferred agent for detecting G2BR protein is an antibody capable of binding to a G2BR polypeptide, conjugate, or fusion protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab¢) ₂ ) can be used. The term“labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end- labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect G2BR mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of G2BR mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of G2BR protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of G2BR genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a G2BR protein include introducing into a subject a labeled anti-G2BR antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[00107] The invention also encompasses kits for detecting the presence of G2BR or UBE2G2 in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting G2BR protein or mRNA or UBE2G2 protein in a biological sample; means for determining the amount of G2BR protein or mRNA or UBE2G2 protein in the sample; and means for comparing the amount of G2BR protein or MRNA or UBE2G2 protein in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect G2BR protein or mRNA, or UBE2G2 protein. Methods of Modulating Protein Degradation or Methods of Treatment

[00108] Also disclosed is a method of modulating expression of a protein in a eukaryotic cell. The method comprises delivering a G2BR polypeptide, fusion protein, or conjugate disclosed herein to a eukaryotic cell expressing a protein under conditions such that expression of the protein is modulated. The modulation can be an increase or a decrease in expression of the protein. The protein can be, for example, WT or mutant CFTR in a patient with cystic fibrosis, or a polypeptide being over-expressed for large-scale production. Examples of a polypeptide being over-expressed for large-scale production include an enzyme, a cytokine, an antibody, and a therapeutic polypeptide.

[00109] Also disclosed is a method of modulating degradation of a protein in a eukaryotic cell. The method can comprise delivering a G2BR polypeptide, fusion protein, or conjugate disclosed herein to a eukaryotic cell comprising a protein under conditions such that degradation of the protein is modulated. The modulation can be an increase or a decrease in degradation of the protein. The protein can be, for example, a CFTR or a polypeptide being over- expressed for large-scale production. Examples of a polypeptide being over-expressed for large- scale production include an enzyme, an antibody, a cytokine, and a therapeutic polypeptide.

[00110] A method of reducing degradation of an over-expressed protein in a eukaryotic cell is disclosed. The method comprises delivering a G2BR polypeptide, fusion protein, or conjugate disclosed herein to a eukaryotic cell overexpressing a protein under conditions such that degradation of the protein is reduced. The eukaryotic cell can be overexpressing a protein for large- scale production. Examples of the overexpressed protein include an enzyme, a cytokine, an antibody, and a therapeutic polypeptide.

[00111] Examples of eukaryotic cells suitable for any of the methods disclosed herein include a mammalian cell, a fish cell, a yeast cell, or an insect cell. A preferred mammalian cell is a human cell.

[00112] Also disclosed is a method of inducing cell death. The method comprises delivering a G2BR polypeptide, fusion protein, or conjugate disclosed herein to a eukaryotic cell under conditions such that degradation of endoplasmic reticulum proteins is inhibited and cell death is induced. The cell is preferably a mammalian cell, more preferably a human cell. The mammalian cell can be an epithelial cell, a muscle cell, a fat cell, a lung cell, a skin cell, a pancreatic cell, an adrenal cell, a liver cell, a blood cell, a nerve cell, a bone cell, or a cancer cell. In preferred embodiments, the cell is a cancer cell. The cancer can be a primary solid tumor cell, a leukemia cell, a lymphoma cell, a myeloma cell, or a cancer cell undergoing metastasis.

[00113] Delivering a G2BR polypeptide, fusion protein, or conjugate to a eukaryotic cell can comprise ex vivo delivery into the cell of the G2BR polypeptide, fusion protein, or conjugate in a form in which the G2BR polypeptide, fusion protein, or conjugate can penetrate into the cell, transfection of the cell with an expression vector for the G2BR polypeptide or fusion protein, or can be co-expression of a stably transfected polynucleotide within the cell which encodes the G2BR polypeptide or fusion protein within the cell, preferably inducible co-expression.

[00114] Also provided are prophylactic and therapeutic methods of treating a subject at risk of, susceptible to, or having an ERAD-associated disorder. As used herein, the term“ERAD- associated disorder” includes a disorder, disease, or condition which is caused, affected, and/or associated with ERAD. ERAD-associated disorders can detrimentally affect cellular functions associated with up- or down-regulation of protein stability, e.g., the stability of a particular protein, such as components of the T cell antigen receptor (which when not assembled are subject to ERAD), or of proteins in general. ERAD-associated disorders also include disorders where up- or down-regulation of protein stability may be beneficial in treating the disorder. Examples of such ERAD-associated disorders include cancer, disorders of cholesterol and/or triglyceride metabolism, cystic fibrosis, Parkinson's disease, prion diseases of humans or animals, and viral infections (e.g., human cytomegalovirus (CMV), or human immunodeficiency virus (HIV)).

[00115] As used herein,“treatment” of a subject includes the application or administration of an active agent to a subject, or application or administration of an active agent to a cell or tissue from a subject, who has a diseases or disorder, has a symptom of a disease or disorder, or is at risk of (or susceptible to) a disease or disorder, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or disorder, the symptom of the disease or disorder, or the risk of (or susceptibility to) the disease or disorder. An "active agent" is any compound, element, or mixture that when administered to a patient alone or in combination with another agent confers, directly or indirectly, a physiological effect on the patient. As used herein, an“active agent” includes, but is not limited to, small molecules, peptides, polypeptides, peptidomimetics, antibodies, ribozymes, and antisense oligonucleotides. A“therapeutically effective amount” or“effective amount” is that amount of an active agent to achieve a pharmacological effect. The term“therapeutically effective amount” includes, for example, a prophylactically effective amount. An“effective amount” is an amount needed to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. The effective amount of a G2BR composition disclosed herein will be selected by those skilled in the art depending on the particular patient and the disease. It is understood that “an effective amount” or“a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of the G2BR composition, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. A“patient” is a human or non-human animal in need of medical treatment. Medical treatment includes treatment of an existing condition, such as a disorder or injury. In certain embodiments treatment also includes prophylactic or preventative treatment, or diagnostic treatment.

[00116] A method of treating an ERAD-associated disorder comprises administering a G2BR polypeptide, fusion protein, or conjugate to a patient having an ERAD-associated disorder. The amount administered can be an effective amount. The ERAD-associated disorder can be a cellular proliferation, growth, or differentiation disorder, such as cancer; cystic fibrosis; a disorder of cholesterol and/or triglyceride metabolism; Parkinson's disease; a prion disease; or a viral infection. In preferred embodiments the ERAD-associated disorder is cystic fibrosis or cancer. Cellular proliferation, growth, or differentiation disorders include those disorders that affect cell proliferation, growth, or differentiation processes. As used herein, a“cellular proliferation, growth, or differentiation process” is a process by which a cell increases in number, size or content, or by which a cell develops a specialized set of characteristics which differ from that of other cells. In particular, there is evidence that ERAD is involved in regulating tumor invasiveness and metastasis, indicating that the G2BR molecules disclosed herein are useful in modulating invasiveness and metastasis. Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepatic disorders; myelodysplastic syndromes; and hematopoietic and/or myeloproliferative disorders. Particularly preferred disorders include melanoma, sarcoma, breast carcinoma, and multiple myeloma.

[00117] In one aspect, a method for preventing in a subject, an ERAD-associated disorder is provided. The method comprises administering to the subject a G2BR polypeptide, fusion protein, or conjugate, or an agent which modulates at least one activity of endogenous gp78 or AUP1 that is mediated by their G2BR domains (e.g., interaction with UBE2G2), or activity of other, uncharacterized, G2BR-like domains. Subjects at risk for a disease that is caused or contributed to by aberrant or unwanted AUP1 G2BR or gp78 G2BR expression or activity can be identified by, for example, methods known in the art. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the ERAD-associated disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of G2BR or G2BR-like aberrancy, for example, a G2BR molecule, G2BR agonist, or G2BR antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[00118] Another aspect of the invention pertains to methods of modulating G2BR activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves delivering to a cell capable of expressing G2BR an agent that modulates one or more of the activities of the AUP1 G2BR, the gp78 G2BR, or other proteins containing G2BR-like activity associated with the cell, such that these cellular activities are modulated. An agent that modulates the AUP1 G2BR, the gp78 G2BR, or other G2BR-like protein activity can be an agent as described herein, such as a G2BR nucleic acid or a protein, a naturally- occurring target molecule of a G2BR protein (e.g., a G2BR target molecule such as UBE2G2), a G2BR antibody, a G2BR agonist or antagonist, a peptidomimetic of a G2BR agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more G2BR activities. Examples of such stimulatory agents include active G2BR protein and a nucleic acid molecule encoding G2BR that has been introduced into the cell. In another embodiment, the agent inhibits one or more AUP1 G2BR, gp78 G2BR, or other G2BR-like activities. Examples of such inhibitory agents include antisense G2BR nucleic acid molecules, anti-G2BR antibodies, and G2BR inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of the AUP1 G2BR, the gp78 G2BR or other G2BR-like domains within proteins or encoded by nucleic acid molecules. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity of the AUP1 G2BR, the gp78 G2BR, or other G2BR-like domains of proteins. In another

embodiment, the method involves administering a G2BR protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted G2BR expression or activity.

[00119] Stimulation of ERAD is desirable in situations in which increased ERAD is likely to have a beneficial effect. For example, it is desirable to upregulate ERAD in cells where it would be beneficial to decrease the stability of a protein that is normally degraded by ERAD or in cells having defects in ERAD machinery. ERAD can be stimulated by increasing G2BR levels at the ER membrane and thereby recruiting/activating UBE2G2. This could occur through generation and delivery of a G2BR conjugate or fusion that interacts either directly or indirectly with the ER membrane.

[00120] Inhibition of endogenous UBE2G2 binding activity to endogenous AUP1 G2BR, gp78 G2BR, or to another G2BR-like domain is desirable in situations in which decreased UBE2G2 activity is likely to have a beneficial effect. For example, inhibition of UBE2G2 binding to the AUP1 G2BR or the gp78 G2BR is desirable in situations in which it is desirable to downregulate ERAD. For example, it is desirable to downregulate ERAD in cells where it would be beneficial to increase the stability or lifetime of a protein that is normally degraded by ERAD. In another embodiment, it is desirable to downregulate ERAD in cancer cells, because downregulation of ERAD may induce cellular stress, which in turn induces an unfolded protein response and apoptosis of the cancer cells.

[00121] The following examples are merely illustrative of the compositions and methods disclosed herein, and are not intended to limit the scope hereof. EXAMPLES The following experimental procedures were used in the Examples, unless otherwise indicated. Crystallography

[00122] The concentration of UBE2G2:AUP1376-410 complex is 15 mg/ml in 150 mM NaCl, 50 mM Tris-HCl pH7.5. The rod-shaped crystals were obtained from crystallization buffer containing 30% PEG-4000, 0.2M NH ₄ Ac, 0.1 M NaAc pH 4.6 (Q-Classics H2), using sitting drop vapor diffusion method. The diffraction data were collected on a MARMOSAIC 325 detector at beamline BL9-2, SSRL. The crystal belonged to space group P212121 and diffracted to 1.74 Å. The structure of the complex was determined by molecular replacement method, with the starting model of PDB entry 3H8K (UBE2G2:G2BR ^gp78 binary complex). The structure refinement was done with PHENIX.

Isothermal Calorimetry (ITC)

[00123] Protein-protein interactions as assessed by ITC were studied using a VP-ITC Microcalorimeter (MicroCal LLC, Northampton, MA) at 25°C. The typical experiment included injection of 25-27 aliquots (10 mL each) of 0.2 mM ligand into a 0.02 mM protein solution in the ITC cell (volume ~1.4 mL) at stirring speed of 300 rpm. The integrated interaction heat values were fit using one set of sites model with data analysis software provided by MicroCal.

Cells and Constructs

[00124] An HT1080 CRISPR knockout line for AUP1 was generated using a Double Nickase Plasmid kit from Santa Cruz (sc-410699-NIC). Targeting plasmids were transfected using Ultra Cruz transfection reagent and plasmid medium (Santa Cruz) and selected with 2 µg/mL puromycin. Loss of protein expression was confirmed by Western Blot.

[00125] Human fibrosarcoma HT1080 (ATCC Accession No. CCL-121) or AUP1 CRISPR/Cas9 knockout cells (AUP1 k.o.) were maintained in Complete Media, which is composed of DMEM supplemented with 10% fetal bovine serum, GLUTAMAX (Gibco), 100 I.U./mL penicillin, and 100 µg/mL streptomycin. For mammalian cell expression, the NHK variant of alpha- 1-antitrypsin (A1AT) and CD3-d were each cloned into the pCDNA3 vector to contain a C-terminal HA tag. Full-length AUP1 (residues 1-410) with wild-type or mutant G2BR residues were cloned into pCDNA3.1(+) containing a C-terminal FLAG tag.

[00126] HT1080 cells were transfected with pcDNA6/TR to express the tet repressor gene and selected in 5 µg/ml blasticidin. Stable cell lines were screened for doxycycline-inducible expression of GFP by transient transfection with pcDNA5/TO/FRT/GFP plasmid. GFP was subcloned into pcDNA5/TO/FRT (Invitrogen) between BamHI and NotI site. Positive clones (HT1080 TR) were maintained in media containing tetracycline-free fetal bovine serum (FBS) supplemented with 2 µg/ml blasticidin or frozen in liquid nitrogen for future experiments.

[00127] For the in vivo animal survival experiment of Example 7, G2BR gene sequences were first ligated into the pEGFP-C1 expression vector, then the EGFP-G2BR fusion was PCR amplified and re-ligated into pCDNA5/TO/FRT to generate a tet-inducible GFP-G2BR expression construct. HT1080 TR cells were transfected with pcDNA5/TO/FRT/EGFP-G2BR plasmids to generate cells with doxycycline-inducible expression of GFP fused to G2BR ^AUP1 or to G2BR ^SCR . SCR is a scrambled/random order gp78 G2BR sequence, which serves as a negative control. Following selection in media containing tetracycline-free FBS with 2 µg/ml blasticidin and 50 µg/ml hygromycin, individual clones were screened for doxycycline-inducible expression of GFP-G2BR proteins.

[00128] N-terminal GST-tagged constructs were generated by PCR using human AUP1 or gp78 as template and subcloned into pGEX6P1 vector and expressed in the BL21 strain of E.Coli. For crystallization, GST-AUP1 fragment (including aa 376-410, which encompasses the G2BR) and UBE2G2 were subcloned in modified pET-Duet vector to allow simultaneous expression of both protein in BL21 strain of E. coli. The complex containing GST-AUP1 and UBE2G2 was purified with Glutathione Sepharose 4B (GE Healthcare) to capture GST-AUP1 and the tightly associated UBE2G2. Nearly 100% of GST-AUP1 was associated with UBE2G2. The complex was released from the column when the GST tag was cleaved with PRECISSION protease. The complex was used for crystallization.

[00129] N-terminal GFP-tagged constructs were generated by PCR and ligation into EGFP-C1 vector. Example 1. Determination of AUP1 residues involved in interaction with UBE2G2

[00130] A crystal structure of UBE2G2 co-crystallized with a fragment of AUP1 (SEQ ID NO:14), amino acid residues 376-410, was obtained in order to determine AUP1 residues interacting with UBE2G2. FIG.1 presents a schematic representation of the portion of the crystal structure showing the interaction between UBE2G2 and the AUP1 fragment (the helix at the top of the structure in each view). In particular, the residues of AUP1 forming salt bridges or hydrogen bonds with residues of UBE2G2 are illustrated. The UBE2G2-binding region (G2BR) of AUP1 has sequence SSWARQESLQERKQALYEYARRRFTER (SEQ ID NO:2), corresponding to residues 378-404 of full-length AUP1 (SEQ ID NO:14).

[00131] FIG.2 is a schematic representation showing the residues of G2BR ^AUP1 involved in interactions with residues of UBE2G2; and also provides a comparison of the residues of G2BR ^gp78 (SEQ ID NO:15) interacting with the same residues of UBE2G2 based on PDB entry 3H8.

Example 2. Determination of binding affinity AUP1 G2BR with UBE2G2

[00132] The dissociation binding constants (K _d ) were determined for binding of AUP1 G2BR or gp78 G2BR to UBE2G2 by isothermal calorimetry (ITC). Synthetic peptides used for ITC were purchased commercially from GenScript or PEPTIDE 2.0 and correspond to gp78 aa 579-600 and AUP1378-404. UBE2G2 was expressed and purified from the BL21 strain of E. Coli.

UBE2G2 expression was induced with 1 mM IPTG at 37 °C for ~ 3 h. Cells were harvested and UBE2G2 was extracted from cell pellets in 4 M urea, 50 mM TRIS pH 7.2, 4 mM DTT, refolded by stepwise dialysis into buffer containing no urea, and purified by ion-exchange followed by gel filtration chromatography. The dissociation binding constants are shown below in Table 2.

Table 2. K _d for G2BR binding to UBE2G2

[00133] Binding between UBE2G2 and the wild-type AUP1 G2BR is about 16 times stronger than binding between UBE2G2 and the gp78 G2BR.

[00134] Comparison of G2BR ^AUP1 and G2BR ^gp78 binding to UBE2G2 was also assessed by a bead assay. ³⁵ S-labeled UBE2G2 was generated using the E. Coli T7/S30 Extract System for Circular DNA (Promega). 0.5 or 1.0 µg (approximately 15 and 30 pmoles, respectively) of GST- AUP1 (aa 376-410) and GST-gp78 (574-600, SEQ ID NO:15) fusions were pre-bound at 4°C to Gutathione Sepharose 4B beads in 50 mM Tris pH 7.4. After washing, ~50 fmoles ³⁵ S-UBE2G2 was added in binding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% IGEPAL CA-630 (Sigma), 5 mM DTT), incubated overnight at 4 °C. Beads were washed in binding buffer, GST fusions and bound UBE2G2 was eluted from beads in polyacrylamide gel electophoresis (PAGE) sample buffer and resolved on 4-12 % Bis-Tris acrylamide gels in 1X MES. Gels were dried and autoradiographed to assess the amount UBE2G2 retained by each of the GST-G2BR fusions.

Results of these experiments are shown in FIG.11, in which it can be seen that at each amount of bound G2BR the GST-G2BR ^AUP1 bound more UBE2G2 than GST-G2BR ^gp78 . These results are consistent with the higher affinity of a synthetic G2BR ^AUP1 peptide for UBE2G2 as compared to a comparable G2BR ^gp78 peptide as determined by ITC.

Example 3. Mutations of AUP1 G2BR residues

[00135] Residues of AUP1 G2BR identified as involved in forming salt bridges and hydrogen bonds with UBE2G2 were mutated to determine the effect of the mutations on the binding interaction. AUP1 G2BR residues were mutated using the QUIKCHANGE II Site-Directed Mutagenesis Kit (Agilent Technologies) and confirmed by DNA sequencing. In particular, fusion proteins of GST with AUP1 residues 292-410 were made with the mutation of one or more of residues R382, Q383, K390, R398, and R400 to alanine (A) or glutamate (E) to assess effect on binding, in the following examples.

[00136] AUP1 G2BR:UBE2G2 binding was assessed by a bead assay. ³⁵ S-labeled UBE2G2 was generated using the E. Coli T7/S30 Extract System for Circular DNA (Promega). 0.1 µg of GST-AUP1 (292-410) fusions were pre-bound at 4 °C to Glutathione Sepharose 4B beads in 50 mM Tris pH 7.4. After washing, ~50 fmoles ³⁵ S-UBE2G2 was added in binding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% IGEPAL CA-630 (Sigma), 5 mM DTT), incubated overnight at 4 °C. Beads were washed in binding buffer, GST-AUP1 was eluted from beads in PAGE sample buffer and run on 4-12 % Bis-Tris acrylamide gels in 1X MES. Gels were dried and

autoradiographed to assess the amount UBE2G2 retained by each of the GST-AUP1 G2BR fusions. Results of these experiments are shown in FIG.3. In FIG.3, it can be seen that mutation of two of these residues to alanine is not sufficient to disrupt the interaction (compare WT lane 2 to AAKRR lane 3); at least 4 residues have to be mutated to alanine (lanes 5 and 7-8) to weaken or to eliminate a detectable interaction. Even when mutating these residues to Glutamate (lanes 6 and 10-12) or Aspartate (not shown) to reverse the charge of the wild-type residue, 3 or more residues have to be mutated to eliminate detectable binding to UBE2G2. The R398A/R400A mutant (lane 4) showed the greatest apparent affinity for UBE2G2 in this experiment. Therefore, in further experiments, UBE2G2 binding by an R398A mutant or an R400A mutant was compared with binding to the wild type sequence and the R398A/R400A mutant. The results are shown in FIG.4. Binding of the double mutant (lane 5) is stronger than binding of wild type (lane 2), and each of the single mutants (lanes 3 and 4) also binds substantially stronger than wild type (lane 2). Thus, substitution of A for R in position 398, 400 or both increases binding of the AUP1 G2BR to UBE2G2.

[00137] ITC analyses are performed to quantify the binding affinity to UBE2G2 of each of the mutants R398A, R400A and R398A/R400A and to corroborate that these mutant peptides have stronger binding to UBE2G2 than wild type AUP1 G2BR.

Example 4. AUP1 expression is required for degradation of the NHK mutant of A1AT.

[00138] The NHK mutant variant of alpha1-antitrypsin (A1AT) has a frame-shift mutation resulting in a C-terminal truncation of 61 amino acids. The NHK mutant is degraded rapidly by ER-associated degradation (ERAD), resulting in deficiency of the protein.

[00139] HT1080 WT or AUP1 k.o. cells were grown to 60-70% confluency and transfected with plasmid encoding HA-tagged NHK and either pCDNA empty vector (EV, i.e. no protein-encoding insert cloned into the plasmid) or plasmids encoding FLAG-tagged full-length AUP1 WT or an AUP1 mutant with G2BR (residues 386-397) deleted from the full-length AUP1 sequence using JETPRIME transfection reagent (Polyplus) for 24 hours. Cells were incubated at 37°C in Complete Media to which 50 µg/mL cycloheximide (CHX) was added to prevent new protein synthesis for the indicated times and thereby allow an assessment of the degradation of pre- existing protein. Cells were collected in 1X PBS, 1% Triton X-100, 0.5 % deoxycholate, 50 µM ALLN (Calbiochem), supplemented with COMPLETE Protease Inhibitor Cocktail (Sigma-Aldrich). Lysates were run on 10% Bis-Tris acrylamide gels in 1X MOPS and analyzed by Western blotting. Proteins levels were analyzed using rat anti-HA-HRP (for NHK), mouse anti-FLAG (for AUP1), or mouse anti-Hsp60 (for Hsp60 gel loading control) antibodies and detected with chemiluminescence.

[00140] The immunoblot results are shown in FIG.5. The control lanes (lanes labeled EV, empty vector containing no protein-encoding insert as control) show that degradation of NHK is inhibited in HT1080 cells lacking AUP1 (AUP1 knock out (k.o.) cells) as compared to in the wild-type HT1080 cells. When the AUP1 k.o. cells are transfected with a plasmid expressing AUP1 wild-type (AUP1 ^WT lanes), degradation of NHK is restored in the AUP1 k.o. cells. The G2BR is required for restoration of this function as transfection of the AUP1 k.o. cells with a plasmid expressing an AUP1 mutant having an internal deletion in the G2BR (amino acids 386-397;

AUP1 ^DG2BR lanes) does not restore NHK degradation.

[00141] Further experiments were performed in which AUP1 k.o. cells are transfected with a plasmid expressing the AUP1 R398A/R400A mutant. Immunoblot results comparing NHK degradation in AUP1 k.o. cells transfected with a plasmid expressing wild-type AUP1, the AUP1 R398A/R400A mutant, or the AUP1 G2BR deletion mutant are shown in FIG.6. Expression of wild-type AUP1 or the AUP1 R398A/R400A mutant (AUP1 ^RR-AA ) in the AUP1 k.o. cells restores NHK degradation compared to the AUP1 G2BR deletion mutant (AUP1 ^∆G2BR ), consistent with the ability of both to bind UBE2G2.

[00142] Additional experiments were performed in which AUP1 k.o. cells were transfected with a plasmid expressing an AUP1 mutant in which residues 90-337 were deleted (AUP1 ^∆90-337 ). Immunoblot results comparing NHK degradation in AUP1 k.o. cells transfected with a plasmid expressing wild-type AUP1 or with the plasmid expressing the AUP1 ^∆90-337 mutant shows that the AUP1 ^∆90-337 mutant can restore NHK degradation as well as wild type AUP1. Thus, an AUP1 including only its membrane anchor and its G2BR is sufficient to restore ERAD in AUP1 knockout cells.

Example 5. AUP1 G2BR overexpression inhibits degradation of CD3-d

[00143] CD3-d deficiency is a rare and fatal form of severe combined

immunodeficiency. CD3-d is a subunit of the T cell receptor, the degradation of which can limit T cell receptor assembly and expression and thereby the ability of an individual to mount an immune response. CD3-d is also a substrate of the UBE2G2/gp78 complex, which results in CD3-d degradation. CD3-d is extensively employed in non-T cells as a test substrate for ERAD as in this example.

[00144] Expression vectors were constructed in the pEGFP-C1 plasmid to express the fusion proteins of GFP with the UBE2G2 binding regions (G2BR) of wild-type gp78 (574-600, SEQ ID NO:15; G2BR ^gp78 ), wild-type AUP1 (378-404 SEQ ID NO:2 G2BR ^{AUP1 WT} ), the AUP1 R398A mutant (G2BR ^{AUP1 R398A} ), the AUP1 R400A mutant (G2BR ^{AUP1 R400A} ), or a scrambled sequence (G2BR ^SCR ).

[00145] HT1080 WT cells were transfected with HA-tagged CD3-d and the GFP fusions of G2BR ^SCR , G2BR ^gp78 , G2BR ^{AUP1 WT} , G2BR ^{AUP1 R398A} or G2BR ^{AUP1 R400A} mutants using JETPRIME transfection reagent (Polyplus) for 24 hours to allow for expression of the fusion proteins. This was followed incubation in Complete Media containing 50 µg/mL cycloheximide (CHX) to prevent new protein synthesis for 0, 3, or 6 hours as indicated in FIG.7 or FIG.8. Cell lysates were collected and analyzed by Western blotting using rat anti-HA-HRP (for CD3-d) and mouse anti-GFP (for GFP-G2BR fusions) antibodies and detected using chemiluminescence.

[00146] FIG.7 shows degradation of HA-tagged CD3-d in the presence of cells expressing G2BR ^SCR , G2BR ^gp78 , or G2BR ^{AUP1 WT} when cycloheximide treatment was used to prevent new protein synthesis. FIG.7 shows that G2BR ^AUP1 overexpression stabilizes the

UBE2G2/gp78 substrate CD3-d against degradation to a greater extent than overexpression of G2BR ^SCR or G2BR ^gp78 .

[00147] FIG.8 shows the effect of cycloheximide on levels of HA-tagged CD3-d in the presence of cells expressing G2BR ^SCR , G2BR ^{AUP1 WT} , G2BR ^{AUP1 R398A} , or G2BR ^{AUP1 R400A} .

Inhibition of CD3-d degradation is also seen with expression of G2BR ^{AUP1 R398A} , or G2BR ^{AUP1 R400A} (FIG 8, compare levels of CD3-d-HA at 0 or 6 hours of treatment with cycloheximide for the G2BR ^SCR control relative to G2BR ^{AUP1 R398A} and G2BR ^{AUP1 R400A} ). However, the AUP1 fusion proteins are destabilized compared to that of SCR (FIG.8, compare the GFP-G2BR band showing level of expression of each fusion protein (“GFP-G2BR”), with a longer exposure of the gel expression of G2BR ^{AUP1 R400A} can be seen at 6 hours of cycloheximide treatment). This destabilization suggests that inhibitory ERAD effects of wild type and mutant GFP-G2BR ^AUP fusion proteins could be underestimated.

Example 6. Effect of expression of the AUP1 G2BR on cell stress

[00148] U.S. Patent No.8,420,776 had observed that expression in cells of the gp78 G2BR elicited a UPR in the cells and resulted in apoptotic cell death. Since gp78 G2BR is a lower affinity G2BR than the AUP1 G2BR, the inventors hypothesize that expression of the significantly tighter binding AUP1 G2BR will similarly result in a UPR and lead to apoptotic cell death. The ability of AUP1 G2BR expression to inhibit ERAD suggests that expression of the AUP1 G2BR might elicit an UPR.

[00149] The effect of overexpression of G2BR on ER stress in HT1080 cells was examined by analyzing the levels of three different cell stress markers, phospho-eIF2a, BiP, and CCAAT/enhancer-binding protein homologous protein (CHOP) in cell lysates. FIG.9A-C are immunoblots showing levels of cell stress markers in HT1080 cell lysates expressing GFP fusions of G2BR ^gp78 , G2BR ^AUP1 , or G2BR ^SCR . The immunoblot shown in FIG.9A demonstrates that each of the AUP1 and gp78 G2BRs increases the level of phospho-eIF2a above the baseline control level (SCR). However, G2BR ^AUP1 is more effective in inducing this stress marker. In FIG.9B, when BiP is assessed, an increase above baseline is only observed with the G2BR ^AUP1 . In FIG.9C, when assessed for induction of CHOP expression, which is an indicator of a pro-apoptotic response to stress, G2BR ^AUP1 was found to be more effective in inducing this protein than G2BR ^gp78 .

[00150] Increased levels of the cell stress markers phospho-eIF2a and BiP correlate with increased inhibition of ERAD, resulting in increased levels of cell stress. An inability to compensate for severe cell stress ultimately results in death of the cells. The increase in CHOP is consistent with a response that will ultimately result in cell death and suggests a greater potential of the G2BR ^AUP1 to induce cell death in a cancer cell line than the G2BR ^gp78 .

[00151] Similar experiments are performed to assess induction of other cell stress markers resulting from overexpression of G2BR ^AUP1 , G2BR ^AUP1 mutants, G2BR ^gp78 , or G2BR ^SCR , respectively, in the HT1080 cells. The additional stress markers include cleavage of ATF6.

Immunoblots show increased cleavage of ATF6 induced by overexpression of the G2BR binding to UGE2G2. Example 7. Animal survival in a mouse fibrosarcoma lung metastasis model after treatment by in vivo expression of a G2BR

[00152] HT1080 cells harboring inducible GFP-G2BR ^AUP1 or GFP-G2BR ^SCR were treated with 1 µg/ml doxycycline overnight to induce expression of GFP-G2BR ^AUP1 or GFP- G2BR ^SCR . The following day, 1x10 ⁶ cells/animal were injected intravenously into athymic nu/nu mice (Charles River; n=10 for each group) via tail vein to induce lung metastasis using a well- accepted metastasis model system (Tsai et al. (2007) Nat Med 13(12):1504-9). Animals were fed 200 mg/kg doxycycline (in food pellets) daily to induce expression of GFP-G2BR proteins.

[00153] A survival curve showing the results of this experiment is shown in FIG.10. All of the animals expressing the GFP-G2BR ^AUP1 survived this tumor cell load until the experiment was terminated at over 100 days. All of the control animals (GFP-G2BR ^SCR ) were deceased by 65 days. These results correlate with the ability of GFP-G2BR ^AUP1 to block ERAD and thereby induce a severe UPR in tumor cells in vivo. Tumor cells and metastasizing cells are subject to ongoing ER stress. By blocking ERAD via the expression of the G2BR ^AUP1 construct, the ER stress cannot be compensated for, resulting in the death of the tumor cells and survival of the mice.

[00154] G2BR expression can also be induced after the cells have established metastases. HT1080 cells harboring inducible GFP-G2BR ^AUP1 or GFP-G2BR ^SCR (1x10 ⁶ cells/animal) are injected intravenously into athymic nu/nu mice (Charles River; n=20 for each group) and allowed to grow metastases for different amount of time. For example, 21 days after injection of HT1080 cells, mice are fed food pellets with or without 200 mg/kg doxycycline daily to induce expression of GFP-G2BR proteins. Doxycycline-induced expression of GFP-G2BR ^AUP1 increases median survival of the animals whereas doxycylcine-induced expression of GFP-G2BR ^SCR has no effect on animal survival.

Example 8. Animal survival in mouse breast cancer, prostate cancer, or melanoma models after treatment by in vivo G2BR expression

[00155] Similar experiments to those described in Example 7 are performed in mouse models for breast cancer, prostate cancer, and melanoma.

[00156] The breast cancer model uses the transplantable 4T1 mammary carcinoma cell line transfected with a plasmid that permits constitutive or inducible in vivo expression of a G2BR, including G2BR ^AUP1 , G2BR ^AUP1 mutants, G2BR ^gp78 , and G2BR ^SCR . Expression of G2BR ^AUP1 and the G2BR ^AUP1 mutants with higher apparent affinity UBE2G2 in the mice increases median survival.

[00157] The prostate cancer xenograft model uses the PC3 cell line transfected with a plasmid that permits constitutive or inducible in vivo expression of a G2BR, including G2BR ^AUP1 , G2BR ^AUP1 mutants, G2BR ^gp78 , and G2BR ^SCR . Expression of G2BR ^AUP1 and the G2BR ^AUP1 mutants with higher apparent affinity for UBE2G2 in the mice increases median survival.

[00158] The melanoma model uses either the B16 cell line or the 1205LU cell line transfected with a plasmid that permits constitutive or inducible in vivo expression of a G2BR, including G2BR ^AUP1 , G2BR ^AUP1 mutants, G2BR ^gp78 , and G2BR ^SCR . Expression of G2BR ^AUP1 and the G2BR ^AUP1 mutants with higher apparent affinity for UBE2G2 in the mice increases median survival.

[00159] This disclosure further encompasses the following aspects.

[00160] Aspect 1. An isolated polypeptide comprises the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1), wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ , and X ₂₄ are each independently selected from any amino acid, or an amino acid sequence having at least 85% identity to residues 5-24 of SEQ ID NO:1, with the proviso that the amino acid sequence does not have the sequence of residues 5 to 24 of SEQ ID NO:2; preferably X ₅ is R, A, E, K, or H, X ₆ is Q, A, H, E, K, or R, X ₁₃ is K, A, E, R, or H, X ₂₁ is G, V, L, I, F, D, R, A, or E, X ₂₃ is G, V, L, I, F, D, R, A, or E, and X ₂₄ is F or Y; more preferably X ₅ is R, A, or E, X ₆ is Q, A, or E, X ₁₃ is K, A, or E, X ₂₁ is R, A, or E, X ₂₃ is R, A, or E, and X ₂₄ is F; even more preferably X ₂₁ is A, X ₂₃ is A, and X ₂₄ is F.

[00161] Aspect 2. The isolated polypeptide of aspect 1, wherein the amino acid sequence is selected from the sequence of residues 5 to 24 of SEQ ID NO:3

(AAESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:4

(RQESLQERKQALYEYAARAF); the sequence of residues 5 to 24 of SEQ ID NO:5

(RQESLQERKQALYEYAARRF); and the sequence of residues 5 to 24 of SEQ ID NO:6

(RQESLQERKQALYEYARRAF).

[00162] Aspect 3. The isolated polypeptide of aspect 1 or 2, wherein the polypeptide is bound to a label, a solid support, a lipid monolayer, a heterologous polypeptide sequence, or a combination thereof, preferably the binding is covalent.

[00163] Aspect 4. The isolated polypeptide of aspect 3, wherein the label comprises a radiolabel, a fluorescent label, a chemiluminescent label, an enzymic label, an immunogenic label, or a combination thereof.

[00164] Aspect 5. The isolated polypeptide of aspect 3 or 4, wherein the heterologous polypeptide sequence is a cell-penetrating peptide sequence, preferably the cell-penetrating peptide sequence comprises Tat, Antennapedia, polyarginine (R9), or NM1

(KVRVRVRVpPTRVRV*RVK, where non-capital letters denote D-amino acids and * denotes peptide backbone N-methylation).

[00165] Aspect 6. The isolated polypeptide of any one of aspects 1 to 5 comprising a stapled polypeptide.

[00166] Aspect 7. A fusion protein or conjugate comprising a first moiety, and a second moiety, wherein the first moiety consists of a G2BR domain consisting of the amino acid sequence X ₅ X ₆ ESLQ ERX ₁₃ QALYEYA X ₂₁ RX ₂₃ X ₂₄ (residues 5-24 of SEQ ID NO:1), wherein X _5, X ₆ , X ₁₃ , X ₂₁ , X ₂₃ , and X ₂₄ are each independently selected from any amino acid or an amino acid sequence having at least 85% identity to residues 5-24 of SEQ ID NO:1; preferably X ₅ is R, A, E, K, or H, X ₆ is Q, A, H, E, K, or R, X ₁₃ is K, A, E, R, or H, X ₂₁ is G, V, L, I, F, D, R, A, or E, X ₂₃ is G, V, L, I, F, D, R, A, or E, and X ₂₄ is F or Y; more preferably X ₅ is R, A, or E, X ₆ is Q, A, or E, X ₁₃ is K, A, or E, X ₂₁ is R, A, or E, X ₂₃ is R, A, or E, and X ₂₄ is F; even more preferably X ₂₁ is A, X ₂₃ is A, and X ₂₄ is F, and preferably, the first moiety is covalently bound to the second moiety. [00167] Aspect 8. The fusion protein or conjugate of aspect 7, wherein the amino acid sequence of the G2BR domain is selected from the sequence of residues 5 to 24 of SEQ ID NO:2 (RQESLQERKQALYEYARRRF), the sequence of residues 5 to 24 of SEQ ID NO:3

(AAESLQERKQALYEYARRRF); the sequence of residues 5 to 24 of SEQ ID NO:4

(RQESLQERKQALYEYAARAF); the sequence of residues 5 to 24 of SEQ ID NO:5

(RQESLQERKQALYEYAARRF); and the sequence of residues 5 to 24 of SEQ ID NO:6

(RQESLQERKQALYEYARRAF

[00168] Aspect 9. The fusion protein or conjugate of aspect 7 or 8, wherein the second moiety comprises a label, a solid support, a lipid monolayer, a heterologous polypeptide sequence, or a combination thereof.

[00169] Aspect 10. The fusion protein or conjugate of any one of aspects 7 to 9, wherein the label comprises a radiolabel, a fluorescent label, a chemiluminescent label, an enzymic label, an immunogenic label, or a combination thereof.

[00170] Aspect 11. The fusion protein or conjugate of any one of aspects 7 to 10, wherein the heterologous polypeptide sequence comprises a cell-penetrating peptide sequence.

[00171] Aspect 12. The fusion protein or conjugate of any one of aspects 7 to 11 wherein the heterologous polypeptide sequence further comprises an amino acid linker.

[00172] Aspect 13. The fusion protein or conjugate of any one of aspects 7 to 12 comprising a stapled polypeptide.

[00173] Aspect 14. An isolated polynucleotide encoding the isolated polypeptide of any one of aspects 1 to 6 or the fusion protein or conjugate of any one of aspects 7 to 13.

[00174] Aspect 15. A recombinant vector comprising the isolated polynucleotide of aspect 14.

[00175] Aspect 16. A host cell transfected with the vector of aspect 15 or 11 or the polynucleotide of aspect14.

[00176] Aspect 17. A method of detecting the ubiquitin conjugating enzyme E2G2 (UBE2G2) in a sample, wherein the method comprises: contacting a sample with the polypeptide of any one of claims 1 to 6 or the fusion protein or conjugate of any one of aspects 7 to 13, the vector of aspect 15, or the polynucleotide of aspect 14; and determining binding of the polypeptide, fusion protein, or conjugate to the UBE2G2 in the sample to detect the presence of UBE2G2 in the sample.

[00177] Aspect 18. A method of modulating expression of a protein in a cell, comprising delivering the polypeptide of any one of aspects 1 to 6 or the fusion protein or conjugate of any one of aspects 7 to 13 to a eukaryotic cell expressing a protein under conditions such that expression of the protein is modulated.

[00178] Aspect 19. A method of modulating degradation of a protein in a cell, comprising delivering the polypeptide of any one of aspects 1 to 6 or the fusion protein or conjugate of any one of aspects 7 to 13, the vector of aspect 15, or the polynucleotide of aspect 14 to a eukaryotic cell comprising a protein under conditions such that degradation of the protein is modulated.

[00179] Aspect 20. The method of aspect 18 or 19, wherein the modulation is an increase

[00180] Aspect 21. The method of aspect 18 or 19, wherein the modulation is a decrease.

[00181] Aspect 22. The method of any one of aspects 19 to 21, wherein degradation is decreased by reducing ubiquitination of the protein

[00182] Aspect 23. A method of reducing degradation of an over-expressed protein in a cell, comprising delivering the polypeptide of any one of aspects 1 to 6 or the fusion protein or conjugate of any one of aspects 7 to 13, the vector of aspect 15, or the polynucleotide of aspect 14 to a eukaryotic cell expressing a protein under conditions such that degradation of the protein is reduced.

[00183] Aspect 24. The method of any one of aspects 18 to 23, wherein the protein is an enzyme, a cytokine, an antibody, a receptor, or a therapeutic polypeptide

[00184] Aspect 25. The method of any one of aspects 15 to 21, wherein the protein is an endoplasmic reticulum-associated degradation (ERAD) substrate targeted for ubiquitination by UBE2G2 and an ERAD E3 ubiquitin ligase that uses UBE2G2 as an E2 enzyme.

[00185] Aspect 26. A method of inducing cell death, comprising delivering the polypeptide of any one of aspects 1 to 6 or the fusion protein or conjugate of any one of aspects 7 to 13, the vector of aspect 15, or the polynucleotide of aspect 14 to a eukaryotic cell under conditions such that endoplasmic reticulum-associated degradation (ERAD) of proteins is inhibited and cell death is induced.

[00186] Aspect 27. The method of any one of aspects 18 to 26, wherein the cell is a cancer cell.

[00187] Aspect 28. A composition comprising the polypeptide of any one of aspects 1 to 6, the fusion protein or conjugate of any one of aspects 7 to 13, the vector of aspect 15, or the polynucleotide of aspect 14 for treatment of an endoplasmic reticulum-associated degradation (ERAD)-associated disorder.

[00188] Aspect 29. The composition of aspect 28, wherein the ERAD-associated disorder is cystic fibrosis or cancer.

[00189] Aspect 30. A method of treating an ERAD-associated disorder in a patient comprising administering the polypeptide of any one of aspects 1 to 6, the fusion protein or conjugate of any one of aspects 7 to 13, the vector of aspect 15, or the polynucleotide of aspect 14 to a patient having an endoplasmic reticulum-associated degradation (ERAD)-associated disorder.

[00190] Aspect 31. The method of aspect 30, wherein the ERAD-associated disorder is cystic fibrosis or cancer.

[00191] The terms“a” and“an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term“or” means“and/or”. The terms “comprising”,“having”,“including”, and“containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to”). The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes the degree of error associated with measurement of the particular quantity).

[00192] Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. In a list of alternatively useable species,“a combination thereof” means that the combination can include a combination of at least one element of the list with one or more like elements not named. Also,“at least one of” means that the list is inclusive of each element individually, as well as combinations of two or more elements of the list, and combinations of at least one element of the list with like elements not named.

[00193] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[00194] All references are incorporated by reference herein.

[00195] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.