EP4648786A1 - Targeted protein degradation - Google Patents

Abstract

Provided herein are compositions, systems, kits, and methods for tagging and/or degrading a target protein (or a protein in a protein complex with the target protein (PIPCWTP)) comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50. In certain embodiments, the degron is attached to a target protein (viral or bacterial protein) or a target protein binding or interacting moiety (TPBOIM), which allows cell-mediated degradation of the target protein and/or PIPCWTP. In some embodiments, a CRISPR system in employed to tag a target gene (e.g., that expresses a disease causing mis-folded protein) with a nucleic acid sequence encoding the degron.

Description

Attorney Docket No.: UM-41598.601 TARGETED PROTEIN DEGRADATION The present application claims priority to U.S. Provisional application serial number 63/479,796, filed January 13, 2023, which is herein incorporated by reference in its entirety. This invention was made with government support under 5F32GM136020, T32- GM145470, and R35GM128592 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD Provided herein are compositions, systems, kits, and methods for tagging and/or degrading a target protein (or a protein in a protein complex with the target protein (PIPCWTP)) comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50. In certain embodiments, the degron is attached to a target protein (viral or bacterial protein) or a target protein binding or interacting moiety (TPBOIM), which allows cell-mediated degradation of the target protein and/or PIPCWTP. In some embodiments, a CRISPR system in employed to tag a target gene (e.g., that expresses a disease causing mis- folded protein) with a nucleic acid sequence encoding the degron). BACKGROUND Protein degradation plays an essential role in regulating diverse cellular processes including cellular signaling, metabolic adaptation, and cell cycle regulation. The ubiquitin proteasome system is the primary cellular degradation route, accounting for over 80% of protein degradation (Collins and Goldberg, 2017). Since its discovery, the ubiquitin proteasome system was posited to be a valuable therapeutic target but has only recently been leveraged for degradation of physiologically important proteins previously thought to be “undruggable.” The first class of intentionally designed targeted protein degradation molecules were called protein targeting chimeras (PROTACs) and over the last two decades these compounds have been continuously refined to the point of entering into clinical trials (Békés et al., 2022; Sakamoto et al., 2001). These first-in-class molecules induce ubiquitination and proteasomal degradation of pathophysiologically relevant proteins, in contrast to traditional methodologies which commonly inhibit an enzymatic “active” site. Unlike these traditional inhibitor technologies, PROTACS can bind their targets via a wide range of surfaces and subsequently induce their degradation to prevent activity. Since the Attorney Docket No.: UM-41598.601 advent of PROTACS, many different technologies have been designed to drive degradation of cytosolic proteins through the ubiquitin proteasome system (MADTACs, PHOTACs, ArMED, dTAG, deGradFP, Trim-Away, etc.) (Alabi and Crews, 2021; Caussinus et al., 2012; Clift et al., 2017; Ibrahim et al., 2020; Nabet et al., 2018; Reynders et al., 2020). A fundamental limitation with these technologies is that their activity is restricted to cytosolic interfaces; only soluble cytosolic or membrane proteins with unique cytosolic domains can be targeted. This prevents >50% of current therapeutic targets that are secretory or membrane proteins from being PROTAC targets (Liu et al., 2022; Santos et al., 2017). Lysosomal targeting chimeras (LYTACs) are a recently described technology designed to induce degradation of proteins exposed on the cell surface or secreted into the extracellular space (Banik et al., 2020). While this has shifted targeted protein degradation away from just the cytoplasm, with LYTACs targets are limited to proteins accessible at the cellular surface. Importantly, this leaves luminal proteins, and most integral membrane proteins without unique cytoplasmic domains, from the endoplasmic reticulum, secretory pathway, and endo/lysosomal system unavailable for targeted degradation. Targeting proteins for degradation within organelles requires a detailed understanding of local organellar protein quality control systems. The ER represents the organelle with the largest flux of proteins, with over 40% of proteins translocated into the endoplasmic reticulum prior to trafficking to other organelles or secretion from the cell. Both soluble luminal proteins and integral membrane proteins are folded in the ER and undergo quality control before being released into the secretory pathway. At the ER, the primary protein quality control pathways are, collectively, referred to as endoplasmic reticulum associated degradation (ERAD) (Carvalho et al., 2006; Denic et al., 2006; Foresti et al., 2014; Huyer et al., 2004; Khmelinskii et al., 2014; Vashist and Ng, 2004). Proteins not passing quality control are recognized by the Hrd1-centric ERAD complex and retrotranslocated from the ER lumen to the cytoplasm for ubiquitin-mediated proteasomal degradation. In Saccharomyces cerevisiae, the Hrd1 complex comprises 5 proteins: Hrd1, Hrd3, Usa1, Der1, and Yos9. In the cytoplasm, this pathway requires a highly conserved AAA-ATPase (Cdc48), it’s cofactors (Ufd1 and Npl4), and the ubiquitination proteasome system to degrade ERAD substrates (Bays et al., 2001; Jarosch et al., 2002; Rabinovich et al., 2002; Ye et al., 2001). Soluble, luminal ERAD substrates are retrotranslocated by hetero-oligomers of Hrd1/Der1 (Mehnert et al., 2014; Pisa and Rapoport, 2022; Wu et al., 2020), or in some cases, homo-oligomers of Hrd1 (Baldridge and Rapoport, 2016; Carvalho et al., 2010). Hrd1 is sufficient for all aspects of ERAD but without the other complex components loses the specificity that normally Attorney Docket No.: UM-41598.601 defines the system (Denic et al., 2006). This system has broad specificity and seems to be able to distinguish folded and unfolded proteins. Perhaps most surprisingly, despite nearly 3 decades of study, degrons (neither sequences nor features) that allow degradation through the Hrd1-centric ERAD pathway remain a complete mystery (Needham et al., 2019). SUMMARY Provided herein are compositions, systems, kits, and methods for tagging and/or degrading a target protein (or a protein in a protein complex with the target protein (PIPCWTP)) comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50. In certain embodiments, the degron is attached to a target protein (viral or bacterial protein) or a target protein binding or interacting moiety (TPBOIM), which allows cell-mediated degradation of the target protein and/or PIPCWTP. In some embodiments, a CRISPR system in employed to tag a target gene (e.g., that expresses a disease causing mis- folded protein) with a nucleic acid sequence encoding the degron (e.g., such that the expressed protein is degraded once expressed in a cell, allowing the wild-type allele to dominate expression). In some embodiments, provided herein are methods of degrading a target protein, and/or a protein in a protein complex with the target protein (PIPCWTP), in a cell comprising: contacting the cell, in vitro or in a subject, with a composition comprising a degradation construct, or a nucleic acid sequencing encoding the degradation construct if it is encodable by nucleic acids, wherein the degradation construct comprises: a) a target protein binding or interacting moiety (TPBOIM), b) a degron comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50, and c) optionally one or more linkers and/or one or more N-terminal amino acids; wherein the degron is optionally attached to the TPBOIM directly or via the one or more linkers, and wherein the target protein and/or the PIPCWTP is degraded in the cell. In certain embodiments, provided herein are compositions, systems, and kits comprising: a composition comprising a degradation construct, or a nucleic acid sequencing encoding the degradation construct if it is encodable by nucleic acids, wherein the degradation construct comprises: a) a target protein binding or interacting moiety (TPBOIM), b) a degron comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50, and c) optionally one or more linkers and/or one or more N-terminal amino acids, wherein the degron is attached to the TPBOIM directly or via the one or more linkers. In certain embodiments, the compositions, systems, and kits further comprising: d) an aqueous Attorney Docket No.: UM-41598.601 buffer. In particular embodiments, the compositions further comprises a cell. In other embodiments, the systems or kits further comprise a device for injecting the composition, or a capsule, wherein the composition is inside the capsule. In some embodiments, the degradation construct further comprises an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence (e.g., HDEL). In other embodiments, the TPBOIM is selected from: a target protein specific antibody or target protein binding portion thereof, a small molecule binding protein that binds the target protein; an aptamer that binds the target protein, or at least a portion of a protein that naturally interacts with, or binds to, the target protein in the cell. In certain embodiments, the target protein specific antibody or target protein binding portion thereof is selected from: a monoclonal antibody, a nanobody, a fab fragment, a f(ab)2, scFv, and dAb. In certain embodiments, the at least one copy of the amino acid sequence is at least two, three, or four copies. In further embodiments, the contacting results in a reduction in the level of just the target protein, or just the, or both the target protein and the PIPCWTP, in the cell. In further embodiments, the contacting results in: i) degradation of the target protein by the endoplasmic reticulum associated protein degradation (ERAD) pathway in the cell, and/or ii) degradation in a lysosome of the cell. In some embodiments, the contacting results in degradation of the target protein at a rate greater than the wild-type rate for the target protein in the cell and/or wherein the target protein is located in the lumen or membrane of the endoplasmic reticulum of the cell. In further embodiments, the contacting is via administration of the composition to the subject, and optionally wherein the administration is performed orally, intravenously, intranasally, optically, vaginally, or rectally. In particular embodiments, the target protein is associated with a proteinopathy disease or condition in the subject, or wherein the target protein is a viral or bacterial protein and the subject is, or is not, infected with a virus or bacteria (e.g., treatment is prophylactic or therapeutic). In additional embodiments, the target proteinopathy disease or condition, and/or the target protein, is one or more shown in Table 4. In additional embodiments, the one or more N-terminal amino acids are present, and optionally comprise a serine and an alanine. In further embodiments, the amino acid sequence comprises, consists essentially of, or consists of: IHPYW (SEQ ID NO:1). In other embodiments, the methods further comprise contacting the cell with a dimerization or other agent that causes the target protein and the PIPCWTP to form a complex. In some embodiments, the amino acid sequence comprises, Attorney Docket No.: UM-41598.601 consists essentially of, or consists of: IWRGR (SEQ ID NO:2), IYTLE (SEQ ID NO:3), LHQLY (SEQ ID NO:4), LWGNH (SEQ ID NO:5), or LYTMR (SEQ ID NO:6). In additional embodiments, the cell is a mammalian cell, and optionally a human cell, and wherein the subject is a mammal, and optionally a human. In some embodiments, the cell is a type selected from: red blood cells, white blood cells, platelets, bone cells, brain cells, egg cells, sperm cells, muscle cells, fat cells, and nerve cells. In additional embodiments, the compositions comprise the degradation construct. In some embodiments, the compositions comprise the nucleic acid sequencing encoding the degradation construct (e.g., as part of an expression vector, such as a plasmid or adeno-associated virus), and the encoded degradation construct comprises amino acids. In certain embodiments, the contacting is performed in vitro. In other embodiments, the target protein is selected from: an ER luminal protein, an integral membrane protein, a secretory pathway protein, and an endo/lysosomal protein. In other embodiments, the TPBOIM comprises a protein, and wherein the degron is attached to the N-terminus of the TPBOIM. In some embodiments, provided herein are compositions comprising: a degron construct, or a nucleic acid sequence encoding the degron construct, wherein the degron construct comprises: a) a degron comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50, and b) a target protein, wherein the target protein is not naturally associated with the degron. In further embodiments, the degron construct further comprises an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence. In particular embodiments, the degron construct further comprises one or more linkers, and wherein the degron is attached to the target protein via the one or more linkers or wherein the degron is directly conjugated to the target protein. In particular embodiments, the composition comprises the degron construct. In certain embodiments, the composition comprises a nucleic acid sequence encoding the degron construct. In other embodiments, the nucleic acid sequence comprises RNA or DNA. In additional embodiments, the degron construct further comprises a linker and/or one or more N-terminal amino acids. In other embodiments, the at least one copy of the amino acid sequence is at least two, three, or four copies. In some embodiments, the target protein is associated with a proteinopathy disease or condition in the subject. In other embodiments, the target protein, is one or more shown in Table 4. In some embodiments, the amino acid sequence comprises IHPYW (SEQ ID NO:1). Attorney Docket No.: UM-41598.601 In further embodiments, the amino acid sequence consists essentially of, or consists of, IHPYW (SEQ ID NO:1). In additional embodiments the amino acid sequence comprises, consists essentially of, or consists of: IWRGR (SEQ ID NO:2), IYTLE (SEQ ID NO:3), LHQLY (SEQ ID NO:4), LWGNH (SEQ ID NO:5), or LYTMR (SEQ ID NO:6). In particular embodiments, the compositions further comprise a cell. In other embodiments, the cell is a mammalian cell, and optionally a human cell. In certain embodiments, the cell is a type selected from: red blood cells, white blood cells, platelets, bone cells, brain cells, egg cells, sperm cells, muscle cells, fat cells, and nerve cells. In certain embodiments, the compositions comprise the degron construct. In other embodiments, the compositions comprise the nucleic acid sequencing encoding the degron construct. In other embodiments, the target protein is selected from: an ER luminal protein, an integral membrane protein, a secretory pathway protein, and an endo/lysosomal protein. In some embodiments, provided herein are systems for tagging a target nucleic acid sequence with a degron comprising: i) a synthetic guide ribonucleic acid (sgRNA), wherein the sgRNA is at least partially complementary to a target nucleotide sequence in or near a target gene; ii) a repair template nucleotide sequence encoding at least one copy of a degron with an amino acid sequence selected from any of SEQ ID NOs:1-50; and iii) at least one of the following: A) a clustered regularly interspaced short palindromic repeat (CRISPR)- associated nuclease, and/or B) first nucleotide sequence, or complement thereof, encoding the CRISPR-associated nuclease. In other embodiments, the repair template further comprises a nucleic acid sequence encoding an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence. In some embodiments, the systems further comprise: i) a nucleic acid vector, wherein the at least two, or all three, of the sgRNA, the repair template nucleotide sequence, and the first nucleotide sequence if present, are present on the nucleic acid vector; or ii) first, second, and third nucleic acid vectors, wherein the sgRNA is present on the first vector, the repair template nucleotide sequence is present on the second vector, and the first nucleotide sequence is present on the nucleic acid vector. In particular embodiments, the at least one copy of the amino acid sequence is at least two, three, or four copies. In other embodiments, the target gene is associated with a proteinopathy disease or condition. In further embodiments, the target proteinopathy disease or condition, and/or the target protein, is one or more shown in Table 4. In other embodiments, the amino acid sequence comprises, consists essentially of, or consists of: IHPYW (SEQ ID NO:1). In other embodiments, the amino acid sequence comprises, consists essentially of, or consists of: IWRGR (SEQ ID NO:2), IYTLE (SEQ ID NO:3), Attorney Docket No.: UM-41598.601 LHQLY (SEQ ID NO:4), LWGNH (SEQ ID NO:5), or LYTMR (SEQ ID NO:6). In additional embodiments, the systems further comprise a cell. In further embodiments, the cell is a mammalian cell, and optionally a human cell. In particular embodiments, the cell is a type selected from: red blood cells, white blood cells, platelets, bone cells, brain cells, egg cells, sperm cells, muscle cells, fat cells, and nerve cells. In additional embodiments, the target protein is selected from: an ER luminal protein, an integral membrane protein, a secretory pathway protein, and an endo/lysosomal protein. In further embodiments, provided are methods for tagging a target gene in a cell in vitro or a cell in a subject comprising: introducing into a cell or subject the systems described above. In certain embodiments, provided herein are methods of tagging a target gene in a cell with a nucleotide sequence encoding a degron comprising: a) introducing into a cell: i) a first nucleotide sequence encoding a synthetic guide ribonucleic acid (sgRNA), wherein the sgRNA is at least partially complementary to a target nucleotide sequence in or near a target gene; ii) a second nucleotide sequence encoding a clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease; iii) a repair template comprising a nucleotide sequence encoding at least one copy of a degron with an amino acid sequence selected from any of SEQ ID NOs:1-50; and b) expressing the sgRNA and Cas9 nuclease in the presence of the repair template in the cell, thereby tagging the target gene with the nucleotide sequence encoding the degron. In other embodiments, the CRISPR-associated nuclease comprises Cas9. In other embodiments, the cell is part of a subject, optionally wherein the subject is a human. In additional embodiments, the repair template further comprises a nucleic acid sequence encoding an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence. In certain embodiments, the CRISPR tagging systems and methods employ methods described in U.S. Pat. Publication US20170009242, which is herein incorporated by reference. DESCRIPTION OF THE FIGURES Figure 1. Identification of endoplasmic reticulum localized degrons. A) Cartoon depicting the ER-tFT and KHN-tFT constructs, which contain an ER-targeting signal sequence (SS), mCherry protein (pink), GFP (green), and the HDEL ER-retention sequence. KHN-tFT functions as a positive control for a quickly degraded ERAD substrate. B) Yeast strains expressing the constructs described in (A) were treated with cycloheximide (CHX) for 0, 30, 60, or 90 minutes, harvested, and protein levels were assessed by western blot against Attorney Docket No.: UM-41598.601 GFP and normalized against total protein in gel using stain-free technology (Loading). C) Flow cytometry of yeast strains expressing the constructs in (A) treated with cycloheximide for 2 hours. The mCherry/GFP fluorescence intensity ratio of each cell was calculated and plotted. D) Quantification of the mean mCherry/GFP ratio of four biological replicates as in (C). E) Overview scheme of pentapeptide library generation and isolating of unstable variants by FACS. A DNA fragment containing the pentapeptide-ER-tFT library was electroporated with digested ER-tFT plasmid. The resulting yeast library contains a mixture of more less stable variants, which can be separated from one another by FACS, with more stable variants having increased mCherry fluorescence intensity compared to unstable variants. F) Heatmap of overall amino acid enrichments at each position within the pentapeptide library, displayed relative to either the frequency based on codon usage, or relative to the input library. G) Flow cytometry of yeast strains expressing either ER-tFT, KHN-tFT, unstable selected pentapeptide-ER-tFT sequences or randomly selected pentapeptide-ER-tFT sequences. The cells were analyzed after treatment with cycloheximide for 2 hours. H) Quantification of 3 biological replicates conducted as in (G). I) As in (C) but with yeast strains expressing ER-tFT, KHN-tFT, and IHPYW (1X), 2x repeat of IHPYW (2X), or 4x repeat of IHPYW at the N-terminus of ER-tFT. J) Quantification of 3 biological replicates of (J). Figure 2. DegV1 is an ERAD-dependent degron degraded by the cytosolic proteasome. (A) The degradation of ER targeted anti-GFP nanobodies (SS-NbGFP-Flag- HDEL) either with, or without, DegV1 were monitored following addition of cycloheximide (CHX). Loading controls were visualized in gel by stain-free technology. (B) The degradation of a nanobody with DegV1 encoded replacing the CDR3 region was analyzed as in (A). (C) The degradation of a nanobody with DegV1 located either directly preceding the C-terminal ER-retention signal (HDEL) or directly at the C-terminus of the nanobody was analyzed as in (A). (D) The degradation of ER-targeted GFP proteins (GFP (top panel), DegV1GFP (middle panel), or Control sequence GFP (ConV1-GFP)) were analyzed by flow cytometry following either ethanol (EtOH) or cycloheximide (CHX) treatment for 2 hours. Where indicated, cells were pretreated with bortezomib (Btz) for 2 hours. (E) As in (G), but in a hrd1Δpdr5Δ strain. (F) Degradation of DegV1-containing nanobody proteins were followed in the absence (top set) or presence of bortezomib (middle set) or in a hrd1Δpdr5Δ strain (bottom set) using a cycloheximide chase. (G) Quantification of at least three independent experiments with biological replicates, including results in (J); error bars represent the standard deviation. (H) The degradation of an endogenous secretory protein Attorney Docket No.: UM-41598.601 with a C-terminal flag-HDEL (Suc2-Flag-HDEL) containing either DegV1 or a control sequence was followed as in (A). (I) The degradation of a single membrane spanning ER resident protein (Big1) either with DegV1 or a control sequence was followed as in (A). (J) The degradation of polytopic integral membrane ER resident protein (Elo1) either with a DegV1 or a control sequence was followed as in (A). (K) The degradation of Big1 with DegV1 or a control sequence was followed after treatment with either ethanol or bortezomib in a pdr5Δ strain. (L) The degradation of Elo1 with DegV1 or a control sequence was followed after treatment with either ethanol or bortezomib in a pdr5Δ strain. All panels in this figure are representative of at least three independent experiments with biological replicates. Figure 3. ERAD allows “piggybacking” degradation of interacting proteins. (A) Suc2-ALFA was coexpressed with nanobodies to differing epitope tags (NbGFP, NbALFA, NbVHH05) with the nanobodies either containing or lacking DegV1. Degradation of both Suc2 and nanobody were followed by cycloheximide chase and immunoblotting. Proteins were detected using anti-V5 and anti-Flag antibodies and the protein loading was observed with stain-free dye (Loading). (B) Quantification of three independent experiments with biological replicates, including results in (A). (C) As in (A), but with Suc2-VHH05. (D) Quantification of three independent experiments with biological replicates, including results in (D). (E) ALFA-Elo1 was coexpressed with nanobodies to differing epitope tags (NbGFP, NbALFA, NbVHH05) with the nanobodies either containing or lacking DegV1. Degradation of both Elo1 and nanobody were followed by cycloheximide chase and immunoblotting. (F) Quantification of three independent experiments with biological replicates, including results in (E). (G) As in (E), but with VHH05-Elo1. (H) Quantification of three independent experiments with biological replicates, including results in (G). (I) Steady state levels of Suc2-ALFA co-expressed with the indicated nanobodies (+/- DegV). (J) Quantification of three independent experiments with biological replicates, including results in (I). (K) As in (I), but with Suc2-VHH05. (L) Quantification of three independent experiments with biological replicates, including results in (K). (M) As in (I), but with ALFA-Elo1. (N) Quantification of three independent experiments with biological replicates, including results in (M). (O) As in (I), but with VHH05-Elo1. (P) Quantification of three independent experiments with biological replicates, including results in (O). Figure 4. Hypothetical non-limiting Model for modes of DegV1 mediated degradation Summary model demonstrating the different modes of DegV1 degradation through ERAD. Attorney Docket No.: UM-41598.601 Figure 5. (A) We targeted the tFT to the ER with two different signal peptides, either that of mating factor alpha or of Ost1. The GFP and mCherry fluorescence were plotted for each cell in the left panel. The mCherry/GFP fluorescence intensity ratio of each cell was calculated and plotted in the right panel. Based on the superior brightness of cells expressing the mating factor alpha signal sequence, we selected this signal peptide for further experimentation. (B) Flow cytometry of yeast strains expressing an ER-tFT and ERAD- substrate KHN-tFT treated with cycloheximide (CHX) for 2 hours. (C) Schematic of the PCR-mediated library generation (left) using degenerate primers (upper right) and homologous recombination in yeast (lower right). (D) Heatmap of input library amino acid enrichments at each position displayed relative to codon frequency. (E) Gating strategy for analyzing single cells by flow cytometry. (F) Sorting bins defined relative to ER-tFT and KHN-tFT. (G) Comparison of mCherry/GFP ratios for alternate signal sequences on ER-tFT and IHPYW-tFT. Figure 6. (A) Degradation of ER-targeted GFP with an ER retention signal (HDEL) with, or without DegV1 was followed in a hrd1Δ strain complemented with either an empty vector or with Hrd1 on a centromeric plasmid. Using a cycloheximide chase and immunoblotting, we found in the absence of Hrd1, we observe transport of the proteins to the vacuole, where free GFP accumulates. The asterisk indicates the vacuolar localized GFP fragment. (B) As in (A), except with CPY*-GFP-HDEL. (C) Degradation of an anti-GFP nanobody protein was followed by cycloheximide chase (top set) in the presence of bortezomib (middle set) or in a hrd1Δpdr5Δ strain (bottom set) using a cycloheximide chase. (D) The degradation of ER-targeted GFP proteins (GFP (top panel), DegV1GFP (middle panel), or Control sequence GFP (ConV1-GFP)) were analyzed by flow cytometry following either ethanol (EtOH) or with bortezomib (Btz) for 2 hours. (E) As in (D), but in a hrd1Δpdr5Δ strain. Figure 7A shows the amino acid sequence (SEQ ID NO:51) and nucleic acid sequence (SEQ ID NO:52) for construct SS_DegV1_NbALFA_3xFLAG_HDEL. Figure 7B shows the amino acid sequence (SEQ ID NO:53) and nucleic acid sequence (SEQ ID NO:54) for construct SS_ConV1_NbALFA_3xFLAG_HDEL. Figure 7C shows the amino acid sequence (SEQ ID NO:55) and nucleic acid sequence (SEQ ID NO:56) for construct SS_DegV1_NbGFP_3xFLAG_HDEL. Figure 7D shows the amino acid sequence (SEQ ID NO:57) and nucleic acid sequence (SEQ ID NO:58) for construct SS_ConV1_NbGFP_3xFLAG_HDEL. Figure 7E shows the amino acid sequence (SEQ ID NO:59) and nucleic acid sequence (SEQ ID NO:60) for construct Attorney Docket No.: UM-41598.601 SS_DegV1_NbVHH05_3xFLAG _HDEL. Figure 7F shows the amino acid sequence (SEQ ID NO:61) and nucleic acid sequence (SEQ ID NO:62) for construct SS_ConV1_NbVHH05_3xFLAG _HDEL. Figure 8A shows the amino acid sequence (SEQ ID NO:63) and nucleic acid sequence (SEQ ID NO:64) for construct SS(Ost1)_DegV1_NbALFA_3xFLAG_HDEL. Figure 8B shows the amino acid sequence (SEQ ID NO:65) and nucleic acid sequence (SEQ ID NO:66) for construct SS(Ost1)_ConV1_NbALFA_3xFLAG_HDEL. Figure 8C shows the amino acid sequence (SEQ ID NO:67) and nucleic acid sequence (SEQ ID NO:68) for construct SS(Ost1)_DegV1_NbGFP_3xFLAG_HDEL. Figure 8D shows the amino acid sequence (SEQ ID NO:69) and nucleic acid sequence (SEQ ID NO:70) for construct SS(Ost1)_ConV1_NbGFP_3xFLAG_HDEL. Figure 8E shows the amino acid sequence (SEQ ID NO:71) and nucleic acid sequence (SEQ ID NO:72) for construct SS(Ost1)_DegV1_NbVHH05_3xFLAG _HDEL. Figure 8F shows the amino acid sequence (SEQ ID NO:73) and nucleic acid sequence (SEQ ID NO:74) for construct SS(Ost1)_ConV1_NbVHH05_3xFLAG _HDEL. Figure 9. DegV1 functions as an ER degron for soluble proteins in mammalian cells. (A) ER-targeted mNeonGreen (ER-HA-mNG) was expressed alone (-), or with a ConV1, or a DegV1 in U2OS cells by transient transfection. The degradation of ER-mNG was followed by immunoblotting with anti-HA antibody after treatment with 50μM emetine. β-actin was used as a loading control. (B) Anti-HA band intensities from (A) were quantified and normalized to the corresponding β-actin level. (C) As in (A) but after treatment with 50nM bortezomib (Btz) for the indicated times. (D) Quantification of (C) normalized to the control protein (ER-HA-mNG). (E) As in (A) but after treatment with 1µM CB5083, a p97 inhibitor, for the indicated times. (F) Quantification of (E) normalized to the control protein (ER-HA- mNG). (G) ER-HA-mNG with either ConV1 (left panel) or DegV1 (right panel) were expressed in U2OS pretreated with either 50nM bortezomib or 1µM CB5083 for 16 hours prior to an emetine chase. (H) The degradation of ER-HA-mNG with either ConV1 or DegV1 was followed in HEK293T cells or HRD1-/- cells using an emetine chase. (I) Quantification of (H). All panels in this figure are representative of at least three independent biological replicates and the quantification is presented as the mean +/- standard deviation. Figure 10. DegV1 functions as an ER degron for integral membrane proteins in mammalian cells. (A) Epitope tag used on integral membrane proteins within this figure. (B) Schematic of proteins from this figure. The signal sequence from BiP was appended to the N- terminus of proteins, followed by either DegV1 or ConV1 and the 3xV5 tag. Proteins were Attorney Docket No.: UM-41598.601 transduced by lentiviral and expressed from a tetracyline-inducible promoter. (C) Progesterone membrane receptor (PGRMC1) with either DegV1 or ConV1 was expressed in U2OS cells. Degradation was followed by immunoblotting after bortezomib treatment and washout (3 hours and 5 hours) and an emetine chase. (D) Quantification of three independent biological replicates from (C). (E) As in (C), but with K562 cells. PGRMC1 expression was induced using 0.1 ug/ml Doxycycline and pretreated with 10nM bortezomib before washout. (F) Quantification of three independent biological replicates from (E). (G) As in (C), but with HEK293 cells and only 1 hour bortezomib pretreatment before washout. (H) Quantification of three independent biological replicates from (G). (I) As in (C), but with the Sigma 1 Opioid Receptor (S1R). (J) Quantification of three independent biological replicates from (J). (K) As in (C), but with but with overnight bortezomib treatment and no washout, demonstrating the proteasome is required for degradation of the target containing DegV1. (L) As in (K), but with HEK293 cells. (M) As in (K), but with K562 cells. (N) Quantification of three independent biological replicates from (M). (O) As in (K), but with Sigma 1 opioid receptor (S1R). (P) Quantification of three independent biological replicates from (O). Target protein expression was induced using 1ug/ml Doxycycline for 24 hours prior to other treatments, unless otherwise noted. For these experiments, DegV1 proteins were turned over rapidly and were difficult to detect without pre-treatment of cells with bortezomib and subsequent washout. Emetine was used at 50µM and Bortezomib was used at 50nM, unless otherwise noted. *<.05, **<.01, ***<.001 Figure 11. DegV1 functions as a cytosolic degron, independent of Hrd1. (A) Degradation of a cytosolically-localized anti-GFP nanobody (cytosolic NbGFP) or with DegV1 (cytosolic DegV1-NbGFP) was followed in pdr5Δ or hrd1Δpdr5Δ strains with, or without bortezomib (Btz) using a cycloheximide (CHX) chase. Note that Hrd1 is not required for the degradation of cytosolic DegV1-NbGFP. (B) Quantification of three independent biological replicates from (A). The error bars represent the standard deviation. Figure 12. Viral genome structures and protein targets. Left) Dengue viral genome and the single open reading frame topology. Small black arrows indicate signal peptidase cleavage sites and the small yellow arrows indicate viral protease NS3 cleavage sites. Degrons herein, for example, can be engineered at signal peptidase sites. Nanobodies currently available for targeting individual viral currently available are indicated with solid lines, and nanobodies for development are indicated with dashed lines. Right) SARS-CoV-2 genome and viral integral membrane proteins. Gray-colored genes encode cytosolic viral proteins that are not generally targetable. Attorney Docket No.: UM-41598.601 DETAILED DESCRIPTION Provided herein are compositions, systems, kits, and methods for tagging and/or degrading a target protein (or a protein in a protein complex with the target protein (PIPCWTP)) comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50. In certain embodiments, the degron is attached to a target protein (viral or bacterial protein) or a target protein binding or interacting moiety (TPBOIM), which allows cell-mediated degradation of the target protein and/or PIPCWTP. In some embodiments, a CRISPR system in employed to tag a target gene (e.g., that expresses a disease causing mis- folded protein) with a nucleic acid sequence encoding the degron (e.g., such that the expressed protein is degraded once expressed in a cell, allowing the wild-type allele to dominate expression). Targeted protein degradation utilizes proteins and small molecules to facilitate degradation of diverse physiological and pathophysiological targets. Current protein degradation technologies are limited in scope to degrade cytosolic or cell surface-accessible targets. Work conducted during development of embodiments described herein identified degrons of the ERAD pathway that allows one to expand the types of proteins that can be targeted for degradation. We have designed, in certain embodiments, a single component system, composed of degron-tagged nanobodies targeted to endogenous ER proteins. It is believed that this is the first method of targeted protein degradation of both soluble, luminal, and integral membrane ER proteins. With this system, we also discovered new biology of the ERAD system, finding that ERAD can “piggyback” degradation of otherwise stable ER proteins, merely through interactions with degron-containing proteins. This provides a framework to target proteins for degradation from the previously unreachable ER lumen and enables novel therapeutic approaches that exploit the highly conserved ERAD system. In medicine, proteinopathy or proteopathy, protein conformational disorder, or protein misfolding disease refers to a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Often the proteins fail to fold into their normal configuration and in this misfolded state, the proteins can become toxic in some way (a toxic gain-of-function) or they can lose their normal function. The proteinopathies include such diseases listed in Table 4 below along with their associated proteins (all of which are target proteins herein). In certain embodiments, the degrons herein (and associated methods and compositions) is important for targeted protein degradation and drug discovery as it facilitates degradation of proteins unreachable through conventional methods. The scope of this Attorney Docket No.: UM-41598.601 technology generally extends broadly because the ER is a hub for critical processes in human physiology. The successful implementation of embodiments of this degron technology would have a broad impact on human health through pathologies related to the ER, including, for example, diabetes (insulin folding and maturation in the ER), cardiac irregularities (ER regulated calcium equilibrium), obesity and cancer (lipid biosynthesis and metabolism at the ER), cancer immunology (regulation of critical secretory and checkpoint factors), autoimmune diseases (related to host immune proteins ER biogenesis), viral infections, bacterial infections, and more (see, e.g., Table 4 for exemplary target proteins). The versatile potential of embodiments of this degron technology provides framework to address a spectrum of human pathologies associated with ER-related processes by simply identifying a target binder to a protein of interest (e.g., those in table 4 and those known in the art). In cancer, in many cases, cancer cells express cell-surface proteins to allow escape from immune cells. These proteins are currently inaccessible to conventional therapies that require either active sites, cytoplasmic proteins, or are only accessible once exposed outside of the cell (by secretion / or cell surface exposure). Embodiments of the degron methods and compositions herein allows targeted degradation of soluble luminal proteins and integral membrane proteins from the extracellular/luminal side. In certain embodiments for treating cancer, the focus can be on three very different sets of proteins: first, proteins secreted by the cancer cells to allow immune evasion (or promote progression and metastasis); second, integral membrane proteins on cancer cells that allow immune evasion (or promote growth, progression, and metastasis); and third, proteins within the host immune cells themselves that suppress recognition of cancerous cells. With secreted proteins, as an example for particular embodiments, one can target TGF-β (see, e.g., Massagué and Sheppard 2023) for degradation. TGF-β plays a bevy of roles, all centered around allowing cancer cells to evade the immune system. Reducing, or eliminating secretion of, TGF-β by developing an available anti-TGF-β nanobody (Henry, Hussack et al.2016) and targeting with the degron composition and methods herein could open a new avenue of therapies. In some embodiments, one can target additional secretory proteins including VEGF (Sangro, Sarobe et al.2021), IL-10 (Itakura, Huang et al.2011), and others. On the cell surface, many solid tumors express checkpoint proteins to allow immune evasion (including the integral membrane proteins PD-L1 (Iwai, Ishida et al.2002) and CD47 (Ingram, Blomberg et al.2017)). By targeting these proteins with the degron methods and compositions herein before surface expression, could prevent surface exposure of these Attorney Docket No.: UM-41598.601 proteins altogether. A major advantage of this strategy is that the degron methods and compositions herein expressed within cancer cells are not subject to competition from other cells with the same protein exposed (as when inhibitory nanobodies are used in the bloodstream). In addition, by targeting proteins within their folding environment, a wider range of nanobody reagents (or other targeting moieties) will be effective compared to currently available “neutralizing” affinity-based reagents that are only effective outside of cells (Farajpour, Rahbarizadeh et al.2014, Ingram, Blomberg et al.2017, Zhang, Wei et al. 2017, Khodabakhsh, Norouzian et al.2018, Wan, Liu et al.2018). In some embodiments, one can target other integral membrane proteins to prevent cancer progression (including CTLA-4 (Wan, Liu et al.2018), HLA-G (Jiang, Yu et al.2023), ENPP1 (Ritchie, Carozza and Li 2022, Solomon, Bracken et al.2024), and others). In certain embodiments, viral and bacterial proteins are targeted by the degron methods and compositions herein. In some embodiments, to determine the optimal host and viral protein targets for degradation, one can directly attach degrons to both host and viral proteins. In certain embodiments, three exemplary viral families can be targeted: flavivirus and coronavirus represent enveloped, positive single-stranded RNA viruses, while polyomavirus represents a non-enveloped, double-stranded DNA virus. From a public health perspective, flaviviruses (including DENV and Zika virus) and coronaviruses (such as SARS- CoV-2) pose significant health threats and polyomaviruses are known to cause cancer, as well as kidney and neurological diseases (Boothpur and Brennan 2010, Ambalathingal, Francis et al.2017). There are a number of, for example, critical host ER factors and viral membrane proteins for these three virus families essential for supporting virus infection. Notably, in the case of the host ER factors, genetic depletion (via either knockdown or knockout methods) did not trigger ER stress or impair cellular integrity. Thus, degrade these host ER proteins will likely maintain overall cell health, which could otherwise confound results. Under many circumstances, surface residues on viral proteins undergo more rapid evolution than internally-oriented proteins allowing “escape” from preexisting immunity (Dolan, Whitfield and Andino 2018). By targeting these proteins within their folding environment with the degron compositions and methods herein, one could expect a different range of nanobody reagents (or other targeting moieties) that will be effective compared to currently available “neutralizing” affinity-based reagents that are only effective outside of cells. In contrast to these “neutralizing” antibodies or nanobodies that would be delivered outside of cells, in certain embodiments, with the degron constructs herein, nanobodies (or other targeting moieties) that recognize unfolded or immature spike, envelope, or even non- Attorney Docket No.: UM-41598.601 structural proteins could also be effective. These additional targets would greatly expand the range of targetable epitopes even within the same proteins. For example, with SARS-CoV-2, viral fitness is impaired by spike protein mutations that would be buried within the folded protein, whereas solvent-exposed residues undergo rapid evolution to help escape preexisting immunities (Starr, Greaney et al.2020, Starr, Greaney et al.2022). TABLE 4 Proteinopathy Major aggrega^ng protein Attorney Docket No.: UM-41598.601 Pelizaeus-Merzbacher disease proteolipid protein (PLP) Attorney Docket No.: UM-41598.601 Cutaneous lichen amyloidosis^[69] Kera^ns L- Attorney Docket No.: UM-41598.601 Flavivirus proteins: M protein, E protein, NS1, NS2A, NS2B, NS4 i l i f ^ , In certain embodiments, the amino acid sequence of the degrons herein comprises, consists essentially of, or consists of, IHPYW (SEQ ID NO:1), IWRGR (SEQ ID NO:2), IYTLE (SEQ ID NO:3), LHQLY (SEQ ID NO:4), LWGNH (SEQ ID NO:5), or LYTMR (SEQ ID NO:6), or those shown in Table 5 below (including SEQ ID NOs:7-50). Each of these degrons may be constructed with longer, shorter, or mutated versions of the sequences shown in SEQ ID NOS: 1-50. For example, one could change one, two, three amino acids in these sequences. For example, one could make conservative changes to a particular amino acid sequence. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. In certain embodiments, provided herein are peptides that have substantial identity to at least a portion of the amino acid sequences shown in SEQ ID NOs:1-50. In certain embodiments, the amino acid sequences of the degrons in SEQ ID NOs: 1- 50 (in table 5 below) or such a peptide with one, two, or three (or more) N-terminal or C- terminal additions, subtractions or mutations therein. One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acid sequences shown in Table 5. Attorney Docket No.: UM-41598.601 TABLE 5 Amino acid sequences (where “x” is any amino acid) SEQ ID NO: IHPYW 1 Attorney Docket No.: UM-41598.601 LGR13xx R13= I or Y ³⁹ LGxR₁₃x R₁₃= I or Y ⁴⁰ EXAMPLES EXAMPLE 1 TARGETED PROTEIN DEGRADATION FROM THE ENDOPLASMIC RETICULUM LUMEN This Example has identified the first bona fide luminal degron of the Hrd1-centric ERAD pathway. We demonstrate that this degron can be functionalized into a single- component system that drives targeted protein degradation from the ER lumen, a previously inaccessible cellular location. We show that this technology can drive degradation of both soluble, luminal ER proteins, and integral membrane ER proteins. Strikingly, this new tool has illuminated fascinating new biology of the ERAD system for “piggybacking” degradation of interacting proteins without their own degron, merely by interacting with other proteins containing the degron. This Example provides an exciting and simple method of targeting proteins for degradation from within the ER by exploiting the highly conserved ERAD system. Attorney Docket No.: UM-41598.601 Methods Yeast strains and plasmids Yeast were cultured at 30°C in synthetic complete medium (SC) supplemented with the appropriate amino acids. The hrd1Δ and pdr5Δ strain used in this study were derivatives of BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) or BY4742 (MATα his3Δ1 leu2Δ0505lys2Δ0 ura3Δ0). The hrd1Δpdr5Δ strain was generated by crossing hrd1Δ and pdr5Δ strains, sporulating the diploids, and screening the appropriate loci by PCR. For a list of yeast strains used in this Example, see Table 1. TABLE 1 Strain Strain name Genotype Source b l, For a list of plasmids used in this study, see Table 2. TABLE 2

Attorney Docket No.: UM-41598.601 Plasmid short name Plasmid Name Figures pBGP134 pBGP134_pRS415_GPD_ss-GFPfast-Cpy*-HDEL_pgk1 6B p_RB869 ^{pFA6 3xALFA 3xV5 HDEL TDH1term NatNT2} _{3A-3B, 3I-3J} 1F Plasmids were constructed using restriction enzyme cloning or NEB HiFi assembly. Plasmids used in this study were either centromeric (Sikorski and Hieter, 1989) or custom integrating plasmids. For a list of primers used to generate the pentapeptide library, see Table 3. TABLE 3 Primer Description SEQ Sequence ID Attorney Docket No.: UM-41598.601 prRP29 Fw primer for library 79 GAATCCCAGCACCAAA amplification (-500 bp/in GPD promoter) CG Schiestl, 2007). Following transformation into yeast, 3-4 independent transformants were passaged 1-2 times on selection media before using in experiments. Yeast pentapeptide library generation We designed an in vivo gap repair strategy for cloning our pentapeptide libraries into the ER-tFT. The plasmid backbone was based on pRS416 (Christianson et al., 1992) and contained a TDH3 promoter (also known as GPD or GAPDH), the signal sequence from mating factor alpha, mCherry, GFP, HDEL ER retention signal, and the CYC1 terminator. To prevent the peptide library from potentially disrupting signal sequence cleavage, two amino acids (Ala and Ser) after the signal sequence cleavage site were left upstream of the library. The final N-terminal amino acid sequence of the ER-tFT library after translocation and signal peptide cleavage is AS-Degron. To generate the pentapeptide DNA library fragment with homology arms to ER-tFT, four PCRs were performed with Phusion polymerase (New England Biolabs, M0530S). We started by generating a linear DNA template to reduce bias in our PCR reactions. Using pRP01 and primers prRP07 and prRP08, we amplified a 1025 bp fragment with 524 bp upstream (overlapping the TDH3 promoter and signal sequence) and 495 bp downstream (overlapping mCherry) of the library insertion (EcoRI) site in pRP01. This fragment (fragment 1) was gel-purified (QIAquick DNA cleanup, Qiagen) to remove any residual plasmid and contain only a linear template. Fragment 2, containing the upstream homology arm, was generated through PCR using fragment 1 (the linear DNA template) with primers prRP10 and prRP29 to amplify a 512 bp fragment, which included homology with both the TDH3 promoter and signal sequence and contained the random DNA library insertions. Fragment 3, containing the downstream homology arm, was generated in a PCR reaction using fragment 1 (the linear DNA template) from the first PCR reaction with primers prRP09 and prRP51 to generate a 146 bp fragment, which included random DNA in the library position along with homology Attorney Docket No.: UM-41598.601 arms in mCherry. Only 102 bp of homology was included in the mCherry coding region to limit the number of mutations found in mCherry included by homologous recombination. When we included longer homology arms into mCherry, we found that our screening procedure was selective enough to identify mutations in mCherry which could lead to false “unstable” hits. To generate the PCR product for homologous recombination containing overlapping homology arms to the target plasmid (pRP01), we gel-purified fragments 2 and 3 and mixed the DNA in an equimolar ratio. Using primers prRP29 and prRP51, we used 25 PCR cycles to generate a 605 bp fragment 4 (containing the pentapeptide library and homology arms covering 488bp upstream and 111 bp downstream of the library cut site). Fragment 4 was purified and mixed at a 30:1 molar ratio with purified pRP01 (digested with EcoRI) immediately before yeast electroporation. Yeast library transformation was performed according to the protocol outlined in Benatuil et al., 2010 (Benatuil et al., 2010) into yRB203. Cells were grown overnight to stationary phase in YPD media, shaking at 225 rpm and 30°C. An aliquot of the overnight culture was used to inoculate 400 mL of YPD media at 0.3 OD600/mL. Cells were grown for approximately 5 hrs until 1.6 OD₆₀₀/mL was reached and collected by centrifugation at 3200 x g for 5 minutes. The cell pellet was washed twice by 200 mL of ice-cold water and once by 200 mL of electroporation buffer (1 M Sorbitol/ 1 mM CaCl₂, sterile filtered). The cell pellet was then resuspended in 80 mL of 100 mM LiAc/ 10 mM DTT, split into two aliquots of 40 mL, and each was incubated in a 250 mL culture flask for 30 minutes at 30°C, shaking at 225 rpm. Next, cells were collected by centrifugation, washed once with 200 mL of ice-cold electroporation buffer, and re-suspended to 2.4 mL in electroporation buffer. The cell resuspension was evenly divided into 6 pre-chilled BioRad GenePulser cuvettes (0.2 cm electrode gap) and kept on ice for 10 min with DNA. One reaction was used as a no DNA control, one reaction received digested vector only, and four cuvettes received 3 µg of digested vector and 9 µg Fragment 4. Cells were electroporated at 2.5 kV and 25 µF, with time constants varying from 4.0-4.3 milliseconds. Cells were gently transferred from each cuvette into 8 mL of a 1:1 mix of 1M sorbitol:YPD in culture tubes (25m diameter) and incubated at 30°C, with shaking at 220 rpm. After 1 hour, cells were pelleted by centrifugation and inoculated into SC dropout media (-URA) at 0.2 OD600/mL. Dilutions from the electroporated cells were also plated on SC dropout plates and grown for two days at 30°C to determine the library transformation size (approximately 1.2 million for the library described here). Attorney Docket No.: UM-41598.601 Electroporated cells were grown for ~18 hrs while shaking at 30°C until reaching 0.5 OD₆₀₀/mL. Control strains expressing ER-tFT (pRP01) and KHN-tFT (pRP08) were also cultured in parallel. The strains and library were then treated with DMSO only (Sigma- Aldrich, D2650) or 50 µg/mL cycloheximide (EMD Millipore, 239763) for 2 hrs. After treatment, the cells were pelleted, washed once in 1x PBS, resuspended in 1x PBS containing 1 µM Sytox Blue (Invitrogen, S11348), and incubated at 4°C prior to cell sorting. Fluorescence-activated cell sorting Cells were sorted on a MoFlo Astrios Cell Sorter (Beckman Coulter) running Summit software. The instrument was set with a 100 µm tip, 405 nm laser with 448/59 nm bandpass filter, 488 nm laser with 514/20 nm bandpass filter, and 561 nm laser with 620/29 nm bandpass filter. Events were gated to select for yeast cell-sized events, single cells, live cells, and mCherry-GFP positive cells (Figure 5E). The mCherry/GFP ratio for each cell in the final gated population was displayed as a histogram. The ER-tFT and KHN-tFT controls were used to help define where to draw the low mCherry/GFP Ratio (unstable) bin for sorting (Figure 5F). In total, 13 million mCherry-GFP positive events were sorted from the pentapeptide-ER-tFT library, >10 times over the library size. The sorted cells were grown at 30°C with shaking in 5 mL of SC dropout media for 24 hrs and expanded to 25 mL cultures overnight.10 OD₆₀₀ of both the “unstable” bin and unsorted pentapeptide-ER-tFT library were pelleted and flash frozen in liquid nitrogen and stored at -80°C prior to DNA extraction. DNA extraction and amplicon sequencing prep DNA extraction was performed as previously described (Kats et al., 2018). The frozen 10 OD₆₀₀ pellets were resuspended in 500 µL of S-buffer (10 mM K₂HPO₄ pH 7.2, 10 mM EDTA, 50 mM 2-mercaptoethanol) and incubated with 50 mg/ml zymolyase 100T (AMSBIO) at 37°C for 30-60 minutes until the mixture became clear.100 µL of lysis buffer (25 mM Tris-HCl pH 7.5, 25 mM EDTA, 2.5% SDS (w/v)) was added and the suspension was incubated at 65°C for 45 min. Proteins were precipitated by adding 166 µL of 3M potassium acetate and incubating on ice for 10 min. Samples were then centrifuged at 21,000 x g for 10 minutes at 4°C. The supernatant containing DNA was collected and the DNA was precipitated by the addition of 800 µL of 100% ethanol, followed by centrifugation at 21,000 x g for 10 minutes at 4°C. Precipitated DNA was washed with 70% (v/v) ethanol and resuspended in 80 µL of water. Attorney Docket No.: UM-41598.601 Next, 5 µL of the isolated DNA solution to amplify a 217 bp fragment encompassing the pentapeptide sequences. Partial adapters for NGS analysis were added by 25 cycles of PCR using primers prRP37 and prRP38 (annealing temperature of 60°C using Phusion DNA polymerase). PCR products were purified (QIAquick DNA cleanup, Qiagen), normalized to 20 ng/µl using a QuBit 3 (Invitrogen dsDNA HS assay, catalog Q32854), and sent for amplicon sequencing using Genewiz Amplicon-EZ (now Azenta Life Sciences). Amplicon-EZ analysis Sequences from the two Amplicon-EZ samples (unstable bin and input library) were analyzed for quality, trimmed, aligned, and translated by Genewiz (now Azenta Life Sciences). The amino acid count was the sum of occurrences for each amino acid at each position (Supplementary data 5, AA Analysis Sheet). We then divided the amino acid frequency at each position for the library or unstable sorted bin by the expected amino acid frequency based on the number of codons that encode a given amino acid. This gave us the relative enrichment of each amino acid at each of the five positions (Fig 1F). Flow cytometry based degradation assays For each experiment, two biological replicates were transferred into SC dropout media in a 96 well plate (Fisherbrand, 12566611) sealed with gas-permeable membranes (Sigma-Aldrich, Z763624) and grown overnight shaking at 1000 rpm at 30°C. Overnight cell density was typically around ~4-5 OD₆₀₀/mL. In the morning, cells were diluted to 0.2 OD600/mL in SC dropout media and grown at 30°C shaking at 1000 rpm for ~5 hrs or until the OD₆₀₀/mL of the cultures was >= 0.5. Cells were pelleted at 3,200 x g and the supernatant was removed by aspiration prior to resuspension in SC dropout media at 2 OD600/mL. For experiments using cells treated with 50 µg/mL cycloheximide or 50 µM bortezomib, cells were transferred directly into new 96 well plates (Grenier Bio-One, 650185). To account for slowed growth by these treatments, in the same plates the untreated/DMSO treated cells were diluted by ⅓ with fresh media. During the treatment periods, cells were incubated at 30°C while shaking at 600 rpm. After treatment, cells were pelleted at 3,200 x g, washed once with 1x PBS, and resuspended in 1x PBS with 1 µM Sytox Blue (Invitrogen, S11348). Cells were maintained at 4°C during flow cytometry analysis on a MACSQuant VYB (Miltenyi) running MACSQuantify software (version 2.13.2). Sytox Blue was followed using the 405 nm laser and 452/45 nm emission filters. GFP was followed using the 488 nm laser and 452/45 nm emission filters. mCherry was followed using the 561 nm laser 615/20 nm emission filters. Attorney Docket No.: UM-41598.601 Downstream analyses were performed in FlowJo (version 10.7.1) with event gating to select yeast cell-sized events, single cells, live cells, and mCherry-GFP positive cells (Figure 5E). The mCherry/GFP ratio of mCherry-GFP positive cells was calculated in FlowJo and compared. Immunoblotting based degradation assays Cycloheximide-chase degradation assays were performed as described previously (Gardner et al., 1998) with the following modifications. Starter cultures were grown overnight in SC dropout media while shaking at 30°C. Cultures were diluted to 0.2 OD₆₀₀/mL in SC dropout media and grown for 4 - 5 hours to mid-log phase (0.4 - 1.0 OD600/mL). Cultures were pelleted at 3,200 x g for 5 minutes and resuspended to 2.0 OD₆₀₀/mL in fresh media before treatment with 50 µg/ml cycloheximide. Samples were incubated at 30°C with shaking and, at the indicated time points, shifted to 4°C and collected by centrifugation at 21,000 x g for 5 min. The supernatant was removed and cell pellets were incubated on dry ice prior to storage at -80°C or cell lysis. For experiments with bortezomib treatment, cells were pre-treated with 50 µM bortezomib for 15 min prior to cycloheximide addition. Cells were resuspended in SUME lysis buffer (1% SDS, 8M urea, 10 mM MOPS, pH 6.8, 10mM EDTA) at 20 OD₆₀₀/mL with acid-washed glass beads (0.1 mm, Bio-Spec). Cells were vortexed for 2 min and an equal volume of sample buffer (4% SDS, 8M urea, 125 mM Tris pH 6.8, 10% β-mercaptoethanol, 0.02 % bromophenol blue) was added and briefly vortexed. The samples were incubated at 65°C for 5 min, separated by SDS-PAGE, transferred to a PVDF membrane, immunoblotted with antibodies (anti-GFP from GeneScript, anti- DYKDDDK (SEQ ID NO:83) from Genscript, anti-HA from Roche, anti-V5 from Genscript, HRP-linked ECL rabbit-IgG and mouse-IgG from Amersham, Goat anti-Mouse IgG Alexa800 from Invitrogen), and detected by chemiluminescence (ECL Select Western blotting detection reagent, Amersham) using a ChemiDoc MP (Bio-Rad). For quantification of the immunoblot band intensities, we used ImageLab version 6.1 (Bio-Rad). Band intensities were normalized to total protein in the sample quantified within each lane using Stain-Free Dye Imaging (Bio-Rad). Attorney Docket No.: UM-41598.601 Mammalian cell culture and transfection U2OS cells were cultured in DMEM containing 4.5 g/liter glucose and L-glutamine, and supplemented with 10% fetal bovine serum (Corning) at 37°C and 5% CO2. Cells at 60- 80% confluence were transiently transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocols. After 48 hours, cells were used for emetine-chase or chemical treatment assays. Mammalian cell chemical treatments and lysis. Cells were treated with a translation inhibitor (50μM emetine) for the indicated time periods. For Bortezomib or CB5083 treatment, cells were mock treated or treated with either Bortezomib at a final concentration of 20 nM for 16 hours, or with CB5083 at 1 μM for 8 hours before harvest. The cells were collected and washed in PBS before lysis in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, protease inhibitor cocktail) for 10 min at 4°C. The lysates were cleared by centrifugation at 20,000 × g for 10 min at 4°C. Protein concentrations were determined using a BCA assay (Thermo Fisher Scientific). Cell lysates were normalized to the same concentrations in Laemmli sample buffer and heated to 65°C for 5 min prior to separation by SDS-PAGE. Immunoblotting was performed as described above. Results Identification of endoplasmic reticulum-localized degrons The endoplasmic reticulum is the primary location for protein quality control within the secretory pathway, but how the protein quality control systems distinguish folded from unfolded proteins is unclear. We wanted to understand the degrons recognized within the ER lumen and started by designing an ER-targeted reporter of protein stability. We targeted a well-characterized tandem fluorescent protein timer (tFT) to the ER to function as a reporter of protein stability (ER-tFT, Figure 1A). tFTs contain a fast-maturing fluorescent protein (GFP) and a slower-maturing fluorescent protein (mCherry) and have been used effectively to identify N-terminal degrons that function in the cytosol and nucleus (Kats et al., 2018; Khmelinskii et al., 2012). By measuring the ratio of mCherry to GFP fluorescence, a protein’s stability can be assessed; the lower the ratio, the more unstable the protein. To test whether the ER-tFT could successfully distinguish stable from unstable proteins, we Attorney Docket No.: UM-41598.601 compared the ER-tFT to a well-characterized, unstable, luminal ERAD substrate, KHN, tagged with the tFT (KHN-tFT, Figure 1A). Using a cycloheximide chase followed by immunoblotting, we found the ER-tFT was quite stable, while the KHN-tFT was degraded with a half-life of less than 30 minutes (Figure 1B), consistent with previous reports (Vashist et al., 2001). Using flow cytometry, the two proteins were also distinguishable following cycloheximide treatment (Figures 1C, 1D and 5A,B). After establishing the tFT reporter could distinguish protein stability within the ER, we turned our attention to identifying luminal degrons. To identify degrons that function within the ER lumen, we generated a library of short, linear peptide sequences embedded into the ER-tFT. Using PCR with degenerate primers, we generated an unbiased pentapeptide library encoded in a DNA fragment to use for homologous recombination in cells (Figure 1E, 5C). The theoretical amino acid diversity of a pentapeptide library is 3.2 million (20^5). We transformed the library into wild-type yeast and obtained 1.2 million transformants. Using fluorescence-activated cell sorting (FACS) we separated cells expressing the pentapeptide-ER-tFT by their mCherry/GFP ratio (Figure 5F). We collected an “unstable” bin (exhibiting a low mCherry/GFP ratio), which encompassed 4% of the sorted cells, for sequencing (Figure 5F). The sequencing results of the unstable sorted bin illuminated an enrichment of pentapeptides beginning with isoleucine and leucine. Specifically, isoleucine at the first position was present in 17.7% of unstable pentapeptides while leucine was present in 14.8%. Based on codon usage in a random sampling, isoleucine was predicted to appear 4.7% of the time (3/64 codons) giving a 3.8 fold enrichment in our dataset. Leucine was predicted to appear at a specific position 9.4% of the time (6/64 codons) giving a 1.6 fold enrichment in our dataset (Figure 1F, 5D). When compared to the input library abundance, enrichment corresponds to 2.7 and 1.5 fold, respectively (Figure 1F, 5D). Rather than build a consensus sequence, we selected pentapeptide sequences present in the unstable bins that broadly represented the enrichment trends we observed to individually clone and characterize (Figure 1F). We compared the stability of six different pentapeptides with isoleucine or leucine at position one to ER-tFT alone, to KHN-tFT, and to a set of randomly selected pentapeptides. KHN-tFT was the least stable, followed by pentapeptides selected from the unstable bin and containing isoleucine or leucine at position one. Several of the randomly selected pentapeptides exhibited a slight reduction in stability, relative ER-tFT alone, but pentapeptides selected from the unstable bin were consistently less stable (Figure 1G, 1H). Attorney Docket No.: UM-41598.601 Encouraged by our results, we sought to find a peptide sequence that would match KHN-tFT instability. We found that repeats of IHPYW (SEQ ID NO:1), one of the most unstable sequences, dramatically decreased protein stability, with a 20 amino acid 4x(IHPYW) repeat successfully resembling the KHN control (Figure 1I, 1J). On the other hand, simply repeating a stable control sequence (GNRWG; SEQ ID NO:84) did not alter protein stability (control variant 1 (ConV1), Figure 1J). Based on these results, we concluded that, for example, the 4x(IHPYW) sequence, which we called DegV1 (Degron Variant 1), functions as an ER- localized degron. DegV1 is an ERAD-dependent degron degraded by the cytosolic proteasome in yeast. Next, we tested whether DegV1 functioned as the first ER luminal degron. As with the original ER-tFT reporter (Figure 1), we started by targeting the LaG16 anti-GFP nanobody (Fridy et al., 2014) to the ER using the mating factor alpha signal sequence and an ER retention signal (Figure 2A, ss-NbGFP-Flag-HDEL). The NbGFP protein alone was quite stable, but embedding DegV1 after the N-terminal signal sequence destabilized the NbGFP protein with a half-life of approximately 30 minutes (Figure 2A). This successfully validated DegV1 as the first degron sequence that can be used for targeted ER luminal substrate degradation. We next tested if DegV1 could also function as an internal or C-terminal degron. To test whether the DegV1 sequence worked within internal loops, we used the same nanobody scaffold but replaced the complementary determining region 3 of the anti-GFP nanobody with DegV1 and found that the presence of an internal DegV1 made no difference in protein stability when compared to the nanobody alone (Figure 2B). Testing DegV1 at the C-terminus of the NbGFP was complicated by the requirement for an ER retention signal. We integrated the DegV1 sequence immediately before the HDEL retention signal, and, again, this resulted in no change in the stability of the NbGFP target protein (Figure 2C). The ER retention signal is an important component of this construct’s innate stability, because loss of the HDEL signal, with or without the DegV1 sequence at the extreme C-terminus, results in rapid degradation (Figure 2C). Together, these results suggest that the DegV1 degron was capable of acting as an ER luminal degron, but, in these particular experiments, only when positioned at the N-terminus of ER-localized proteins. Given that DegV1 was able to actively target proteins for degradation from the ER lumen, we tested whether its degradation was proteasome-dependent. To determine if DegV1-tagged proteins were degraded by the cytosolic proteasome, we tested the stability of the fluorescent ER-localized construct GFP-HDEL alone, or with DegV1 or ConV1. We Attorney Docket No.: UM-41598.601 treated cells for 2 hours with the proteasomal inhibitor bortezomib and/or cycloheximide. The stability of GFP-HDEL alone was similar either in the presence (dashed outline) or absence (solid outline) of bortezomib (panel 1, Figure 2D). Appending DegV1 immediately after the signal sequence resulted in degradation of GFP-HDEL (solid line), which was inhibited by adding bortezomib (dashed line, panel 2 Figure 2D). This indicated DegV1 targets the ER luminal GFP-HDEL for proteasomal degradation. As expected, appending a control sequence (ConV1) of the same length was similarly stable to GFP-HDEL alone and stability was not affected by bortezomib (solid line versus dashed line, panel 3, Figure 2D, see also 6D). Therefore, DegV1-targeted luminal ER substrates were degraded by the cytoplasmic proteasome. Proteasomal degradation of proteins from the lumen of the ER is mediated by the Hrd1-ERAD system (Bordallo et al., 1998). Therefore, we suspected that Hrd1-centered ERAD mediates DegV1-targeted proteasomal degradation. Again, we tested the stability of ER-GFP in cells lacking the central component to the Hrd1-ERAD system, the ubiquitin ligase Hrd1. The steady state levels of GFP-HDEL alone or with ConV1 were similar in the absence of Hrd1 and remained unaffected by proteasome inhibition (panels 1 and 3, Figure 2E). In contrast, DegV1-containing GFP was stable in a hrd1Δ strain, and bortezomib did not further stabilize DegV1-containing GFP (panel 2, Figure 2E). In the absence of Hrd1, we observed some ER leakage of DegV1-GFP-HDEL to the vacuole and appearance of a degradation resistant GFP fragment that resembled the known luminal ERAD substrate, CPY* (Figure 6A, B). Altogether, these results are consistent with a role for Hrd1 in the degradation of DegV1-GFP-HDEL. To further test the role of Hrd1 in DegV1-targeted degradation of luminal ER substrates, we tested two more soluble ER proteins (Figure 2F) that are otherwise relatively stable in the ER lumen (Figure 6C). As expected, when DegV1 was appended to either the anti-ALFA nanobody or the anti-GFP nanobody, we found that both proteins were unstable, with half-lives of approximately 30 minutes following cycloheximide chase (Figure 2F, quantification in 2G). Consistently, degradation of each of these DegV1 nanobodies was inhibited by either treating cells with bortezomib or deleting Hrd1. When the anti-GFP nanobody containing DegV1 was targeted to the cytoplasm, the degradation was proteasome- dependent but was independent of Hrd1 (Figure 11A, quantification in Figure 11B). In these experiments, we confirmed that DegV1 targets heterologously expressed ER luminal proteins for ERAD-mediated proteasomal degradation. We next tested whether DegV1 could target endogenous S. cerevisiae proteins for degradation. We transplanted DegV1 onto three different classes of endogenous, ER-localized proteins. First, we used the Attorney Docket No.: UM-41598.601 endogenous protein Suc2. We used the Suc2 signal sequence followed by either DegV1, or ConV1, an HA tag, the Suc2 coding sequence, a Flag tag, and an HDEL (Figure 2H). With ConV1, Suc2 was stable over several hours, and, based on the modified glycosylation pattern, even appeared to be partially trafficked from the ER. In contrast, with DegV1, Suc2 was dramatically destabilized (Figure 2H). Next, we attached DegV1 to a type I integral membrane ER protein, called Big1, that contained a single transmembrane segment (Azuma et al., 2002). We replaced the signal sequence of Big1 with the signal sequence of mating factor alpha followed by either DegV1 or a control sequence, an HA tag, and the Big1 coding sequence. DegV1 was also able to destabilize the integral membrane protein Big1 (Figure 2I). Finally, we attached DegV1 or the control sequence to the N-terminus of Elo1, a multispanning integral membrane protein with 7 probable transmembrane segments (Nie et al., 2021; Toke and Martin, 1996). We found that DegV1 was capable of driving degradation for the multi-spanning membrane protein Elo1 (Figure 2J). These results support that DegV1 is an N-terminal degron for endogenous proteins with a range of topologies. Soluble, luminal DegV1-tagged proteins are targeted to the proteasome by the Hrd1- ERAD pathway (Figures 2E-G). To test whether DegV1-targeted integral membrane proteins are also degraded by the proteasome, we followed Big1 and Elo1 degradation after treatment with cycloheximide and bortezomib. Both membrane proteins were significantly stabilized upon treatment with bortezomib (Figures 2K & L). Therefore, DegV1 targets both luminal and integral membrane ER-proteins for recognition by ERAD and subsequent degradation by the cytosolic proteasome. DegV1 is a functional degron in mammalian cells. We turned our attention to using DegV1 as a tool in mammalian cells. To determine that DegV1 functioned as an ER degron in mammalian cells, we generated an ER-targeted mNeonGreen by appending an N-terminal BiP signal sequence, the HA epitope tag, and the C-terminal ER retention peptide (KDEL) (ER-HA-mNG). We transfected U2OS cells with the ER-mNG containing either ConV1 or DegV1. The addition of DegV1, but not ConV1, reduced the steady-state ER-mNG levels compared to the control (Figure 9A, compare lanes 1, 4, and 7). When we inhibited translation with emetine, we found that ER-mNG and ER- ConV1-mNG were quite stable (Figure 9A, B). In contrast, ER-DegV1-mNG was unstable, with a half-life of approximately 4 hours (Figure 9A, B). In S. cerevisiae, DegV1 degradation was dependent on the cytosolic proteasome and mediated through ERAD. First, we tested whether degradation of DegV1 was proteasome- Attorney Docket No.: UM-41598.601 dependent in U2OS cells. We treated cells with bortezomib to inhibit proteasomal degradation and found with an 8 hour treatment, ER-mNG and ER-ConV1-mNG levels remained largely unchanged, but after 24 hours we observed a 1.5-fold increase. In contrast, for ER-DegV1-mNG proteasomal inhibition resulted in a dramatic accumulation of protein within 8 hours of treatment (Figure 9C, D). Remarkably, at 24 hours of bortezomib treatment ER-DegV1-mNG accumulated to similar levels compared to ER-ConV1-mNG, suggesting that the low steady-state level of ER-DegV1-mNG was caused by continuous degradation, rather than a general expression problem. ERAD-dependent proteasomal degradation also requires the AAA-ATPase p97/VCP (Cdc48 in yeast). Therefore, we tested the stability of the ER-mNG proteins after treatment with the VCP inhibitor CB5083. Similar to the bortezomib treatment, we found that treatment with CB5083 resulted in stabilization of ER-DegV1-mNG (Figures 9E, F). As the CB5083 incubation length increased, we found that the levels of ER-DegV1-mNG approached those of the stable, control proteins. To confirm that bortezomib and CB5083 were preventing the active degradation of ER-DegV1-mNG, rather than just improving expression, we analyzed the degradation of the ER-mNG proteins in the presence of bortezomib or CB5083. We confirmed that, without addition of bortezomib or CB5083, ER-ConV1-mNG was stable while ER-DegV1-mNG was degraded (Figure 9G). When we pretreated cells with either bortezomib or CB5083 and inhibited translation with emetine, we found that the degradation of ER-DegV1-mNG was completely inhibited. Finally, we tested whether Hrd1 was required for degradation of ER-DegV1-mNG in mammalian cells. Using either wild-type or Hrd1 knockout cells, we followed degradation of ER-DegV1-mNG and found that the degron- containing protein was stabilized in the absence of Hrd1 (Figure 9H, I). Next, we turned our attention to testing whether DegV1 would function on mammalian integral membrane proteins that pass through the ER. We started with Progesterone Membrane Receptor (PGRMC1) used lentiviruses to transduce U2OS cells and with a tetracycline-inducible PGRMC1 system. We found that inclusion of DegV1, but not ConV1, caused the degradation of PGRMC1 in U2OS cells (Figure 10C, D), K562 suspension cells (Figure 10E, F), and HEK293 cells (Figure 10G, H). Similar to PGRMC1, DegV1 caused degradation of the integral membrane protein Sigma 1 Opioid Receptor (S1R, Figure 10I, J). Finally, we tested whether the DegV1-mediated degradation of PGRMC1 and S1R were dependent on the proteasome and found that, as expected, the proteasomal was required for degradation (Figure 10K-P). Taken together, these data indicate that DegV1 functions as a Hrd1-dependent ER-localized degron in mammalian cells. Attorney Docket No.: UM-41598.601 ERAD allows “piggybacking” degradation of interacting proteins. DegV1 is the first example of a degron facilitating degradation from the ER lumen and also represents the first short, portable degron tag (<180 amino acids (Carvalho et al., 2010)) identified for the Hrd1-ERAD system. We turned our attention to the possibility of using DegV1 as a tool to illuminate new biology of the well-studied Hrd1-ERAD system. Our results suggest that the Hrd1-ERAD system is capable of efficiently retrotranslocating otherwise stable proteins across the membrane for cytosolic degradation (Figure 2). We wondered whether proteins that interact with a DegV1-tagged protein, but lack the DegV1 sequence themselves, are also degraded, thus “piggybacking” degradation by association. We designed a system that exploits interaction of two stable in the ER lumen. We used the ER luminal protein Suc2 including an ALFA-epitope tag and co-expressed one of three different nanobody constructs (NbGFP, NbALFA, NbVHH05) either with or without the DegV1 sequence. When the non-DegV1 containing nanobodies were able to interact with Suc2, we saw no change in the stability of Suc2 (Figure 3A). Quite remarkably, when the nanobodies contained DegV1 and were able to interact with Suc2, we observed degradation of Suc2 in addition to the degron containing interacting nanobody. In contrast, the presence of DegV1 on a non-interacting nanobody did not stimulate degradation of Suc2 (Figures 3A, B). Similarly, when we switched the Suc2 epitope tag from ALFA to VHH05, only NbVHH05 with DegV1 could stimulate degradation of Suc2 (Figure 3C, D). Together, this indicates that DegV1 enables “piggybacking” degradation of stable, soluble, ER-luminal protein merely by interaction with a targeted Hrd1-ERAD substrate. Next, we asked whether DegV1-associated piggybacking works with endogenous integral membrane proteins. We inserted an ALFA-epitope tag into the luminal region of the polytopic integral membrane protein, Elo1. As with Suc2, the specificity was quite remarkable. With ALFA-Elo1, we only observed degradation when NbALFA contained DegV1 (Figure 3E, F). Similarly, with VHH05-Elo1, we only observed degradation when NbVHH05 contained DegV1 (Figure 3G, H). Therefore, soluble luminal DegV1-tagged proteins are capable of triggering Hrd1-ERAD degradation of untargeted interacting membrane proteins from the ER, simply by recognition and interactions occurring on the luminal side of the ER. Finally, we tested whether DegV1 nanobodies depress the overall steady-state levels of interacting proteins. We found that the co-expression of Suc2-ALFA with DegV1- containing NbALFA depressed steady-state Suc2-ALFA to about 20% of the levels in the untargeted controls (Figure 3I, J). Similarly, DegV1-containing NbVHH05 depressed the Attorney Docket No.: UM-41598.601 steady-state levels of its target, Suc2-VHH05, to around 35% of the untargeted controls (Figure 3K, L). We also confirmed reduced steady-state levels for integral membrane protein targets by co-expressing targeting or non-targeting NbALFA with ALFA-Elo1. We found that DegV1-containing NbALFA specifically depressed ALFA-Elo1 steady-state levels to about 35% of untargeted controls (Figure 3M, N) and the results were similar using NbVHH05 and VHH05-Elo1 (Figure 3O, P). Taken together, our results demonstrate that the Hrd1-ERAD system can “piggyback” degradation of partner proteins from both the ER lumen and the ER membrane and provides a novel method for highly selective targeted protein degradation from the ER. In this example, we identified a novel short peptide motif (DegV1) that targets proteins for degradation (degrons) from the endoplasmic reticulum (Figure 1). We found that DegV1 works with luminal and completely soluble proteins as well as integral membrane proteins with differing topologies. It works with both exogenous and endogenous proteins. This degron appears, in this Example, to only work at the N-terminus of nascent proteins, rather than internally or C-terminally. Importantly, DegV1 is degraded through the Hrd1 ERAD axis using the cytosolic proteasome (Figure 2). We found that interacting proteins can also be degraded when one partner contains an ERAD-dependent degron and represents an unexpected “piggybacking” function of the ERAD system (Figures 3). Our data demonstrate that we have developed a robust, highly selective, and modular system for targeted protein degradation from the ER lumen and membrane. Despite a growing understanding of how the ERAD system functions, the fundamental question of degrons recognized by ERAD remains unanswered. Here, we identified a number of different degrons that remain to be characterized completely, but focused on a single degron (DegV1). The sequence “IHPYW” forming the basis of DegV1 appears to be relatively uncommon in nature and is not present in other proteins encoded in the S. cerevisiae genome. In fact, DegV1 is absent from the currently annotated and available fungal genomes in the Saccharomyces Genome Database (Cherry et al., 2012) and any available genomic sequences by NCBI BLAST. Based on our selection criteria, IHPYW alone is unlikely to be the most potent ER degron. In fact, we were able to demonstrate that just by increasing the length of our short degron, we could improve the degron to be more like that of a full ERAD substrate protein. It is somewhat surprising that DegV1, in these experiments, seems to only work as an N-terminal degron. Attorney Docket No.: UM-41598.601 We were able to demonstrate the utility of DegV1 by illuminating, for the first time, that the ERAD system can piggyback degradation of interacting proteins. This is particularly interesting in light of ERAD substrates like the oligosaccharyltransferase complex or T cell antigen receptors, which have multiple subunits and are degraded efficiently when components are missing from the complex (Bonifacino et al., 1989; Lippincott-Schwartz et al., 1988; Mueller et al., 2015). Here, the assumption has long been that each “orphaned” subunit must be individually recognized by the degradation machinery (Juszkiewicz and Hegde, 2018), but our results suggest that this isn’t necessarily the case and piggybacking might be more efficient methods for recognizing and degrading multiple components in the same unassembled complex. It should be noted that the degradation rates of the piggybacking partners are not only similar to those of the protein containing the degrons, but also to the rates of other well-characterized ERAD substrates. We interpret these results to mean that DegV1 is degraded in a manner consistent with that of endogenous ERAD substrates. Furthermore, similar rates for the piggybacking partners mean that this process is not just a peculiarity of these experiments, but represents a normal mode of action for ERAD. The retrotranslocation process itself appears to function by unfolding, or mostly unfolding, its substrates. Few protein transport systems can transport fully-folded proteins across a lipid bilayer with the notable exceptions being the twin arginine transporter (TAT) system (for review see (Berks, 2015)) and peroxisomal import machinery (Glover et al., 1994; McNew and Goodman, 1994; Romano et al., 2019; Skowyra and Rapoport, 2022). However, previous studies in the ERAD field provide conflicting accounts over the ability to transport fully-folded proteins from the ER to cytoplasm (Bhamidipati et al., 2005; Shi et al., 2019; Tirosh et al., 2003). What is certain is that glycosylated ERAD substrate proteins are retrotranslocated across the ER membrane, representing a similar steric challenge compared to secondary structure or smaller folded proteins (Grotzke et al., 2013). In this new ERAD- based piggybacking transport mechanic, it is interesting to speculate that the piggybacking proteins are transported in a fully-folded and interacting state, but it remains to be seen whether cotransport might result from each substrate being engaged by the ERAD system and transported separately. However, while not necessary to understand or practice the present invention, we favor the idea that the targeted proteins are likely recognized and transported in an unfolded state, disrupting the interactions between the two proteins after ERAD complex engagement. Perhaps each substrate would occupy adjacent ERAD complexes, or this process could require other unidentified proteins. Attorney Docket No.: UM-41598.601 REFERENCES: Alabi, et al., (2021). Major advances in targeted protein degradation: PROTACs, LYTACs, and MADTACs. J. Biol. Chem.296. Azuma, et al., (2002). Saccharomyces cerevisiae Big1p, a putative endoplasmic reticulum membrane protein required for normal levels of cell wall β-1,6-glucan. Yeast 19, 783-793. Baldridge and Rapoport (2016). Autoubiquitination of the Hrd1 Ligase Triggers Protein Retrotranslocation in ERAD. Cell 166, 394-407. Banik et al., (2020). Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 584, 291-297. Bays, et al., (2001). HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. Mol. Biol. Cell 12, 4114-4128. Békés et al., (2022). PROTAC targeted protein degraders: the past is prologue. Nature Reviews Drug Discovery 21, 181-200. Benatuil et al., (2010). An improved yeast transformation method for the generation of very large human antibody libraries. Protein Engineering, Design and Selection 23, 155- 159. Berks, B.C. (2015). The Twin-Arginine Protein Translocation Pathway. Annu. Rev. Biochem.84, 843-864. Bhamidipati et al., (2005). Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Mol. Cell 19, 741-751. Bonifacino et al., (1989). Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly. Journal of Cell Biology 109, 73-83. Bordallo et al., (1998). Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol. Biol. Cell 9, 209- 222. Carvalho et al., (2006). Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126, 361-373. Carvalho et al., (2010). Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p. Cell 143, 579-591. Attorney Docket No.: UM-41598.601 Caussinus et al., (2012). Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nature Structural & Molecular Biology 19, 117-121. Cherry et al. (2012). Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res 40, D700-705. Christianson et al., (1992). Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119. Clift et al. (2017). A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell 171, 1692-1706 e1618. Collins et al., (2017). The Logic of the 26S Proteasome. Cell 169, 792-806. Denic, et al., (2006). A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126, 349-359. Foresti et al., (2014). Quality control of inner nuclear membrane proteins by the Asi complex. Science 346, 751-755. Fridy et al., (2014). A robust pipeline for rapid production of versatile nanobody repertoires. Nat. Methods 11, 1253-1260. Gardner et al., (1998). Sequence determinants for regulated degradation of yeast 3- hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol. Biol. Cell 9, 2611-2626. Gietz and Schiestl (2007). High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2, 31-34. Glover et al., (1994). Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer. Proc. Natl. Acad. Sci. U.S.A.91, 10541-10545. Grotzke et al., (2013). Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation. Proc. Natl. Acad. Sci. U.S.A.110, 3393- 3398. Huyer et al., (2004). Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. J. Biol. Chem.279, 38369-38378. Hwang et al. (2023). The ERAD system is restricted by elevated ceramides. Sci. Adv. Ibrahim et al., (2020). Antibody RING-Mediated Destruction of Endogenous Proteins. Mol. Cell 79, 155-166 e159. Jarosch et al., (2002). Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat. Cell Biol.4, 134-139. Attorney Docket No.: UM-41598.601 Juszkiewicz and Hegde (2018). Quality Control of Orphaned Proteins. Mol. Cell 71, 443-457. Kats et al., (2018). Mapping Degradation Signals and Pathways in a Eukaryotic N- terminome. Mol. Cell 70, 488-501.e485. Khmelinskii et al. (2014). Protein quality control at the inner nuclear membrane. Nature 516, 410-413. Khmelinskii et al. (2012). Tandem fluorescent protein timers for in vivo analysis of protein dynamics. Nat. Biotechnol.30, 708-714. Lippincott-Schwartz et al., (1988). Degradation from the endoplasmic reticulum: Disposing of newly synthesized proteins. Cell 54, 209-220. Liu et al., (2022). Exploration of target spaces in the human genome for protein and peptide drugs. Genomics, Proteomics & Bioinformatics. https:// doi.org/10.1016/j.gpb.2021.10.007. McNew and Goodman (1994). An oligomeric protein is imported into peroxisomes in vivo. Journal of Cell Biology 127, 1245-1257. Mehnert et al., (2014). Der1 promotes movement of misfolded proteins through the endoplasmic reticulum membrane. Nat. Cell Biol.16, 77-86. Mueller et al., (2015). Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly. Mol. Biol. Cell 26, 2596-2608. Nabet et al. (2018). The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14, 431-441. Needham et al., (2019). Chaperoning Endoplasmic Reticulum–Associated Degradation (ERAD) and Protein Conformational Diseases. Cold Spring Harb Perspect Biol 11.10.1101/cshperspect.a033928. Nie et al. (2021). The structural basis of fatty acid elongation by the ELOVL elongases. Nature Structural & Molecular Biology 28, 512-520. Pisa and Rapoport (2022). Disulfide-crosslink analysis of the ubiquitin ligase Hrd1 complex during endoplasmic reticulum-associated protein degradation. J. Biol. Chem., 102373. Rabinovich et al., (2002). AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol. Cell. Biol.22, 626-634. Reynders et al., (2020). PHOTACs enable optical control of protein degradation. Sci Adv 6, eaay5064. Attorney Docket No.: UM-41598.601 Romano et al., (2019). Peroxisome protein import recapitulated in Xenopus egg extracts. J. Cell Biol.218, 2021-2034. Sakamoto et al., (2001). Protacs: Chimeric molecules that target proteins to the Skp1- Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. U.S.A.98, 8554-8559. Santos et al., (2017). A comprehensive map of molecular drug targets. Nature Reviews Drug Discovery 16, 19-34. Shi, et al., (2019). A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum and associated degradation. J. Biol. Chem.294, 20084-20096. Sikorski and Hieter (1989). A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces ceravisiae. Genetics 122, 19- 27. Skowyra and Rapoport (2022). PEX5 translocation into and out of peroxisomes drives matrix protein import. Mol. Cell 82, 3209-3225.e3207. Tirosh et al., (2003). Protein Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation. J. Biol. Chem.278, 6664-6672. Toke and Martin (1996). Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. J. Biol. Chem.271, 18413-18422. Vashist et al., (2001). Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J. Cell Biol.155, 355-368. Vashist and Ng (2004). Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. J. Cell Biol.165, 41-52. Wu, et al., (2020). Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex. Science 368, eaaz2449. Ye et al., (2001). The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, 652-656. ADDITIONAL REFERENCES: Ambalathingal, et al., (2017). "BK Polyomavirus: Clinical Aspects, Immune Regulation, and Emerging Therapies." Clinical Microbiology Reviews 30(2): 503-528. Boothpur, R. and D. C. Brennan (2010). "Human polyoma viruses and disease with emphasis on clinical BK and JC." Journal of Clinical Virology 47(4): 306-312. Attorney Docket No.: UM-41598.601 Dolan, P. T., Z. J. Whitfield and R. Andino (2018). "Mechanisms and Concepts in RNA Virus Population Dynamics and Evolution." Annual Review of Virology 5(1): 69-92. Farajpour, Z., F. Rahbarizadeh, B. Kazemi and D. Ahmadvand (2014). "A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment." Biochemical and Biophysical Research Communications 446(1): 132-136. Henry, K. A., G. Hussack, C. Collins, J. C. Zwaagstra, J. Tanha and C. R. MacKenzie (2016). "Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing." Protein Engineering, Design and Selection 29(10): 439-443. Ingram, J. R., O. S. Blomberg, J. T. Sockolosky, L. Ali, F. I. Schmidt, N. Pishesha, C. Espinosa, S. K. Dougan, K. C. Garcia, H. L. Ploegh and M. Dougan (2017). "Localized CD47 blockade enhances immunotherapy for murine melanoma." Proceedings of the National Academy of Sciences 114(38): 10184-10189. Itakura, E., R.-R. Huang, D.-R. Wen, E. Paul, P. H. Wünsch and A. J. Cochran (2011). "IL-10 expression by primary tumor cells correlates with melanoma progression from radial to vertical growth phase and development of metastatic competence." Modern Pathology 24(6): 801-809. Iwai, Y., M. Ishida, Y. Tanaka, T. Okazaki, T. Honjo and N. Minato (2002). "Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade." Proceedings of the National Academy of Sciences 99(19): 12293-12297. Jiang, N., Y. Yu, D. Wu, S. Wang, Y. Fang, H. Miao, P. Ma, H. Huang, M. Zhang, Y. Zhang, Y. Tang and N. Li (2023). "HLA and tumour immunology: immune escape, immunotherapy and immune-related adverse events." Journal of Cancer Research and Clinical Oncology 149(2): 737-747. Khodabakhsh, F., D. Norouzian, B. Vaziri, R. Ahangari Cohan, S. Sardari, F. Mahboudi, M. Behdani, K. Mansouri and A. Mehdizadeh (2018). "Development of a novel nano-sized anti-VEGFA nanobody with enhanced physicochemical and pharmacokinetic properties." Artificial Cells, Nanomedicine, and Biotechnology 46(7): 1402-1414. Massagué, J. and D. Sheppard (2023). "TGF-β signaling in health and disease." Cell 186(19): 4007-4037. Ritchie, C., J. A. Carozza and L. Li (2022). "Biochemistry, Cell Biology, and Pathophysiology of the Innate Immune cGAS–cGAMP–STING Pathway." Annual Review of Biochemistry 91(1): 599-628. Attorney Docket No.: UM-41598.601 Sangro, B., P. Sarobe, S. Hervás-Stubbs and I. Melero (2021). "Advances in immunotherapy for hepatocellular carcinoma." Nature Reviews Gastroenterology & Hepatology 18(8): 525-543. Solomon, P. E., C. J. Bracken, J. A. Carozza, H. Wang, E. P. Young, A. Wellner, C. C. Liu, E. A. Sweet-Cordero, L. Li and J. A. Wells (2024). "Discovery of VH domains that allosterically inhibit ENPP1." Nature Chemical Biology 20(1): 30-41. Starr et al., (2022). "Shifting mutational constraints in the SARS-CoV-2 receptor- binding domain during viral evolution." Science 377(6604): 420-424. Starr, et al. (2020). "Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding." Cell 182(5): 1295-1310.e1220. Wan et al., (2018). "Screening and antitumor effect of an anti‑CTLA‑4 nanobody." Oncol Rep 39(2): 511-518. Zhang, et al., (2017). "Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade." Cell Discovery 3(1): 17004. All publications and patents mentioned in the specification and/or listed below are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope described herein.

Claims

Attorney Docket No.: UM-41598.601 CLAIMS We Claim: 1. A method of degrading a target protein, and/or a protein in a protein complex with said target protein (PIPCWTP), in a cell comprising: contacting said cell, in vitro or in a subject, with a composition comprising a degradation construct, or a nucleic acid sequencing encoding said degradation construct if it is encodable by nucleic acids, wherein said degradation construct comprises: a) a target protein binding or interacting moiety (TPBOIM), b) a degron comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50, and c) optionally one or more linkers and/or one or more N-terminal amino acids; wherein said degron is optionally attached to said TPBOIM directly or via said one or more linkers, and wherein said target protein and/or said PIPCWTP is degraded in said cell. 2. The method of claim 1, wherein said degradation construct further comprises an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence. 3. The method of claim 1, wherein said TPBOIM is selected from: a target protein specific antibody or target protein binding portion thereof, a small molecule binding protein that binds said target protein; an aptamer that binds said target protein, or at least a portion of a protein that naturally interacts with, or binds to, said target protein in said cell. 4. The method of claim 3, wherein said target protein specific antibody or target protein binding portion thereof is selected from: a monoclonal antibody, a nanobody, a fab fragment, a f(ab)2, scFv, and dAb. 5. The method of claim 1, wherein said at least one copy of said amino acid sequence is at least two, three, or four copies. Attorney Docket No.: UM-41598.601 6. The method of claim 1, wherein: said contacting results in a reduction in the level of just said target protein, or just said, or both said target protein and said PIPCWTP, in said cell. 7. The method of claim 1, wherein said contacting results in: i) degradation of said target protein by the endoplasmic reticulum associated protein degradation (ERAD) pathway in said cell, and/or ii) degradation in a lysosome of said cell. 8. The method of claim 1, wherein said contacting results in degradation of said target protein at a rate greater than the wild-type rate for said target protein in said cell and/or wherein said target protein is located in the lumen or membrane of the endoplasmic reticulum of said cell. 9. The method of claim 1, wherein said contacting is via administration of said composition to said subject, and optionally wherein said administration is performed orally, intravenously, intranasally, optically, vaginally, or rectally. 10. The method of claim 9, wherein said target protein is associated with a proteinopathy disease or condition in said subject, or wherein said target protein is a viral or bacterial protein and said subject is, or is not, infected with a virus or bacteria. 11. The method of claim 10, wherein said target proteinopathy disease or condition, and/or said target protein, is one or more shown in Table 4. 12. The method of claim 1, wherein said one or more N-terminal amino acids are present, and optionally comprise a serine and an alanine. 13. The method of claim 1, wherein said amino acid sequence comprises, consists essentially of, or consists of: IHPYW (SEQ ID NO:1). 14. The method of claim 1, further comprising contacting said cell with a dimerization or other agent that causes said target protein and said PIPCWTP to form a complex. Attorney Docket No.: UM-41598.601 15. The method of claim 1, wherein said amino acid sequence comprises, consists essentially of, or consists of: IWRGR (SEQ ID NO:2), IYTLE (SEQ ID NO:3), LHQLY (SEQ ID NO:4), LWGNH (SEQ ID NO:5), or LYTMR (SEQ ID NO:6). 16. The method of claim 1, wherein said cell is a mammalian cell, and optionally a human cell, and wherein said subject is a mammal, and optionally a human. 17. The method of claim 1, wherein said cell is a type selected from: red blood cells, white blood cells, platelets, bone cells, brain cells, egg cells, sperm cells, muscle cells, fat cells, and nerve cells. 18. The method of claim 1, wherein said composition comprises said degradation construct. 19. The method of claim 1, wherein said composition comprises said nucleic acid sequencing encoding said degradation construct, and said encoded degradation construct comprises amino acids. 20. The method of claim 1, wherein said contacting is performed in vitro. 21. The method of claim 1, wherein said target protein is selected from: an ER luminal protein, an integral membrane protein, a secretory pathway protein, and an endo/lysosomal protein. 22. The method of claim 1, wherein said TPBOIM comprises a protein, and wherein said degron is attached to the N-terminus of said TPBOIM.

Attorney Docket No.: UM-41598.601 23. A composition, system, or kit comprising: a composition comprising a degradation construct, or a nucleic acid sequencing encoding said degradation construct if it is encodable by nucleic acids, wherein said degradation construct comprises: a) a target protein binding or interacting moiety (TPBOIM), b) a degron comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50, and c) optionally one or more linkers and/or one or more N-terminal amino acids, wherein said degron is attached to said TPBOIM directly or via said one or more linkers. 24. The composition, system, or kit of claim 23, wherein said degradation construct further comprises an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence. 25. The composition, system, or kit of claim 23, wherein said TPBOIM is selected from: a target protein specific antibody or target protein binding portion thereof, a small molecule binding protein that binds said target protein; an aptamer that binds said target protein, or at least a portion of a protein that naturally interacts with, or binds to, said target protein in said cell. 26. The composition, system, or kit of claim 25, wherein said target protein specific antibody or target protein binding portion thereof is selected from: a monoclonal antibody, a nanobody, a fab fragment, a f(ab)2, scFv, and dAb. 27. The composition, system, or kit of claim 23, wherein said at least one copy of said amino acid sequence is at least two, three, or four copies and/or wherein said composition further comprises d) an aqueous buffer. 28. The composition, system, or kit of claim 23, wherein said target protein is associated with a proteinopathy disease or condition in said subject. 29. The composition, system, or kit of claim 23, wherein said target protein is one or more shown in Table 4. Attorney Docket No.: UM-41598.601 30. The composition, system, or kit of claim 23, wherein said amino acid sequence comprises IHPYW (SEQ ID NO:1). 31. The composition, system, or kit of claim 23, wherein said amino acid sequence consists essentially of, or consists of, IHPYW (SEQ ID NO:1). 32. The composition, system, or kit of claim 23, wherein said amino acid sequence comprises, consists essentially of, or consists of: IWRGR (SEQ ID NO:2), IYTLE (SEQ ID NO:3), LHQLY (SEQ ID NO:4), LWGNH (SEQ ID NO:5), or LYTMR (SEQ ID NO:6). 33. The composition, system, or kit of claim 23, wherein said composition further comprises a cell. 34. The composition, system, or kit of claim 33, wherein said cell is a mammalian cell, and optionally a human cell, and wherein said subject is a mammal, and optionally a human. 35. The composition, system, or kit of claim 33, wherein said cell is a type selected from: red blood cells, white blood cells, platelets, bone cells, brain cells, egg cells, sperm cells, muscle cells, fat cells, and nerve cells. 36. The composition, system, or kit of claim 23, wherein said composition comprises said degradation construct. 37. The composition, system, or kit of claim 23, which said composition comprises said nucleic acid sequencing encoding said degradation construct, and said encoded degradation construct comprises amino acids. 38. The composition, system, or kit of claim 23, wherein said target protein is selected from: an ER luminal protein, an integral membrane protein, a secretory pathway protein, and an endo/lysosomal protein. Attorney Docket No.: UM-41598.601 39. The composition, system, or kit of claim 23, wherein said TPBOIM comprises a protein, and wherein said degron is attached to the N-terminus of said TPBOIM. 40. The system, or kit of claim 23, further comprising a device for injecting said composition, or a capsule, wherein said composition is inside said capsule. 41. A composition comprising: a degron construct, or a nucleic acid sequence encoding said degron construct, wherein said degron construct comprises: a) a degron comprising at least one copy of an amino acid sequence shown in any of SEQ ID NOs:1-50, and b) a target protein, wherein said target protein is not naturally associated with said degron. 42. The composition of claim 41, wherein said degron construct further comprises an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence. 43. A system for tagging a target nucleic acid sequence with a degron comprising: i) a synthetic guide ribonucleic acid (sgRNA), wherein the sgRNA is at least partially complementary to a target nucleotide sequence in or near a target gene; ii) a repair template nucleotide sequence encoding at least one copy of a degron with an amino acid sequence selected from any of SEQ ID NOs:1-50; and iii) at least one of the following: A) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease, and/or B) first nucleotide sequence, or complement thereof, encoding said CRISPR-associated nuclease. 44. The system of claim 43, wherein said repair template further comprises a nucleic acid sequence encoding an endoplasmic reticulum signal sequence (ERSS) and/or an endoplasmic reticulum retention sequence. Attorney Docket No.: UM-41598.601 45. A method for tagging a target gene in a cell in vitro or a cell in a subject comprising: introducing into a cell or subject the system of claim 43. 46. A method of tagging a target gene in a cell with a nucleotide sequence encoding a degron comprising: a) introducing into a cell: i) a first nucleotide sequence encoding a synthetic guide ribonucleic acid (sgRNA), wherein the sgRNA is at least partially complementary to a target nucleotide sequence in or near a target gene; ii) a second nucleotide sequence encoding a clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease; iii) a repair template comprising a nucleotide sequence encoding at least one copy of a degron with an amino acid sequence selected from any of SEQ ID NOs:1-50; and b) expressing the sgRNA and Cas9 nuclease in the presence of the repair template in the cell, thereby tagging the target gene with the nucleotide sequence encoding the degron.