Protein degradation targeting chimera constructed based on REG gamma and application thereof

Abstract

The invention discloses a protein degradation targeting chimera constructed based on REG gamma and application thereof, the targeting chimera comprises a compound as shown in (I): # imgabs0 #, PL represents a ligand capable of specifically binding to a target protein, REG L represents a ligand capable of recruiting REG gamma, and L represents a linking group for linking PL and REG L. In conclusion, through the unique ubiquitin-independent mechanism, the REGrTAC not only overcomes the limitation of the traditional PROTAC technology, but also provides a new thought and method for targeted protein degradation. The appearance of the gene widens the understanding and application of targeted protein degradation, and provides a new direction for future drug development and disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a protein degradation targeting chimera constructed based on REGγ and application thereof. Background Art

[0002] In eukaryotic cells, protein degradation plays a key role in almost all cellular processes, and the proteasome system mediates the clearance of major misfolded or abnormal proteins. The proteasome system operates through two modes of protein degradation: ubiquitin-dependent and ubiquitin-independent. In the ubiquitin-proteasome system (UPS), the 26S proteasome serves as the main mediator of selective protein degradation, which operates through an ATP- and ubiquitin-dependent pathway. The ubiquitination process is coordinated by E1, E2, and E3 enzymes, involving more than 800 E3 ligases. These ligases physically interact with specific target substrates, ensuring the precise targeting and degradation of damaged or unnecessary regulatory proteins for subsequent protease degradation. The ability of the proteasome to recognize ubiquitin chains and loosely folded regions forms the basis of its selective protein degradation. Certain proteins have specific sequence or structural motifs, called degradation motifs, that mark them for degradation within the cell. These degradation motifs share certain amino acid sequence motifs that have been gradually identified and defined over time.

[0003] Degradation motifs are defined as the minimal elements in a substrate protein sufficient to be recognized by the degradation machinery. A fundamental property of degradation motifs is their transferability. In most cases, engineering a degradation motif element from an unstable protein into another long-lived protein should be sufficient to destabilize the latter. Degradation motifs are mainly composed of short linear motifs containing 2 to 10 amino acid residues and are usually located in disordered regions of proteins. They can be constitutive, leading to continuous degradation, or conditional, initiated by post-translational modifications (PTMs) or exposure after protease cleavage. Extensive studies have revealed that degradation motifs can be present at the N-terminus, C-terminus, or any fragment of the substrate, depending on the reciprocal binding regions recognized by the E3 and substrate. The discovery of degradation motifs has led to targeted protein degradation via the proteasome or autophagosome.

[0004] Based on this discovery, PROTAC (Protein Degradation Targeting Chimera) was developed to exploit the ubiquitin-proteasome system to degrade target proteins. PROTAC consists of three components: a ligand that binds to the protein of interest (POI), a connector, and another ligand that targets the E3 ubiquitin ligase, collectively referred to as a "triplet". PROTAC promotes the simultaneous binding of POI and ligase, leading to ubiquitination of POI and subsequent degradation by the ubiquitin-proteasome system (UPS). PROTAC is then recycled to bind another set of POI molecules. CRBN- / VHL-based PRAOTACs, such as dBET1, have been shown to be effective in targeting BRD2, BRD3, and BRD4. The VHL E3 ligase complex is also known for degrading hypoxia-inducible factor α (HIF). Advances in high-throughput genetic technologies have driven significant progress in the study of degradation motifs, however, studies focusing on degradation motifs in ubiquitin-independent systems remain limited.

[0005] The REGγ-20S proteasome operates in an ATP- and ubiquitin-independent manner, a feature that has been greatly understood over the past few decades. REGγ, also known as PSME3, PA28γ, or Ki antigen, is a member of the 11S family of proteasome activators. Our research group and others have established that REGγ mediates the degradation of various downstream targets. This process contributes to the progression of multiple diseases and is implemented through ubiquitin-independent proteasomal degradation pathways. The structure of the REGγ heptamer and its binding mechanism to the 20S proteasome have been documented to elucidate its proteolytic activity. However, due to the transient binding affinity between REGγ and its substrates, studies on the mechanism of REGγ substrate recognition remain limited. To date, no studies on the degradation motif of REGγ have been reported. Summary of the invention

[0006] The purpose of the present invention is to provide a protein degradation targeting chimera constructed based on REGγ and its application in view of the deficiencies in the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect is to provide a protein degradation targeting chimera constructed based on REGγ, wherein the targeting chimera comprises a compound as shown in (I):

[0009]

[0010] Among them, PL represents a ligand that can specifically bind to the target protein, REG L represents a ligand that can recruit REGγ, and L (Linker) represents a linker group connecting PL and REG L.

[0011] Furthermore, the REG L is a degradation sub polypeptide sequence for REGγ to recognize and degrade substrates, and the REG L contains one or more polypeptide sequences as shown in formula (II):

[0012] R( X )K( X )RR(K)

[0013] Formula (II);

[0014] Among them, () means yes or no; X represents any amino acid.

[0015] Furthermore, the specific amino acid sequence of REG L is GRKRRQ (SEQ ID No. 1) or GRKRRRKRRQ (SEQ ID No. 2).

[0016] Furthermore, the PL is a ligand that can specifically bind to a target protein with therapeutic potential.

[0017] Furthermore, the PL is selected from small molecule ligands that can target bromodomain protein 4 (BRD4), androgen receptor (AR), cyclin-dependent kinase 4 / 6 (CDK4 / 6) or protein arginine transferase 5 (PRMT5).

[0018] Furthermore, the Linker includes C 1-12 Alkyl, C 1-12 Alkoxy or aliphatic ring composed of 4-7 atoms.

[0019] Furthermore, the Linker includes C 1-12 Alkoxy groups and connected to the N-terminus / C-terminus of PL and REG L, respectively, through amide condensation.

[0020] The second aspect is to provide the use of the protein degradation targeting chimera constructed based on REGγ as described above in ubiquitin-independent protein degradation.

[0021] The third aspect is to provide a degradation sub polypeptide sequence for REGγ to recognize degradation substrates and to degrade non-REGγ natural substrate proteins, wherein the degradation sub polypeptide sequence comprises one or more polypeptide sequences as shown in formula (II):

[0022] R( X )K( X )RR(K)

[0023] Formula (II);

[0024] Among them, () means yes or no; X represents any amino acid.

[0025] Furthermore, by connecting a non-REGγ natural substrate protein to the N-terminus or C-terminus of the degrader polypeptide sequence, the non-degradable protein can be labeled to become a degradation target of the REGγ-20S protease.

[0026] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0027] The targeted protein degradation method of REGrTAC (REGγ targeted degradation technology) of the present invention shows significant advantages over existing technologies such as PROTAC (Proteolysis Targeting Chimeras). PROTAC is a widely used protein degradation technology that achieves degradation of specific targets by inducing the ubiquitin-proteasome pathway. However, REGrTAC directly recruits REGγ to degrade proteins in a mechanism that is not dependent on ubiquitin, which provides a new solution for dealing with many targets that are difficult to degrade by traditional methods. Secondly, REGrTAC can effectively deal with the drug resistance problem of PROTAC. Studies in recent years have shown that PROTAC resistance is often due to changes in E3 ligases or disorders of the ubiquitination pathway, rather than dysfunction of the proteasome itself. Because REGrTAC is independent of E3 ligases, it exhibits greater adaptability and potential in the face of these resistance mechanisms. This gives REGrTAC a clear advantage in the treatment of drug-resistant cancers and other complex diseases.

[0028] In addition, REGrTAC also shows flexibility in design and optimization. Researchers can adjust the ligands bound by REGγ as needed to efficiently induce the degradation of different targets. This flexibility allows REGrTAC to be used not only for the degradation of a single target, but also to be expanded to multiple targets in future studies to achieve multiple therapeutic effects.

[0029] In terms of application, the advantages of REGrTAC are not only reflected in basic research, but also show great potential for clinical application. The targeted degradation strategy can be used to treat a variety of diseases, especially cancer, autoimmune diseases and neurodegenerative diseases. In these diseases, the overexpression or abnormal stability of specific proteins is often closely related to the pathological process, and REGrTAC can provide an effective means of intervention.

[0030] In summary, the REGrTAC of the present invention not only overcomes the limitations of traditional PROTAC technology through its unique ubiquitin-independent mechanism, but also provides new ideas and methods for targeted protein degradation. Its emergence has broadened the understanding and application of targeted protein degradation and provided a new direction for future drug development and disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 (A) Western blot image showing the binding affinity between p21 truncations and REGγ; (B) Modular display of the interaction between p21 truncations and REGγ; (C) Degron sequence of REGγ; (DE) Western blot images showing the interaction between p21 degron sequence mutations and REGγ; (F) BLI experiment showing the KD value between REGγ and p21 determined by using synthetic biotin-p21 peptide and purified REGγ protein; (G) Native binding of REGγ-p21 peptide complex PAGE and SDS-PAGE analysis; REGγ effectively binds to p21 (WT) peptide, resulting in gel migration, while the mutant peptide has no such interaction; (H) Streptavidin bead pull-down experiment analyzes the affinity of REGγ to p21 peptide; When bound to biotin-p21 (WT) peptide, REGγ was effectively pulled down by streptavidin beads, while no interaction with the mutant peptide was observed; where * indicates the binding affinity between p21 and REGγ; an increase in * indicates a stronger interaction between the two proteins, while a decrease in * indicates a weaker interaction; "-" indicates that there is no interaction between p21 truncation and REGγ; protein molecular weight (kDa) is as shown in the figure (A, D, E, G, H); all data shown represent representative results of two (F) or three independent experiments (A, D, E, G, H).

[0032] Figure 2(A) Statistical analysis of IP-MS of Flag-REGγ or empty vector from REGγKO 293T cells; the volcano plot shows the proteins in the Flag-REGγ transient expression group and the control group, and the red dots indicate the proteins that were upregulated at least 1.5 times after Flag-REGγ transient expression (p value < 0.05); (B) TSPYL1 intensity in the Flag-REGγ or empty vector expression group; the purple box indicates the enrichment of 293T cell lysate overexpressing REGγ, and the gray box indicates the enrichment of overexpressing empty plasmid ; The Y axis indicates the protein intensity detected by mass spectrometry, *** indicates p value = 0.0001; (CD) are Western blot images showing the physical association between TSPYL1 and REGγ, after overexpression of Flag-TSPYL1, HA-REGγ or control empty vector in 293T cells, mutual co-immunoprecipitation and Western blot analysis were performed; (E) is a Western blot image showing that REGγ directly promotes the degradation of TSPYL1 in vitro; using purified REGγ , 20S protease and in vitro transcribed TSPYL1 protein were subjected to in vitro proteolysis analysis at 30°C for 6 h; (F) is the quantitative analysis of TSPYL1 protein level corresponding to (E), ** indicates p value ≤ 0.0032; (G) is a Western blot image showing that REGγ regulates TSPYL1 protein stability in 293T cells, control and REGγ knockout 293T cells were treated with 100 μg / ml cycloheximide (CHX) for the indicated time, and then Western blot was collected. ern blot analysis; (H) is the quantitative analysis of TSPYL1 protein stability corresponding to (G), * indicates significant difference, p value = 0.031; wherein, actin level is used as a control for protein loading (E, G); representative immunoblot results from three independent experiments are shown (C, D, E, G); protein molecular weight (kDa) is as shown (C, D, E, G); data are presented as mean ± SD, from independent experiments (n = 4 or 3), and analyzed by Student's t-test (B, F, H).

[0033] Figure 3(A) Top and side views of the cryo-EM map of the REGγ-p21 peptide; here the p21 peptide consists of amino acids G135 to P164, REGγ is shown in maize blue, the p21 peptide is in medium purple-red, and the enlarged central slide view illustrates the binding of the p21 peptide on top of the central plug density; (B) Overlay map between REGγ (color) and the available cryo-EM map of independent REGγ (EMDB-34024, transparent gray); both maps show the central plug density at the entrance of the channel (indicated by a dark red border); (C) XL-MS results of the REGγ-p21 peptide complex, showing their interaction; (D) Figure 3DVA motion showing two extremes in variance; the central plug density appears to move within the channel (indicated by the green dashed circle), occasionally contacting different sides of the channel wall; (EF) are Western blot images showing that the K195A mutation in REGγ reduces the degradation ability in 293T cells; the degradation ability of REGγ was assessed by detecting p21 overexpression (E) and endogenous (F) levels in cells transfected with WT or K195AREGγ; actin levels were used as a control for protein loading; protein molecular weight (kDa) is indicated; blue arrows indicate endogenous REGγ protein in 293T cells; representative immunoblot results from three independent experiments are shown (E, F).

[0034] Figure 4(A and F) Western blot images showing the physical binding affinity of REGγ to N2-p27 (A) / MP3B2-C2 (F) in 293T cells; after overexpression of Flag-REGγ, GFP-N2-27, GFP-MP3B2-C2 or control empty vector in 293T cells, mutual co-immunoprecipitation and Western blot analysis were performed; (B and G) Western blot images showing REGγ regulating N2-p27 (B) / MP3B2-C2 (G) protein stability in 293T cells; control and REGγ knockout 293T cells were treated with 100 μg / ml cycloheximide (CHX) for the indicated time, and then Western blot analysis was collected; (C and H) Quantitative analysis of N2-p27 (C) / MP3B2-C2 (H) protein levels corresponding to (B, G); (D and I) shows Western blot images showing that REGγ directly promotes the degradation of N2-p27(D) / MP3B2-C2(I) in vitro; in vitro proteolysis analysis was performed at 30°C for 4 hours (D) or 3 hours (I) using purified REGγ, 20S protease and in vitro transcribed N2-p27(D) / MP3B2-C2(I) protein; (E and J) quantitative analysis of N2-p27(E) / MP3B2-C2(J) protein stability corresponding to (D, I); where actin levels were used as protein loading control (B, D, G, I); representative immunoblot results from three independent experiments are shown (A, B, D, F, G, I); protein molecular weight (kDa) is shown in the figure (A, B, D, F, G, I); data are presented as mean ± SD, from three independent experiments, and analyzed by Student's t test (C, E, H, J).

[0035] Figure 5The target protein BRD4 is degraded through the REGγdegron pathway; (AB) Western blots (A) and quantitative analysis (B) of BRD4 levels in A549 cells treated with X1, X2, JQ1 or MG132 (20 μM) for 24 hours; (CD) Western blots (C) and quantitative analysis (D) of BRD4 levels in A549 cells treated with X1 or MG132 (20 μM) for different time periods; (EF) Western blot (E) and quantitative analysis (F) of BRD4 levels after different concentrations of G132 (12 hours); (GH) Western blot (G) and quantitative analysis (H) of BRD4 levels over time after A549 cells were treated with X1 (20 μM); (I, K, L) Western blot analysis of BRD4 levels in control or REGγ knockout cell lines after X1 (20 μM) treatment; (J) Western blot analysis of BRD4 levels in control or REGγ knockout cell lines after X1 (20 μM) treatment. Pearson analysis of the correlation between the relative BRD4 degradation rate and REGγ level in various cell lines after treatment with X1 (20μM, 24 hours); the data were plotted and analyzed by GraphPadPrism software, R2=0.4539, p value=0.0327; the central line in the figure represents the best fit line of the two variables in the Pearson correlation coefficient model, and the dotted line indicates the 95% confidence band; (MP) is a Western blot analysis of PRMT5 levels in various cell lines after X3 treatment; (Q) shows a proposed model for REGγTAC, illustrating its ability to degrade the protein of interest; among them, actin levels are used for protein loading control (A, C, E, G, I); representative immunoblot results from three independent experiments are shown (A, C, E, G, I); protein molecular weight (kDa) is shown in the figure (A, C, E, G, I); data are presented as mean ± SD, from three independent experiments, and analyzed by Student's t test (B, D, F, H).

[0036] Figure 6(A, B, D, E, F) show Western blot images of the binding affinity between AID, HCV truncations and REGγ; (C, G) are modular displays of the interactions between AID, HCV truncations and REGγ; (H) is an alignment of the key binding sequences of REGγ substrates; wherein, * indicates the binding affinity between the substrate and REGγ; an increase in the number of * indicates a stronger interaction between the two proteins, while fewer * indicate a weaker interaction; "-" indicates that there is no interaction between AID truncations and REGγ; sequence alignment was performed using NCBI-COBALT (https: / / www.ncbi.nlm.nih.gov / tools / cobalt / ); protein molecular weight (kDa) is shown in the figure (A, B, D, E, F); all presented Western blot data represent three independent experiments (A, B, D, E, F).

[0037] Figure 7 (AB) Western blot images showing the interaction between AID degron sequence mutations and REGγ; (CD) Western blot images showing the interaction between HCV degron sequence mutations and REGγ; (E) The sequence linked to the N-terminus of wild-type or mutant p21 peptide and the sequence of biotin; (F) Native PAGE and SDS-PAGE analysis of REGγ-p21 peptide complex; p21 (WT) peptide effectively binds to REGγ, resulting in gel migration, but no interaction with BSA or 293T cell lysate; g, Streptavidin beads pull-down analysis for analyzing the affinity of REGγ to p21 peptide. When bound to biotin-p21 (WT) peptide, REGγ was efficiently pulled down by Streptavidin beads, but no interaction with BSA; where the protein molecular weight (kDa) is as shown in the figure (A, B, C, D, F, G); all presented data represent three independent experiments (A, B, C, D, F, G).

[0038] Figure 8 (AC) Mass spectrometry analysis of wild-type or mutant biotin-p21 peptide; (DF) HPLC analysis of wild-type or mutant biotin-p21 peptide.

[0039] Fig. 9(A) Western blot images showing the efficiency of REGγ knockout (KO) in 293T cells; actin levels were used as a control for protein loading, and protein molecular weights (kDa) are shown in the figure. Representative blot results from three independent experiments are shown; (B) Workflow for performing IP-MS of Flag-REGγ or empty vector from REGγKO 293T cells; (C) The number of significantly upregulated proteins in IP-MS, red squares indicate proteins upregulated by at least 1.5-fold in the Flag-REGγ overexpression group (p value < 0.05), gray squares indicate proteins not regulated, blue squares indicate proteins downregulated by at least 1.5-fold in the Flag-REGγ overexpression group (p value < 0.05), and the numbers in the squares indicate the number of protein clusters; (D-E) Intensity of Flag-REGγ (D), p21, and NIP30 (E) in Flag-REGγ or empty vector expression groups; orange (D ) and green (E) boxes indicate the enrichment of 293T cell lysates overexpressing REGγ, the gray box indicates the enrichment of overexpressed empty vector, the Y-axis indicates the protein intensity detected by mass spectrometry, and the data are presented as mean ± SD from four independent experiments and analyzed by Student's t-test (D, E); (F) is a Venn diagram of upregulated and REGγ interacting proteins at the protein level, protein upregulation can be observed from the red squares in (C), and interacting proteins indicate REGγ interacting proteins obtained from BioGRID (version 4.4) (https: / / thebiogrid.org / ).

[0040] Fig.10 (A) Gel filtration of purified wild-type (WT) REGγ; (B) Sequence of wild-type p21 peptide with or without biotin attached to the N-terminus; (CD) Mass spectrometry and HPLC analysis of p21(135-164) peptide; (E) Native PAGE and SDS-PAGE analysis of REGγ-p21 peptide complex; p21 peptide and Biotin-p21 peptide effectively bind to REGγ, resulting in gel shift, while there is no such interaction with BSA or 293T cell lysate; wherein, protein molecular weight (kDa) is indicated (A, E); the data presented are representative of three independent experiments (A, E)

[0041] Fig.11(A) is a typical original micrograph of the REGγ-p21 peptide complex; (B) is the reference-free 2D class average of the REGγ-p21 peptide complex; (C) is the data processing workflow for the structure determination of the REGγ-p21 peptide complex; the mask of the additional density used for focused 3D classification is shown as a yellow transparent sphere, the process performed in CryoSPARC is shown in green font, and the process performed in RELION is shown in light blue font; (D) is the map resolution assessment at the FSC standard of 0.143.

[0042] Fig.12 (AB) Euler angle distribution (A) and local resolution assessment (B) of the REGγ-p21 map; (C) shows that a single subunit of REGγ consists of four α-helices, and the model map fit of each α-helix, illustrating the high-resolution structural features; (D) shows that K195 of one subunit can form contacts with the central plug density, suggesting that K195 may be a key residue for forming / stabilizing the plug; (E) Positions of residues K188 / K192 / K195 facing the central channel of REGγ; (F) Fit of the REGγ model predicted by AlphaFold2, including the loop 64-102 region, in our map (transparent gray); The figure shows a model of one REGγ subunit (magenta), and the other models are shown in cornflower blue; it appears that the central plug may be composed of several amino acids within the loop 64-102 region (indicated by the black dashed ellipse).

[0043] Fig.13 (AB) Western blot images showing that REGγ does not directly promote the degradation of p27 (A) or MP3B2 (B) in vitro; in vitro protease assays were performed using purified REGγ, 20S proteasome, and in vitro transcribed p27 (A) or MP3B2 (B) proteins at 30°C for 4 hours (A) or 3 hours (B); actin levels were used as a control for protein loading; protein molecular weights (kDa) are indicated; data are representative of three independent experiments.

[0044] Fig.14 (AC) Chemical structure (A), HPLC analysis (B) and HRMS analysis (C) of the control peptide; (DF) Chemical structure (D), HPLC analysis (E) and HRMS analysis (F) of X1; (GI) Chemical structure (G), HPLC analysis (H) and HRMS analysis (I) of X2; (JK) Chemical structure (J), HPLC analysis (K) and ESI-MS analysis (L) of X3.

[0045] Fig.15The degradation of BRD4 and PRMT5 in cells through the REGγ degradation pathway is shown; (a, b) Western blot (a) and quantitative analysis (b) of BRD4 levels in H1299 cells treated with X1, X2, JQ1 or MG132 (20 μM) for 24 hours; (c, d) Western blot (c) and quantitative analysis (d) of BRD4 levels in H1299 cells treated with X1 or MG132 (20 μM) for different time periods; (e, f) Western blot (e) and quantitative analysis (f) of BRD4 levels in H1299 cells treated with X1 or MG132 (12 hours) at different concentrations; (g, h) Western blot (g) and quantitative analysis (h) of BRD4 levels in H1299 cells in a time-dependent manner after X1 (20 μM) was eluted; (i) Western Blot images showing the efficiency of REGγ knockdown (KD) in A549 and H1299 cells; (j) Western blot analysis of BRD4 levels in control or REGγ knockdown H1299 cells after treatment with X1 (20 μM) for 24 h; (k) Western blot analysis of BRD4 levels in H1299 cells treated with X1 (20 μM) or MZ1 (5 μM) for 24 h, with or without pretreatment with MLN4924 (5 μM, 48 h); (l) Western blot analysis of BRD4 levels in 786-O and A498 cells treated with X1 (10 μM in 786-O cells and 5 μM in A498 cells) or MZ1 (10 μM in 786-O cells and 5 μM in A498 cells) for 24 h blot analysis; (m) Western blot analysis of BRD4 levels in A549 and A498 cells after treatment with X1 (20 μM in A549 cells and 30 μM in A498 cells) or MZ1 (20 μM in A549 cells and 30 μM in A498 cells) for 18 h; (n) Western blot analysis of PRMT5 levels in MCF7 cells after treatment with X3, MS4322 or MG132 (20 μM) for 24 h; (o) Western blot analysis of PRMT5 levels in MCF7 cells after treatment with X1, MS4322 or MG132 (20 μM) for 1 or 3 days, with culture medium changed every day; (p) Western blot analysis of PRMT5 levels in MCF7 cells after treatment with different concentrations of X3 for 24 h; (q) Western blot analysis of time-dependent PRMT5 levels in MCF7 cells after X3 (20 μM) washout Blot analysis; Actin level was used as a control for protein loading (a, c, e, g, i, j, k, l, m, n, o, p, q);Representative Western blot results are from three independent experiments (a, c, e, g, i, j, k) or two independent experiments (l, m, n, o, p, q); the approximate molecular weight (kDa) of the markers is indicated in the figure (a, c, e, g, i, j, k, l, m, n, o, p, q); data are presented as mean ± SD and analyzed by Student's t test (b, d, f, h). ; DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0047] Example 1: Identification of the conserved binding motif (degradation motif) of REGγ

[0048] It has been demonstrated that REGγ-20S proteasome plays a key role in various diseases in degrading multiple substrates. However, the substrate recognition mechanism remains unclear. P21 is a well-known substrate of REGγ-20S proteasome and has been extensively studied. When p21 is truncated to 140 amino acids, the interaction between REGγ and p21 is disrupted.

[0049] To map the interaction domain between REGγ and p21, we transiently overexpressed HA-p21 truncated (1-138) or wild-type HA-p21 in 293T cells and performed co-IP experiments. We found that deletion of amino acids 139-164 in p21 completely abolished the interaction with REGγ ( Figure 1 , A and B). Further refinement of the p21 binding site, involving internal deletions of residues 139-144 / 145-150 / 151-157 / 158-164, showed that the binding affinity was significantly reduced when residues 139-144, 151-157, and 158-164 were deleted in p21. Interestingly, deletion of residues 145-150 of p21 did not affect its interaction with REGγ. Therefore, while the 139-164 region of p21 is critical for the interaction with REGγ, the 145-150 region appears to have less impact.

[0050] For another REGγ-20S proteasome substrate protein AID, the present invention constructed a GFP-tagged AID full-length mammalian cell expression plasmid (1-198), as well as a series of truncated mutant plasmids, including AID (1-40), AID (1-77), AID (1-116) and AID (78-198). Then, the interaction of these AID truncations with REGγ was examined using co-IP. It was found that, except for AID (78-198) which showed a disrupted interaction with REGγ, all truncations interacted with REGγ almost as well, compared with the full-length AID ( Figure 6 A). According to the amino acid sequence region (attached Figure 6 C), it was inferred that the key REGγ binding site on AID may be located between amino acids 1-40. Similar to p21, specific sequences on AID were deleted to further examine its interaction with REGγ. Co-IP experiments showed that each deleted region contributed to binding ( Figure 6 , B and C). In the case of HCV, similar to p21 and AID, the present invention determined that the 5-12 / 57-63 / 64-71 / 112-122 region is critical for interaction with REGγ ( Figure 6 , DG).

[0051] In order to identify the conserved REGγ binding motif, the present invention analyzed the key amino acid regions of p21, AID and HCV. It was found that the basic amino acids in these regions were enriched, forming a KR repeating motif. Subsequently, the key binding sequences of these three substrates were compared and a conserved sequence K(R)KRR( Figure 1 C and Figure 6 H). Based on these findings, the present invention proposes that REGγ may use this motif to recognize its substrate, which may serve as a degradation motif for REGγ.

[0052] It is hypothesized that the positive charges on K and R may enhance the affinity between the substrate and REGγ, while mutations in the motif may reduce this affinity. To test this hypothesis, the present invention mutated basic amino acids to acidic or neutral amino acids. In the case of p21, K / R was mutated to A or E in the 139-164 region, and co-IP experiments were performed to examine the binding affinity. It was found that the interaction between the p21 mutant and REGγ was weaker than that between wild-type p21 and REGγ ( Figure 1 D). More specifically, 6E or 7E mutations showed weaker binding ability compared to 3E or 4E mutations ( Figure 1 , D and E). In addition, the 10E mutation almost completely abolished the interaction between p21 and REGγ ( Figure 1E) These results suggest that the charge of the motif may play a key role in enabling REGγ protease to recognize its substrate.

[0053] Subsequently, the present invention introduced mutations in the amino acids of the four K and R-rich regions of AID 1-40. Specifically, RRK was mutated to AAA and EEE, KNVR was mutated to ANVA and ENVE, KGRR was mutated to AGAA and EGEE, and KRR was mutated to AAA and EEE. Subsequent co-IP showed that these eight mutants interacted more weakly with REGγ than full-length AID. Among these mutants, the KNVR mutation showed a relatively small reduction in the strength of the interaction, while several other mutations showed a more significant reduction ( Figure 7 , A and B). Similar results were observed in HCV K / R mutations ( Figure 7 , C and D). Collectively, these findings suggest that elimination of the positive charge in the motif results in a decrease in the binding affinity between REGγ and its substrate.

[0054] To exclude the possibility that deletion of the motif may have altered the conformation of p21, thereby affecting its binding to REGγ, the present invention examined the interaction between REGγ and the p21 motif region. Various p21 motif peptides were synthesized, including biotinylated wild-type (WT) and mutant p21 peptides. These are shown in the attached Figure 7 E. The mass spectrometry and HPLC characterization of these peptides are shown in Figure 8 , AF. The binding affinity between the p21 motif peptide and purified REGγ was measured using biolayer interferometry (BLI) experiments. This resulted in a dissociation constant (Kd) of 4.182 nM ( Figure 1 F). Native PAGE gel shift assay showed that biotin-p21 peptide effectively bound to REGγ. However, complex formation between BSA or 293T cell lysate and REGγ was not affected ( Figure 7 F). In addition, native gel electrophoresis confirmed the high affinity of REGγ for biotin-p21 (WT), but it did not bind to the biotin-p21 mutant peptide ( Figure 1 G). In an in vitro pull-down assay using streptavidin-biotin enrichment for biotin-p21 peptide, streptavidin beads remarkably and specifically captured REGγ by biotin-p21 peptide. There was no evidence of nonspecific binding under the experimental conditions of the present invention ( Figure 7 G). Furthermore, WT p21 peptide efficiently captured REGγ, whereas mutant p21 peptide did not produce similar results ( Figure 1 H).

[0055] In summary, the results of the present invention indicate that REGγ interacts with its degradation motif both in vivo and in vitro. It is noteworthy that the K / R residues were found to play an important role in promoting this interaction.

[0056] Example 2: Combining IP-MS and degron (motif) to explore new degradable substrates of REGγ

[0057] Over the past 20 years, more than a dozen substrates of REGγ have been identified. However, a comprehensive systems-level exploration of REGγ substrates is still lacking. It is known that REGγ substrates should physically interact with REGγ, and many substrates discovered so far contain repeated motif sequences. Therefore, the present invention hypothesized that combining the interactome and the motifs of REGγ may lead to the discovery of new substrates.

[0058] To investigate the interactome of REGγ, we performed IP-MS experiments in REGγKO 293T cells overexpressing Flag-REGγ ( Fig. 9 , A and B). Proteins with an enrichment ratio (REGγ / EV) greater than 1.5 and a p-value less than 0.05 were considered positive hits and marked with red dots ( Figure 2 A). In total, the present invention identified 2,927 proteins, of which 187 were upregulated (Appendix Fig. 9 C). To verify the quality of this IP-MS experiment, upregulated proteins were analyzed and several known binding proteins of REGγ were searched. Both p21 and NIP30 were readily captured by REGγ (see Figure 2). Fig. 9 , D and E), indicating that the interacting group proteins of REGγ were indeed enriched in the up-regulated proteins. We listed these up-regulated proteins as candidate substrates derived from the interactome data.

[0059] The present invention established certain criteria to identify potential substrate proteins. Initially, the list of upregulated proteins obtained from IP-MS was compared with the public interactome derived from the BioGRID database (version 4.4; https: / / thebiogrid.org / ). As of November 24, 2023, this database has identified 401 interacting proteins in human cells. Comparison of the two datasets revealed 42 overlapping proteins (see Appendix Fig. 9 F). Recognizing the key role of K / R residues in REGγ interaction, we conducted a comprehensive analysis of the K / R residue ratios in these proteins. We also ranked the substrate proteins according to their enrichment levels in IP-MS data. These factors led us to turn our attention to TSPYL1.

[0060] TSPYL1, a protein known for enhancing spermatogenesis efficiency, was significantly enriched in cells overexpressing REGγ ( Figure 2 , A and B). Interestingly, the enrichment ratio of TSPYL1 exceeded that of REGγ, indicating a strong affinity between TSPYL1 and REGγ. Upon further investigation, several sequences of TSPYL1 were found to be similar to the motif of REGγ around position 260 and at the C-terminus of the TSPYL1 sequence. In addition, TSPYL1 was observed to be enriched with REGγ at positions 330, 360, and 370. Our reciprocal co-IP experiments demonstrated that TSPYL1 can indeed interact with REGγ ( Figure 2 , C and D). Using CHX tracking experiments, the present invention determined that the half-life of TSPYL1 in 293T REGγKO cells was longer than that in wild-type control cells ( Figure 2 , G and H). In addition, in vitro degradation experiments showed that REGγ and 20S protease could significantly degrade TSPYL1 ( Figure 2 , E and F). Together, these findings suggest that REGγ directly promotes TSPYL1 degradation. Therefore, combining IP-MS and motif analysis can help identify substrates and thus expand the scope of REGγ interactome studies.

[0061] Example 3: The structure of p21 peptide bound to REGγ revealed by cryo-electron microscopy

[0062] The previously reported structure of REGγ exists as a homo-heptamer complex (EMDB:34024). However, the structure of REGγ with its substrates has not been explored. In this study, we purified REGγ and synthesized a p21 peptide ( Fig.10 , A and B). Here the p21 peptide ( Fig.10 B), consisting of amino acids G135 to P164, represents the major interaction motif according to our Co-IP analysis ( Figure 1 A). To avoid the possible interference of biotin in the bound p21 peptide, a biotin-free version was synthesized for structural studies ( Fig.10 , BD), and verified its strong affinity to REGγ ( Fig.10 E). After this, the p21 peptide was incubated with REGγ and analyzed by cryo-EM. To overcome the problem of preferred orientation, tilt data were also collected. After dedicated three-dimensional (3D) classification without symmetry constraints, we obtained a cryo-EM image of the REGγ-p21 peptide complex with a nominal resolution of ( Figure 3 A, Fig.11 and Fig.12AB). We then constructed an atomic model that fits well in the corresponding maps and reveals high-resolution structural features ( Fig.12 C).

[0063] The REGγ-p21 structure also exhibits a funnel-shaped heptamer structure ( Figure 3 A), as in the PA28αβ-iCP immunoprotease in the PA28αβ heteroheptamer structure. Here, the overall REGγ conformation closely resembles the isolated REGγ structure, with both structures showing a plug at the narrow entrance of the central channel ( Figure 3 B). This observation suggests that this plug density is part of REGγ and can probably be attributed to part of the disordered ring 64-107. Notably, an additional density is observed on top of REGγ, and is associated with the central plug, compared to the free REGγ map ( Figure 3 , A and B). This additional density may represent the density of the bound p21 peptide. Although this additional density is of lower resolution, we were unable to construct a model for this part. Taken together these observations suggest that the p21 peptide may bind to the central plug at the entrance of the REGγ channel.

[0064] Our analysis of the REGγ structure revealed that each subunit is composed of four long α-helices, labeled H1-H4 ( Fig.12 C). Among them, we observed that H3 interacts with the central plug through residue K195 ( Fig.12 D), suggesting that K195 may be a key residue for plug formation. Our chemical cross-linking and mass spectrometry (XL-MS) analysis of the REGγ-p21 peptide system indeed detected cross-links between REGγ and p21 peptide ( Figure 3 C), thus confirming their interaction. In addition, the central channel of REGγ faces residues K188 / K192 / K195 ( Fig.12 E) can detect cross-linking with p21 peptide ( Figure 3 C), indicating that p21 may enter REGγ through the entrance of this central channel.

[0065] Furthermore, the present invention demonstrates the intrinsic dynamics of the central plug density through 3D variability analysis (3DVA) of the REGγ-p21 peptide system. Figure 3 D). This density appears to move within the channel, sometimes making contact with different sides of the channel's "inner wall", potentially leaving space on the opposite side of the channel for substrates to enter the REGγ chamber. We then propose that p21 first anchors to the central plug of REGγ, and then, following the intrinsic movement of the plug, REGγ transports substrates through the narrow central channel into the 20S core particle for degradation.

[0066] In this case, the key residue is K195, which appears to be essential for plug formation. To address the effect of K195 on p21 binding, we generated a REGγK195A mutant to assess the degradation capacity of REGγ. Compared with wild-type REGγ, the REGγK195A mutant had reduced degradation capacity in 293T cells ( Figure 3 , E and F). This finding supports the idea that K195 plays a key role in forming the central plug, which may be involved in substrate binding and transport into the REGγ channel.

[0067] Example 4: REGγ degron can be designed to degrade non-native substrate proteins

[0068] A fundamental property of degrons is their transferability. In most cases, the addition of a degron sequence to a non-substrate protein can accelerate its degradation. We speculated that the degron of REGγ might have a similar ability to accelerate the degradation of non-substrate proteins.

[0069] To verify this hypothesis, the present invention designed a series of p27 plasmids, each of which was fused to the N-terminus or C-terminus with one, two or three repeated degron sequences. Taking GFP-N2-p27 as an example, two repeated codons of the degron sequence RKRRRKRR were introduced into the fusion site of the GFP-p27 plasmid. Then there was a GGS sequence to enhance the flexibility between the degron and the p27 protein. Co-IP showed that GFP-N2-p27 did bind to REGγ, although GFP-p27 did not interact with REGγ ( Figure 4 A). Then, the present invention investigated the effect of REGγ on the stability of N2-p27. Cyclohexane dihydrazide (CHX) tracking experiments were performed to determine whether the half-life of N2-p27 was different between control 293T cells and REGγ knockout 293T cells. Figure 4 , B and C, the half-life of N2-p27 in REGγ knockout cells was significantly prolonged compared with that in control cells. It is noteworthy that the degradation rate of Flag-p27 remained consistent in 293T cells with or without REGγ, which is consistent with previous reports, indicating that p27 is not a substrate of REGγ. To further confirm that N2-p27 is directly degraded by REGγ, in vitro translated N2-p27 was incubated with recombinant REGγ and 20S protease. The results showed that N2-p27 was significantly degraded ( Figure 4 , D and E), but p27 was not degraded ( Fig.13 A) Thus, when the degron sequence is attached, REGγ can directly promote the degradation of p27.

[0070] Next, the general applicability of the degron fusion method was explored, focusing on MP3B2, a microtubule-associated protein involved in physiological functions such as mitophagy. The complete MP3B2 consists of 125 amino acids with a molecular weight of 15 kD. An MP3B2 with one, two, or three repeats of the degron sequence was fused to the N-terminus or C-terminus. Co-IP detected a clear GFP signal in cells co-transfected with MP3B2-C2, indicating that MP3B2-C2 can interact with Flag-REGγ, while MP3B2 cannot ( Figure 4 F). Both CHX tracking experiments and in vitro degradation experiments showed that REGγ mediated the degradation of MP3B2-C2 ( Figure 4 , GJ), but not MP3B2( Fig.13 B) Taken together, these experimental results suggest that the degron sequence of REGγ can “tag” non-degradable proteins, making them targets for degradation by the REGγ-20S protease.

[0071] Example 5: Ubiquitin-independent degradation of BRD4 using REGγTAC technology

[0072] In recent years, protein degradation targeting technology has become one of the most promising strategies for eliminating specific disease-related proteins through proteasomes or lysosomes. These technologies include PROTAC, molecular glue, LYTAC (lysosome targeting chimera) and AUTOTAC. Given that REGγ mediates protein degradation through the proteasome and has a strong and selective degron, we hypothesized that binding the degron peptide to the POI (protein of interest) can promote REGγ-proteasome degradation by bringing POI and REGγ close together. To verify this hypothesis, the present invention selected BRD4 as a prototype target because extensive research on BRD4 has involved different BRD4-targeting PROTACs used by a variety of different E3 ligases, and it plays a key role in cancer progression.

[0073] Given that degron peptides can specifically bind to REGγ, and JQ1 (BET inhibitor) is a specific small molecule ligand for BRD4, the present invention designed a bifunctional degrader that connects degron peptides and JQ1. The degron peptide sequence RKRR of REGγ was used as a ligand for REGγ recruitment. JQ1 was linked to the N-terminus or C-terminus of the degron peptide via an amide bond, with PEG as a linker. This process produced two REGγ TAC peptides: X1 and X2 ( Fig.14 , D and G). Meanwhile, the GRKRRQ peptide was synthesized as a negative control for the degrader ( Fig.14A). HPLC purification confirmed the purity of the synthesized degrader peptide to be greater than 95%, which was verified by HRMS ( Fig.14 BC, 14E-F and 14H-I). Then, the present invention evaluated the ability of these degraders to induce the degradation of endogenous BRD4 in A549 cells. A549 cells were incubated with 20 μM of the degraders for 24 hours, and then the cells were collected for immunoblotting. Figure 5 , as shown in A and B, both X1 and X2 significantly reduced BRD4 levels, with X1 being more efficient than X2. Similar results were obtained for H1299 cells ( Fig.15 , A and B). Therefore, X1 was selected for further experiments. To evaluate the time course of BRD4 degradation, the present invention exposed A549 cells to X1 and measured BRD4 levels over time. As expected, MG132, a well-known protease inhibitor, blocked the process of BRD4 degradation by X1 ( Figure 5 C), confirming that X1-mediated BRD4 degradation is carried out through the proteasome pathway. X1 begins to induce BRD4 degradation 6 hours after treatment and continues until 24 hours ( Figure 5 , C and D). However, after 48 h of treatment, a significant recovery of BRD4 was observed, suggesting that cellular hydrolysis of X1 may have occurred during this period. Similarly, X1 induced degradation of BRD4 in A549 cells in a dose-dependent manner ( Figure 5 , E and F). When X1 was washed away, BRD4 levels were observed to recover within 12 hours ( Figure 5 , G and H). This finding suggests that X1-mediated BRD4 degradation is reversible and can be controlled, in contrast to the irreversibility of currently known genetic approaches. The degradation effect of X1 is dependent on REGγ, as BRD4 expression remains unchanged in REGγ-silenced cells, regardless of X1 treatment ( Figure 5 I and Fig.15 I). In addition, when X1 was applied to another cell line H1299, the time course ( Fig.15 , C and D), dose response ( Fig.15 , E and F) and elution experiments ( Fig.15 , G, and H) all produced similar results, indicating that X1-mediated protein degradation is REGγ-dependent and can ubiquitinate ( Fig.15 , J and K). Finally, comparative analysis in multiple cell lines after X1 treatment revealed a positive correlation between REGγ levels and the rate of BRD4 degradation ( Figure 5 J). Taken together, these results confirm that X1 can indeed effectively degrade BRD4 in a REGγ-dependent manner ( Figure 5 K).

[0074] To further demonstrate the versatility of REGγ-mediated protein degradation, we synthesized X3, a conjugate that linked GSK3326595 to the C-terminus of the degradation peptide via an amide bond with a linker of PEG( Fig.14 , JL). We tested the ability of X3 to degrade PRMT5 and surprisingly, in both HeLa and MCF7 cells, after only one day of treatment, its degradation efficiency was significantly better than that of MS4322 ( Figure 5 m, Fig.15 n). In time-dependent experiments ( Figure 5 n, Fig.15 o), dose response experiment ( Figure 5 o, Fig.15 p) and elution experiments ( Figure 5 p, Fig.15 q), further reinforcing the effectiveness of X3 in degrading PRMT5 in these cell models. These findings suggest that the REGγ-mediated degradation system is not limited to BRD4 and provides a general approach for degrading a variety of target proteins. This highlights the importance of REGγTAC ( Figure 5 q) Potential as an innovative protein degradation approach, especially for targets that are resistant to traditional ubiquitin-dependent pathways. The universality and efficiency of this system lay the foundation for expanding its application to a wider range of substrates, thereby providing new strategies for therapeutic interventions that are difficult to achieve with traditional degradation systems.

[0075] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A protein degradation targeting chimera constructed based on REGγ, characterized in that: The targeting chimera comprises the compound shown as (I): Among them, PL represents a ligand that can specifically bind to the target protein, REG L represents a ligand that can recruit REGγ, and L represents a linker group connecting PL and REG L.

2. The protein degradation targeting chimera constructed based on REGγ according to claim 1, characterized in that: The REG L is a degradation sub polypeptide sequence for REGγ to recognize and degrade substrates, and the REG L contains one or more polypeptide sequences as shown in formula (II): R( X )K( X )RR(K) Formula (II); Among them, () means yes or no; X represents any amino acid.

3. The protein degradation targeting chimera constructed based on REGγ according to claim 2, characterized in that: The specific amino acid sequence of REG L is GRKRRQ or GRKRRRKRRQ.

4. The protein degradation targeting chimera constructed based on REGγ according to claim 1, characterized in that: The PL is a ligand that is able to specifically bind to a target protein with therapeutic potential.

5. The protein degradation targeting chimera constructed based on REGγ according to claim 4, characterized in that: The PL is selected from small molecule ligands that can target bromodomain protein 4 (BRD4), androgen receptor (AR), cyclin-dependent kinase 4 / 6 (CDK4 / 6) or protein arginine transferase 5 (PRMT5).

6. The protein degradation targeting chimera constructed based on REGγ according to claim 1, characterized in that: The Linker contains C 1-12 Alkyl, C 1-12 Alkoxy or aliphatic ring composed of 4-7 atoms.

7. The protein degradation targeting chimera constructed based on REGγ according to claim 6, characterized in that: The Linker contains C 1-12 Alkoxy groups and connected to the N-terminus / C-terminus of PL and REG L, respectively, through amide condensation.

8. Use of the protein degradation targeting chimera constructed based on REGγ as described in any one of claims 1 to 7 in ubiquitin-independent protein degradation.

9. Application of a degradation sub polypeptide sequence for REGγ recognition of degradation substrates in the degradation of non-REGγ natural substrate proteins, characterized in that: The degrader polypeptide sequence comprises one or more polypeptide sequences as shown in formula (II): R( X )K( X )RR(K) Formula (II); Among them, () means yes or no; X represents any amino acid.

10. The use according to claim 9, characterized in that: By connecting a non-REGγ natural substrate protein to the N-terminus or C-terminus of the degrader polypeptide sequence, the non-degradable protein can be marked to become a degradation target of the REGγ-20S protease.

Images