Screening method of degradation signal peptide, fusion protein and proteasome inhibitor

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a screening method of a degradation signal peptide, a fusion protein and a proteasome inhibitor. The invention firstly provides a degradation signal peptide. The degradation signal peptide can mediate degradation of target protein directly or indirectly connected with the degradation signal peptide; the degradation signal peptide comprises a sequence which is the same as a sequence formed by 6-200 amino acids in TMEM8B protein, namely, the degradation signal peptide is of a polypeptide structure containing 6-200 amino acids. The degradation signal peptide provided by the invention is coupled with the target protein, so that the target protein can be completely degraded very efficiently; compared with a wild type full-length TMEM8B-a protein, the degradation signal peptide contains less amino acid, is a direct active site, and is not easy to cover due to space folding of the protein; meanwhile, due to the smaller size, in-vitro artificial synthesis is easier, and downstream application is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for screening degradation signal peptides, fusion proteins, and proteasome inhibitors. Background Technology

[0002] The proteasome is a multi-subunit macromolecular protein complex, widely found in eukaryotes and some prokaryotes. The main function of the proteasome is the selective degradation of the vast majority (80–90%) of intracellular proteins. Therefore, proteasome function is crucial for protein homeostasis and regulates almost all vital life activities.

[0003] Proteasome inhibitors block the UPP (ubiquitin-proteasome pathway) by inhibiting proteasome activity, thereby inhibiting the degradation of antitumor protein molecules and thus suppressing the proliferation of various tumor cells and inducing tumor cell apoptosis. The proteasome and its inhibitors have become novel targets for antitumor therapy. Based on the chemical structure of the pharmacodynamic group, proteasome inhibitors can be classified into five classes: peptidaldehyde (MG-132), borate peptide (bortezomib), peptidoxyketone (epoxymycin, epimycin, and carfilzomib), peptidyl sulfone, and β-lactone (lactacystin and its derivatives). Bortezomib (or PS341) was the first proteasome inhibitor approved for marketing by the U.S. Food and Drug Administration (FDA) and is clinically used to treat multiple myeloma and mantle cell lymphoma. The development of proteasome-targeting inhibitors is currently a hot research topic internationally. Currently, proteasome inhibitors are mainly used to treat hematologic malignancies, and their efficacy against solid tumors is not ideal.

[0004] The TMEM8B gene (also known as the NGX6 gene) is a metastasis suppressor gene closely related to nasopharyngeal carcinoma and colorectal cancer, independently cloned by the inventors' team. Our previous studies demonstrated that the TMEM8B gene encodes two protein isoforms: TMEM8B-a and TMEM8B-b. TMEM8B-a is 472 amino acids long, containing one epidermal growth factor (EGF)-like extracellular domain and seven transmembrane domains; while TMEM8B-b is 338 amino acids long, also containing one EGF-like domain, but only two transmembrane domains. The TMEM8B-b protein isoform was identified and cloned earlier than TMEM8B-a.

[0005] In summary, proteasome inhibitors block UPP by inhibiting proteasome activity, thereby inhibiting the degradation of the antitumor protein molecules (both TMEM8B-a and TMEM8B-b proteins are antitumor protein molecules) by the proteasome, which can inhibit the proliferation of various tumor cells and induce tumor cell apoptosis and related cell death.

[0006] The applicant's prior invention patent CN117756947B first provided a novel recombinant engineered protein, comprising a fusion protein formed by TMEM8B-a protein and a reporter protein, wherein the reporter protein is a fluorescent protein, specifically green or red fluorescent protein. Using this recombinant engineered protein, high-throughput screening of novel proteasome inhibitors can be performed. This invention also provides a corresponding high-throughput screening method for proteasome inhibitors, comprising the following steps: Step A, seeding cells stably expressing the fusion protein, wherein the fusion protein is the aforementioned recombinant engineered protein, into a multi-well plate; Step B, adding a small molecule compound to be tested into the cultured cells; Step C, detecting whether the small molecule compound is a proteasome inhibitor using a fluorescence microscope or a fluorescence multi-functional microplate reader. Using this screening method, it is expected to rapidly and effectively screen for next-generation proteasome inhibitors with better efficacy, higher specificity, fewer side effects, and activity against solid tumors. Furthermore, this high-throughput screening method can be completed in a live cell state without cell lysis or the addition of additional chromogenic or luminescent reagents, making the high-throughput screening method simple, rapid, and accurate. The screening system constructed by this invention can conveniently and quickly screen a large number of compounds in a short time with high throughput, and rapidly and accurately obtain new small molecule inhibitors targeting the proteasome.

[0007] To further investigate the molecular mechanism of TMEM8B-a protein degradation, this study aims to identify which sequences in the TMEM8B-a protein are key domains for degradation, and ultimately obtain a degradation signal peptide capable of effectively degrading the target protein. Summary of the Invention

[0008] The present invention first provides a degradation signal peptide that can mediate the degradation of a target protein directly or indirectly linked to it; the degradation signal peptide contains a sequence identical to that of 6 to 200 amino acids in the TMEM8B protein, that is, the degradation signal peptide is a polypeptide structure containing 6 to 200 amino acids.

[0009] In one specific embodiment, the degradation signal peptide is a polypeptide structure containing 7 to 150 amino acids, preferably a polypeptide structure containing 8 to 60 amino acids, and preferably the degradation signal peptide guides the target protein to the proteasome for degradation.

[0010] In one specific embodiment, the degradation signal peptide comprises a sequence identical to that of amino acids 64 to 123 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 60 amino acids, named degradation signal peptide I; or the degradation signal peptide comprises a sequence identical to that of amino acids 185 to 221 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 37 amino acids, named degradation signal peptide II; or the degradation signal peptide comprises a sequence identical to that of amino acids 228 to 273 of the TMEM8B-a protein, i.e., the degradation signal peptide... The degradation signal peptide is a polypeptide structure containing 46 amino acids, named degradation signal peptide III; or the degradation signal peptide contains the same sequence as amino acids 292 to 316 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 25 amino acids, named degradation signal peptide IV; or the degradation signal peptide contains the same sequence as amino acids 317 to 342 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 26 amino acids, named degradation signal peptide V; or the degradation signal peptide contains amino acids 406 to 417 of the TMEM8B-a protein. The degradation signal peptide is named degradation signal peptide VI if it contains the same sequence as amino acids 406 to 413 of the TMEM8B-a protein, i.e., it is a polypeptide structure containing 8 amino acids; or if it contains the same sequence as amino acids 406 to 421 of the TMEM8B-a protein, i.e., it is a polypeptide structure containing 16 amino acids; or if it contains the same sequence as amino acids 406 to 421 of the TMEM8B-a protein, i.e., it is a polypeptide structure containing 16 amino acids. The degradation signal peptide is a polypeptide structure containing 33 amino acids, consisting of the same sequence as amino acids 406 to 438 of the 8B-a protein; or the degradation signal peptide is a polypeptide structure containing 50 amino acids, consisting of the same sequence as amino acids 423 to 472 of the TMEM8B-a protein; or the degradation signal peptide is a polypeptide structure containing 34 amino acids, consisting of the same sequence as amino acids 439 to 472 of the TMEM8B-a protein.

[0011] In this invention, the amino acid sequence of the TMEM8B-a protein is as clearly described in the prior art, and its specific sequence is as follows:

[0012] 1mnmpqslgnq plppeppslg tpaegpgtts ppehcwpvrp tlrneldtfs vhfyiffgps

[0013] 61valpperpav famrllpvld sggvlslelq lnassvrqen vtvfgclthe vplslgdaav

[0014] 121tcskeslagf llsvsattrv arlripfpqt gtwflalrsl cgvgprfvrc rnataevrmr

[0015] 181tflspcvddc gpygqckllr thnylyaace ckagwrgwgc tdsadaltyg fqllstlllc

[0016] 241lsnlmflppv vlairsryvl eaavytftmf fstfyhacdq pgivvfcimd ydvlqfcdfl

[0017] 301gslmsvwvtv iamarlqpvv kqvlyllgam llsmalqldr hglwnllgps lfalgilata

[0018] 361wtvrsvrrrh cypptwrrwl fylcpgslia gsavllyafv etrdnyfyih siwhmliags

[0019] 421vgfllpprak tdhgvpsgar argcgyqlci neqeelglvg pggatvssic as.

[0020] In one specific embodiment, the target protein includes one or more of fluorescent protein, luciferase, P53 protein, K-Ras protein, and HIV-1 gp120 protein. The fluorescent protein, luciferase, and HIV-1 gp120 protein are all exogenous proteins of mammals. The P53 protein and K-Ras protein are both endogenous proteins of mammals.

[0021] The present invention also provides a fusion protein comprising one or more degradation signal peptides as described above and a reporter protein attached to the N-terminus or C-terminus of the degradation signal peptide, wherein the reporter protein comprises a fluorescent protein or a luciferase.

[0022] In one specific embodiment, the reporter protein is a fluorescent protein, preferably a green fluorescent protein or a red fluorescent protein.

[0023] The present invention also provides an application of the fusion protein as described above for screening proteasome inhibitors, preferably by first establishing cells that stably express the fusion protein, and said cells are 293 series cells or BEAS-2B cells; more preferably said cells are BEAS-2B cells.

[0024] This invention also provides a method for screening proteasome inhibitors, comprising the following steps: Step A, seeding cells stably expressing a fusion protein into a multi-well plate, wherein the fusion protein is any of the fusion proteins described above; Step B, adding a small molecule compound to be tested into the cultured cells; Step C, detecting whether the small molecule compound is a proteasome inhibitor using a fluorescence microscope or a fluorescence multi-functional microplate reader; preferably, before Step A, a step of constructing a stable expression cell line of the fusion protein is included, specifically including first constructing a eukaryotic expression vector with a fluorescent protein fused to its N-terminus or C-terminus, and transfecting it into 293 series cells or BEAS-2B cells, thereby constructing a stable expression cell line of the fusion protein.

[0025] The present invention also provides a biological complex comprising a linker structure and one or more degradation signal peptides as described above. The linker structure is used to connect a target protein and a degradation signal peptide. The linker structure is another protein, polypeptide molecule, nucleic acid molecule, or small molecule compound capable of binding the target protein. The biological complex is synthesized extracellularly and is used to degrade the target protein. Preferably, the linker structure is an antibody capable of binding the target protein. Preferably, the biological complex is used to degrade the target protein in a proteasome.

[0026] The present invention also provides another fusion protein, which includes one or more degradation signal peptides as described above and a target protein attached to the N-terminus or C-terminus of the degradation signal peptide. Preferably, the target protein is an endogenous mammalian protein, and more preferably, the target protein is a P53 protein or a K-Ras protein.

[0027] The present invention has at least the following beneficial effects:

[0028] 1. This invention first screens out eleven degradation signal peptides, all of which can be used to rapidly and completely degrade target proteins. These degradation signal peptides, coupled with the target protein, can efficiently drag the target protein into the proteasome for degradation. Compared to the wild-type full-length TMEM8B-a protein, these degradation signal peptides contain fewer amino acids, are direct active sites, and are less likely to be masked by protein spatial folding; furthermore, their smaller size makes them easier to synthesize in vitro, which is highly beneficial for their downstream applications.

[0029] 2. In many cases, the presence of just one degradation signal peptide is sufficient to achieve protein degradation, or two or more degradation signal peptides can be used in combination to achieve the degradation of the target protein. In this invention, the degradation signal peptide can promote the degradation of the target protein simply by being linked to it. This linkage can be a direct covalent link or other types of linkage, such as indirect linkage through other molecules.

[0030] 3. Utilizing these degradation signal peptides, we constructed and optimized a new generation of high-throughput screening system for small-molecule proteasome inhibitors with extremely high signal-to-noise ratio. The principle is to fuse the degradation signal peptide with a reporter protein (such as GFP) in the cell. Since the degradation signal peptide can be effectively degraded by the proteasome in the cell, the reporter protein is simultaneously degraded by the proteasome under the guidance of the degradation signal peptide. Therefore, under normal proteasome function, GFP is fully degraded in the cell, and the cell does not emit green fluorescence. When various small-molecule compounds are added to the cell, if the small-molecule compound can inhibit the function of the proteasome, the degradation signal peptide-GFP fusion protein cannot be degraded, accumulates in the cell, and green fluorescence can be detected; if the small-molecule compound cannot inhibit the function of the proteasome, the degradation signal peptide-GFP fusion protein is degraded by the proteasome, and the cell does not emit green fluorescence. Commonly used reporter proteins include GFP and RFP. If RFP is used, red fluorescence is used instead of the aforementioned green fluorescence; and other reporter proteins are also feasible, including fluorescent proteins of other colors and luciferases.

[0031] 4. Compared with the first-generation screening system provided in patent CN117756947B, the new system provided by the present invention is more sensitive, with a significant increase in positive signal and a better signal-to-noise ratio, because the degradation signal peptide of the present invention is shorter and has higher translational expression efficiency in cells.

[0032] 5. After replacing the host cells from the 293 series cells with BEAS-2B cells, the cells adhered more stably, and the culture operation was more convenient and stable.

[0033] 6. The new generation screening system provided by this invention can achieve a detection limit as low as 5 nM for the proteasome-specific inhibitor bortezomib PS341. Attached Figure Description

[0034] Figure 1 Fluorescence micrographs showing the effect of degradation signal peptide I and peptides in the comparative example on the degradation of green fluorescent protein in cells.

[0035] Figure 2 Fluorescence micrographs showing the effects of degradation signal peptides II and III, as well as peptides in the comparative example, on the degradation of green fluorescent protein in cells.

[0036] Figure 3 Fluorescence micrographs showing the effects of degradation signal peptides IV and V, as well as peptides in the comparative example, on the degradation of green fluorescent protein in cells.

[0037] Figure 4 Fluorescence micrographs showing the effects of degradation signal peptides VI and VII, as well as peptides in the comparative example, on the degradation of green fluorescent protein in cells.

[0038] Figure 5 Fluorescence micrographs showing the effects of degradation signal peptides VIII and IX, as well as peptides in the comparative example, on the degradation of green fluorescent protein in cells.

[0039] Figure 6 Fluorescence micrographs showing the effects of degradation signal peptides X and XI, as well as peptides in the comparative example, on the degradation of green fluorescent protein in cells.

[0040] Figure 7 This is a fluorescence micrograph of the peptide in the comparative example, showing that it cannot degrade green fluorescent protein within cells.

[0041] Figure 8 Fluorescence micrographs illustrating that the degradation signal peptide XI (439-472AA) described in Example 1 of this invention can be expressed more effectively in BEAS-2B cells and degrade green fluorescent protein copGFP compared to the wild-type full-length peptide.

[0042] Figure 9 This is a Western blot diagram used to illustrate that the K-Ras antibody conjugated to the degradation signal peptide in this invention can effectively degrade K-Ras protein.

[0043] Figure 10 This is a Western blot image used to illustrate that the gp120 antibody conjugated with the degradation signal peptide in this invention can effectively degrade the gp120 protein of HIV-1.

[0044] Figure 11 Western blot analysis of the recombinant protein fused with the degradation signal peptide in 293FT cells (Fig. A) and in PC9 lung cancer cells (Fig. B), showing that the degradation signal peptide can effectively degrade human p53 protein.

[0045] Figure 12 Western blot analysis of recombinant proteins fused with the degradation signal peptide, showing that the degradation signal peptide can effectively degrade human K-ras protein in lung cancer cells A549 and H1299 (Fig. A) and normal human 293FT cells (Fig. B). Detailed Implementation

[0046] The present invention is further illustrated by the following embodiments, comparative examples and drawings, but these embodiments, comparative examples and drawings should not be used to limit the scope of protection of the present invention.

[0047] Example 1 and Comparative Example

[0048] To further investigate the molecular mechanism of TMEM8B-a protein degradation and to identify which sequences of the TMEM8B-a protein are key domains for its degradation, we systematically constructed a large number of deletion mutants, totaling 95, and conducted a detailed and comprehensive study of the full protein sequence of TMEM8B-a protein. We discovered that the TMEM8B-a protein does not possess just one degradation domain, but rather multiple highly efficient degradation domains or degradation peptide structures, including: 1. 64-123AA (60 amino acids); 2. 185-221AA (37 amino acids); 3. 228-273 (46 amino acids); 4. 292-316AA (25 amino acids); 5. 317-342AA (26 amino acids); 6. 406-413AA (8 amino acids), 406-417AA (12 amino acids), 406-421 (16 amino acids), or 406-438AA (33 amino acids), all four of these peptides are effective; 7. 423-472 (50 amino acids) or 439-472AA (34 amino acids), both of these two peptides are effective; a total of 7 key degradation domains. These regions are the core of the degradation process. Many other non-core regions lose their function as degradation proteins, such as 1-47, 92-123, 185-205, 343-405, 414-421, 423-438, and 439-469. These peptides cannot constitute degradation signal peptides. For example, compared to 439-472AA (34 amino acids), 439-469AA (31 amino acids) is only missing 3 amino acid residues, yet it completely loses its degradation function. In this invention, these degradation peptides are defined as degradation signal peptides.

[0049] Figures 1-7 This image illustrates that the 11 degradation signal peptides described in Example 1 of this invention can all degrade green fluorescent protein (copGFP) in the proteasome of 293FT cells, and that several peptides in the comparative example cannot degrade copGFP in the proteasome, as shown in the fluorescence microscopy images. The appropriate amount of the proteasome-specific inhibitor bortezomib (PS341) added to the image effectively inhibits the degradation of copGFP in the proteasome. These degradation signal peptides are shorter, have higher translation efficiency in cells, and compared to the wild-type full-length peptides, PS341 treatment significantly increases the positive signal of copGFP and provides a better signal-to-noise ratio.

[0050] Figure 1 Fluorescence micrographs showing the effect of degradation signal peptide I and peptides in the comparative example on the degradation of green fluorescent protein in cells.

[0051] Figure 1 In Figure A, the recombinant protein fusion of full-length TMEM8B-a and copGFP is expressed in 293FT cells. Without treatment with the proteasome-specific inhibitor bortezomib PS341, the green fluorescent protein (GFP) is almost completely degraded in the proteasome of the 293FT cells, with almost no green fluorescence observed. In Figure B, the recombinant protein fusion of full-length TMEM8B-a and copGFP is expressed in 293FT cells. Treatment with 100 nM bortezomib PS341 shows that due to the effective inhibition of proteasome activity by PS341, the recombinant GFP cannot be degraded in the 293FT cells and accumulates in the cells, resulting in a green fluorescent signal. Figure C shows the peptide in the comparative example, where amino acid sequences 1-47 of the TMEM8B-a protein are used instead of the full-length TMEM8B-a protein fused with copGFP in Figure A. Without treatment with the proteasome-specific inhibitor bortezomib PS341, GFP does not show significant degradation in the 293FT cells. Figure D shows the results of treating cells with 100 nM of the proteasome-specific inhibitor bortezomib PS341, with other conditions identical to Figure C. In this case, the expression of green fluorescent protein (GFP) in 293FT cells did not show significant changes. Figures C and D illustrate that the peptide segment consisting of amino acid sequences 1-47 of the TMEM8B-a protein does not constitute a degradation signal peptide, and this peptide cannot degrade the GFP linked to it. Figure E shows the results of using degradation signal peptide I (64-123AA) from this invention instead of the full-length TMEM8B-a protein linked to copGFP in Figure A. Without treatment with the proteasome-specific inhibitor bortezomib PS341, GFP was almost completely degraded in the proteasome of 293FT cells. Figure F shows the results of using 100 nM of the proteasome-specific inhibitor bortezomib PS341, with other conditions identical to Figure E. In this case, GFP was not completely degraded in the proteasome of 293FT cells. Figures E and F illustrate that the degradation signal peptide I (64-123AA) of this invention can almost completely degrade the green fluorescent protein copGFP attached to it in the proteasome of 293FT cells. A comparison of Figure F and Figure B shows that, compared with the full-length TMEM8B-a protein, the degradation signal peptide I (64-123AA) of this invention is more sensitive, produces a stronger signal, and has a higher signal-to-noise ratio when used for screening proteasome inhibitors.

[0052] Figure 2Fluorescence micrographs showing the intracellular degradation effects of signal peptides II and III, as well as peptides in the comparative example, on green fluorescent protein. Figure 1 Similarly, Figure 2 A and B indicate that the peptide segment consisting of amino acid sequences 92 to 123 of the TMEM8B-a protein cannot serve as a degradation signal peptide, and therefore cannot effectively degrade copGFP in the proteasome. Figure 2 Figures C through F indicate that both degradation signal peptide II (185-221AA) and degradation signal peptide III (228-273AA) can effectively degrade copGFP. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 1 As shown in Figure B, degradation signal peptides II (185-221AA) and III (228-273AA) are more sensitive, have stronger signals, and higher signal-to-noise ratios when used to screen proteasome inhibitors compared to the full-length TMEM8B-a protein; and degradation signal peptide II (185-221AA) is more effective than degradation signal peptide III (228-273AA).

[0053] Figure 3 Fluorescence micrographs showing the intracellular degradation effects of signal peptides IV and V, as well as peptides in the comparative example, on green fluorescent protein. Figure 1 Similarly, Figure 3 In A and B, it is explained that the peptide segment consisting of amino acid sequences 185-205 of the TMEM8B-a protein cannot serve as a degradation signal peptide, and therefore cannot effectively degrade copGFP in the proteasome. Figure 3 Figures C through F indicate that both degradation signal peptides IV (292-316AA) and V (317-342AA) can effectively degrade copGFP. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 1 As shown in Figure B, degradation signal peptides IV (292-316AA) and V (317-342AA) are more sensitive, have stronger signals, and higher signal-to-noise ratios when used to screen proteasome inhibitors compared to the full-length TMEM8B-a protein.

[0054] Figure 4 Fluorescence micrographs showing the intracellular degradation effects of signal peptides VI and VII, as well as peptides in the comparative example, on green fluorescent protein. Figure 1 Similarly, Figure 4 A and B indicate that the peptide segment consisting of amino acid sequences 343 to 405 of the TMEM8B-a protein cannot serve as a degradation signal peptide, and therefore cannot effectively degrade copGFP in the proteasome. Figure 4Figures C through F show that both degradation signal peptides VI (406-417AA) and VII (406-413AA) can effectively degrade copGFP. Furthermore, the peptide segments of degradation signal peptides VI (406-417AA) and VII (406-413AA) are extremely short, containing only 8-12 amino acids, making subsequent operations the most convenient.

[0055] Figure 5 Fluorescence micrographs showing the intracellular degradation effects of degradation signal peptides VIII and IX, as well as peptides in the comparative example, on green fluorescent protein. Figure 1 Similarly, Figure 5 E and F indicate that the peptide segment consisting of amino acid sequences 414-421 of the TMEM8B-a protein cannot serve as a degradation signal peptide, and therefore cannot effectively degrade copGFP in the proteasome. Figure 5 Figures A through D indicate that both degradation signal peptides VIII (406-421AA) and IX (406-438AA) can effectively degrade copGFP. Furthermore, they are related to... Figure 1 As shown in Figure B, degradation signal peptides VIII (406-421AA) and IX (406-438AA) are more sensitive, produce stronger signals, and have a higher signal-to-noise ratio when used to screen proteasome inhibitors compared to the full-length TMEM8B-a protein. Figure 5 and Figure 4 In comparison, the fusion proteins corresponding to degradation signal peptides VIII (406-421AA) and IX (406-438AA) are more sensitive, have stronger signals, and higher signal-to-noise ratios when used to screen proteasome inhibitors compared to the fusion proteins corresponding to degradation signal peptides VI (406-417AA) and VII (406-413AA).

[0056] Figure 6 Fluorescence micrographs showing the intracellular degradation effects of degradation signal peptides X and XI, as well as peptides in the comparative example, on green fluorescent protein. Figure 1 Similarly, Figure 6 In A and B, it is explained that the peptide segment consisting of amino acid sequences 423 to 438 of the TMEM8B-a protein cannot serve as a degradation signal peptide, and therefore cannot effectively degrade copGFP in the proteasome. Figure 6 Figures C through F indicate that both degradation signal peptides X (423-472AA) and XI (439-472AA) can effectively degrade copGFP. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 1As shown in Figure B, degradation signal peptides X (423-472AA) and XI (439-472AA) are more sensitive, have stronger signals, and higher signal-to-noise ratios when used to screen proteasome inhibitors compared to the full-length TMEM8B-a protein.

[0057] Figure 7 This is a fluorescence micrograph of the peptide in the comparative example, showing its inability to degrade green fluorescent protein within cells. Figure 1 Similarly, Figure 6 The sequences A through F indicate that the peptides consisting of amino acid sequences 439–469, 446–472, and 457–472 of the TMEM8B-a protein cannot serve as degradation signal peptides, and therefore cannot effectively degrade copGFP in the proteasome.

[0058] Figure 8 To illustrate that the degradation signal peptide XI (439-472AA) described in Example 1 of this invention can be expressed more effectively in the proteasome of BEAS-2B cells and degrade the green fluorescent protein copGFP compared to the wild-type full-length peptide, a fluorescence microscopy image is provided. Figure 8 C~D and Figure 6 As shown in equations E to F, after replacing the 293 series cells with BEAS-2B, the fusion protein corresponding to the degradation signal peptide, when used for screening proteasome inhibitors, exhibited more thorough degradation of GFP without the addition of proteasome inhibitors. However, after treating cells with the proteasome inhibitor PS341, GFP was not degraded by the proteasome, and the green fluorescence emitted by GFP was the strongest. This indicates that the screening system established in BEAS-2B cells is more sensitive and has a higher signal-to-noise ratio. Subsequent experiments revealed that the detection limit of this screening system for the proteasome-specific inhibitor bortezomib PS341 can be as low as 5 nM.

[0059] Furthermore, since experiments cannot be exhaustive, if other degradation signal peptides are found in TMEM8B-a or TMEM8B-b proteins that can achieve the same or similar effects as the degradation signal peptide described in this invention, they also fall within the scope of protection of this invention. The claims of this invention protect the degradation signal peptide to contain a sequence identical to the 6-200 amino acid sequence in the TMEM8B protein because, firstly, the degradation signal peptide needs to be composed of amino acids of a certain length, and its peptide chain length should not be too short; secondly, the length of the degradation signal peptide should not be too long. When it reaches a maximum of 200 amino acids, its peptide chain length is significantly shorter than the original 472 amino acids of the TMEM8B-a protein. Similarly, it is preferred that the degradation signal peptide is a polypeptide structure containing 7-150 amino acids, and more preferably a polypeptide structure containing 8-60 amino acids.

[0060] Example 2

[0061] This invention artificially synthesizes these degradation signal peptides and conjugates them in vitro with specific antibodies. The target protein is captured by the specific antibody, and then the antibody-conjugated degradation signal peptide carries the entire antigen-antibody complex into the proteasome for degradation, thus achieving specific degradation of the target protein of interest. We first used the degradation signal peptide to conjugate a K-Ras-specific antibody, then added purified human K-Ras protein, 26S proteasome extract, and reaction buffer in vitro, and reacted at 37 degrees Celsius for 1 hour. The samples were then subjected to electrophoresis, and the degradation of K-Ras was detected by Western blot. Figure 9 This is a Western blot diagram used to illustrate that the K-Ras antibody conjugated to the degradation signal peptide in this invention can effectively degrade K-Ras protein.

[0062] Example 3

[0063] Similar to Example 2, this invention uses a degraded peptide conjugated with a gp120-specific antibody, and then adds purified HIV-1 gp120 protein, 26S proteasome extract and reaction buffer in vitro, and reacts at 37 degrees for 1 hour; each sample is electrophoresed, and the degradation of gp120 protein is detected by Western blot. Figure 10 This is a Western blot image used to illustrate that the gp120 antibody conjugated with the degradation signal peptide in this invention can effectively degrade the gp120 protein of HIV-1.

[0064] Examples 2 and 3 illustrate that the degradation signal peptide described in this invention can not only degrade GFP in cells, but also degrade other proteins in vitro, such as K-Ras protein and HIV-1 gp120 protein.

[0065] Example 4

[0066] This embodiment verifies that these degradation signal peptides can effectively degrade mammalian endogenous proteins in cells. This invention uses molecular cloning and recombinant technology to select degradation signal peptides 64-123 (degradation signal peptide I), 185-221 (degradation signal peptide II), 292-316 (degradation signal peptide IV), 406-472 (containing degradation signal peptides IX and X), 423-472 (degradation signal peptide X), and 439-472 (degradation signal peptide XI), and constructs a fusion expression plasmid together with the full-length human p53 protein. The coding DNA sequences of each degradation signal peptide and the coding DNA sequence of the full-length p53 protein are amplified using PCR technology. The degradation signal peptide DNA and the p53 coding DNA are then inserted into the same eukaryotic expression plasmid to construct a recombinant protein expression plasmid fused with degradation signal peptides and p53. Thus, under the control of the same promoter, the recombinant protein fused with the full-length p53 can be transcribed and translated; that is, the degradation signal peptide and p53 are on the same protein molecule, directly linked by peptide bonds of amino acids. Such a protein molecule containing both the amino acid sequence of the degradation signal peptide and the full-length amino acid sequence of p53 is called a fusion protein or fusion recombinant protein.

[0067] The constructed recombinant eukaryotic expression plasmids were transfected into 293FT cells and PC9 lung cancer cells, respectively, with or without the proteasome inhibitor PS341. Cells were collected and lysed, total cellular protein was extracted, and the expression of each recombinant protein was detected by Western blot after electrophoresis. Figure 11 Western blot analysis of the recombinant protein fused with the degradation signal peptide in 293FT cells (Fig. A) and in PC9 lung cancer cells (Fig. B), showing that the degradation signal peptide can effectively degrade human p53 protein. Figure 11 The results showed that the recombinant p53 protein containing the degradation signal peptide underwent significant degradation in cells. After treatment of cells with the proteasome inhibitor PS341, the function of the proteasome was inhibited, and the expression level of the recombinant p53 protein containing the degradation signal peptide was significantly increased. Figure 11 A and Figure 11In section B, cells were treated with or without the proteasome inhibitor PS341. "-" indicates no PS341 treatment, and "+" indicates 50 nM PS341 treatment. Cells were collected and lysed, total cellular protein was extracted, and Western blot analysis was performed after electrophoresis to detect the expression of each recombinant protein. "anti-strep" indicates that the primary antibody used for this Western blot was a strep tag antibody. "β-actin and GAPDH" indicates that the same Western blot membrane was used with β-actin and GAPDH primary antibodies to detect the expression levels of β-actin and GAPDH proteins; these are used for sample loading control and serve as internal control molecules. The numbers and letters on the left of the image represent protein standards of different molecular weights added during electrophoresis, used to indicate the migration positions of proteins of different molecular weights.

[0068] Figure 11 In the image, A represents recombinant expression plasmids fused with the degradation signal peptides (64-123, 185-221, 292-316, 406-472, 423-472, 439-472) and the full-length human p53 protein, respectively, transfected into 293FT cells. The resulting plasmids are 64-123-p53, 185-221-p53, 292-316-p53, 406-472-p53, 423-472-p53, and 439-472-p53. 293FT NC represents 293FT cells transfected with the empty vector as a negative control. Figure 11 In the middle B, recombinant expression plasmids fused with the full-length human p53 protein were transfected into PC9 lung cancer cells with degradation signal peptides 185-221 (degradation signal peptide II) and 423-472 (degradation signal peptide X), respectively. These plasmids are 185-221-p53 and 423-472-p53.

[0069] Example 5

[0070] Following the same strategy as in Example 4, we further constructed a fusion expression plasmid using molecular cloning and recombination technology to degrade the signal peptides (185-221, 423-472) and human K-ras protein. The constructed recombinant eukaryotic expression plasmid was transfected into lung cancer cells A549, lung cancer cells H1299, and normal human 293FT cells, respectively. Cells were treated with or without the proteasome inhibitor PS341. Cells were collected and lysed, total cellular protein was extracted, and the expression of each recombinant protein was detected by Western blot after electrophoresis. Figure 12 Western blot analysis of recombinant proteins fused with the degradation signal peptide, showing that the degradation signal peptide can effectively degrade human K-ras protein in lung cancer cells A549 and H1299 (Fig. A) and normal human 293FT cells (Fig. B). Figure 12The results showed that the K-ras recombinant protein containing the degradation signal peptide underwent significant degradation in cells. After treatment of cells with the proteasome inhibitor PS341, the function of the proteasome was inhibited, and the expression level of the K-ras recombinant protein containing the degradation signal peptide was significantly increased.

[0071] Figure 12 In the middle, A represents lung cancer cells A549 and H1299. Figure 12 In Figure B, recombinant expression plasmids fused with the degradation signal peptides (185-221, 423-472) and the full-length human K-ras protein, respectively, were transfected into normal human 293FT cells, namely 185-221-kras and 423-472-kras. NC represents cells transfected with the empty vector as a negative control. In Figures A and B, cells were treated with or without the proteasome inhibitor PS341 (- indicates no PS341 treatment, + indicates 50 nM PS341 treatment). Cells were collected, lysed, and total cellular protein was extracted. The expression of each recombinant protein was detected by Western blot after electrophoresis. "anti-strep" indicates that the primary antibody used for this Western blot membrane is a strep tag antibody; "anti-β-actin" and "anti-GAPDH" indicate that the primary antibodies used for this Western blot membrane are β-actin antibody and GAPDH antibody, respectively; β-actin and GAPDH indicate that the same Western blot membrane was used to detect the expression levels of β-actin and GAPDH proteins, respectively, and are used for sample loading control as internal control molecules. The numbers and letters on the left of the image represent protein standards of different molecular weights added during electrophoresis to indicate the migration positions of proteins of different molecular weights.

[0072] In summary, both the degradation signal peptide and the target protein described in this invention can be rapidly and completely degraded by the proteasome, rather than undergoing controlled degradation. There is no feedback mechanism that prevents further degradation of the target protein when its level falls below a certain value, thus avoiding incomplete degradation of the target protein. Furthermore, the degradation signal peptide described in this invention is not masked or interfered with by fluorescent proteins, and can successfully carry fluorescent proteins to the proteasome for breakdown while undergoing its own degradation.

[0073] The degradation signal peptide provided by this invention, due to its small amino acid count, can be easily added to various sites of the target protein, or more easily and conveniently synthesized artificially in vitro. The degradation signal peptide has a higher degradation efficiency, and can more effectively guide various target proteins to the proteasome for degradation.

[0074] Using the screening system described in this invention, a large number of compounds can be screened in a short time using high throughput, rapidly and accurately obtaining new small-molecule proteasome inhibitors targeting the proteasome. The entire high-throughput screening can be completed in only three steps, and the whole process can be carried out in a live cell state without lysing cells or adding additional chromogenic or luminescent reagents.

[0075] This invention first provides a degradation signal peptide that mediates the degradation of a target protein directly or indirectly linked to it. The degradation signal peptide contains a sequence identical to that of the TMEM8B protein, consisting of 6-200 amino acids; that is, the degradation signal peptide is a polypeptide structure containing 6-200 amino acids. The coupling of the degradation signal peptide and the target protein provided by this invention can efficiently and completely degrade the target protein. Compared to the wild-type full-length TMEM8B-a protein, these degradation signal peptides contain fewer amino acids, are direct active sites, and are less likely to be masked by protein spatial folding. Furthermore, their smaller size makes them easier to synthesize in vitro, which is highly beneficial for downstream applications.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A degradation signal peptide, characterized in that, The degradation signal peptide can mediate the degradation of its target protein, which is directly or indirectly linked to it; the degradation signal peptide contains a sequence identical to that of the TMEM8B protein, consisting of 6 to 200 amino acids, i.e., the degradation signal peptide is a polypeptide structure containing 6 to 200 amino acids.

2. The degradation signal peptide according to claim 1, characterized in that, The degradation signal peptide is a polypeptide structure containing 7 to 150 amino acids, preferably a polypeptide structure containing 8 to 60 amino acids, and preferably the degradation signal peptide guides the target protein to the proteasome for degradation.

3. The degradation signal peptide according to claim 1, characterized in that, The degradation signal peptide contains the same sequence as amino acids 64 to 123 of the TMEM8B-a protein, that is, the degradation signal peptide is a polypeptide structure containing 60 amino acids, and is named degradation signal peptide I. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 185 to 221 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 37 amino acids, and is named degradation signal peptide II. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 228 to 273 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 46 amino acids, named degradation signal peptide III. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 292 to 316 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 25 amino acids, named degradation signal peptide IV. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 317 to 342 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 26 amino acids, named degradation signal peptide V. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 406 to 417 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 12 amino acids, named degradation signal peptide VI. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 406 to 413 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 8 amino acids, and is named degradation signal peptide VII. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 406 to 421 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 16 amino acids, named degradation signal peptide VIII. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 406 to 438 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 33 amino acids, named degradation signal peptide IX. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 423 to 472 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 50 amino acids, named degradation signal peptide X. Alternatively, the degradation signal peptide may contain a sequence identical to that of amino acids 439 to 472 of the TMEM8B-a protein, i.e., the degradation signal peptide is a polypeptide structure containing 34 amino acids, and is named degradation signal peptide XI.

4. The degradation signal peptide according to claim 1, characterized in that, The target proteins include one or more of the following: fluorescent protein, luciferase, P53 protein, K-Ras protein, and HIV-1 gp120 protein.

5. A fusion protein, characterized in that, The fusion protein includes one or more degradation signal peptides as described in any one of claims 1 to 4 and a reporter protein attached to the N-terminus or C-terminus of the degradation signal peptide, wherein the reporter protein includes a fluorescent protein or a luciferase.

6. A fusion protein according to claim 5, characterized in that, The reporter protein is a fluorescent protein, preferably a green fluorescent protein or a red fluorescent protein.

7. The application of the fusion protein as described in any one of claims 5 to 6 for screening proteasome inhibitors; preferably, cells stably expressing the fusion protein are first established, and said cells are 293 series cells or BEAS-2B cells, more preferably said cells are BEAS-2B cells.

8. A method for screening proteasome inhibitors, characterized in that, Includes the following steps: Step A: Seed cells stably expressing the fusion protein into a multi-well plate, wherein the fusion protein is the fusion protein as described in any one of claims 5 to 6; Step B: Add the small molecule compound to be tested into the cultured cells; Step C: Use a fluorescence microscope or a fluorescence multi-functional microplate reader to detect whether the small molecule compound is a proteasome inhibitor; Preferably, before step A, there is a step of constructing a stable expression cell line for the fusion protein. Specifically, this includes first constructing a eukaryotic expression vector with a fluorescent protein fused to the N-terminus or C-terminus, and then transfecting it into 293 series cells or BEAS-2B cells, thereby constructing a stable expression cell line for the fusion protein.

9. A biological complex comprising a linker structure and one or more degradation signal peptides as described in any one of claims 1 to 4, wherein the linker structure is used to link a target protein and a degradation signal peptide, the linker structure being other proteins, polypeptides, nucleic acids, or small molecule compounds capable of binding the target protein, the biological complex being synthesized extracellularly and used to degrade the target protein; preferably, the linker structure is an antibody capable of binding the target protein; preferably, the biological complex is used to degrade the target protein in a proteasome.

10. A fusion protein, characterized in that, The fusion protein includes one or more degradation signal peptides as described in any one of claims 1 to 4 and a target protein attached to the N-terminus or C-terminus of the degradation signal peptide. Preferably, the target protein is an endogenous mammalian protein, and more preferably, the target protein is a P53 protein or a K-Ras protein.

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