Polypeptide for inhibiting RIPK1 protein liquid-liquid phase separation and application thereof in regulating programmed necrosis of cells

Abstract

The invention discloses a polypeptide for inhibiting RIPK1 protein liquid-liquid phase separation and application of the polypeptide in regulating and controlling programmed necrosis of cells, the polypeptide for inhibiting RIPK1 protein liquid-liquid phase separation is a fusion polypeptide of a cell penetrating peptide and an RIPK1 protein SUMO modification inhibition polypeptide, and the RIPK1 protein SUMO modification inhibition polypeptide is a targeting polypeptide targeting RIPK1 protein. The SUMO modification of lysine at the 305th site of the RIPK1 protein is inhibited. The polypeptide for inhibiting liquid-liquid phase separation of the RIPK1 protein can inhibit SUMOylation modification of endogenous RIPK1 protein in cells, so that liquid-liquid phase separation of the RIPK1 protein is limited, phase conversion of the RIPK1 protein is effectively relieved, and programmed cell necrosis mediated by the RIPK1 is remarkably reduced under the condition that the activity of RIPK1 kinase is not changed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a polypeptide that inhibits the liquid-liquid phase separation of RIPK1 protein and its application in regulating programmed cell necrosis. Background Art

[0002] Necroptosis is a caspase-independent form of regulated cell death tightly regulated by the RIPK1 / RIPK3 / MLKL signaling axis, with membrane pore formation mediated by the mixed-lineage kinase domain protein MLKL. Necroptosis typically releases damage-associated molecular patterns, pathogen-associated molecular patterns, and cellular components, leading to a severe secondary inflammatory response. Therefore, the development of necroptosis requires tight regulation by multiple signaling pathways and key molecules. Developing novel approaches to mitigate necroptosis based on the discovery of novel mechanisms holds great promise for treating a variety of inflammatory and infectious diseases. Receptor-interacting protein kinase 1 (RIPK1) is a key determinant of necroptosis initiation, and its function has been previously reported to be primarily regulated by kinase activity and protein stability. Existing technologies and patents disclose compounds that inhibit RIPK1 activity, which can be used to reduce RIPK1-mediated necroptosis. However, altering RIPK1 kinase activity may affect other biological functions beyond RIPK1-mediated cell death, leading to varying degrees of side effects. Therefore, there is an urgent need to provide a new preparation and method that can effectively regulate RIPK1 function without affecting the RIPK1 kinase activity, thereby reducing cell programmed necrosis. Summary of the Invention

[0003] The purpose of the present invention is to overcome the defects and shortcomings of the prior art that require regulating programmed cell necrosis by affecting the activity of RIPK1 kinase, and to provide a polypeptide that inhibits the liquid-liquid phase separation of RIPK1 protein.

[0004] The second object of the present invention is to provide the use of the polypeptide in regulating programmed cell necrosis.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0006] The present invention first provides a polypeptide that inhibits the liquid-liquid phase separation of RIPK1 protein, wherein the polypeptide is a fusion polypeptide of a cell-penetrating peptide and a RIPK1 protein SUMOylation modification inhibitory polypeptide; the RIPK1 protein SUMOylation modification inhibitory polypeptide is a targeted polypeptide targeting the RIPK1 protein, and is used to inhibit the SUMOylation modification of lysine at position 305 of the RIPK1 protein.

[0007] Phase transitions of biomacromolecules play a key role in the orderly regulation of complex reactions in cellular life activities. Common forms of phase separation in cellular life activities include liquid-liquid phase separation and liquid-solid phase separation. Liquid-liquid phase separation of biomacromolecules is a special way for biomacromolecules to accumulate in cells to form membraneless compartments. It has the characteristics of regulating specific molecules to form a certain order in the chaotic intracellular environment. Liquid-liquid phase separation drives specific proteins from a free state to droplets of aggregated state, which not only realizes the functional area division of specific molecules with local high concentrations in the cell, but also maintains the frequent material exchange between specific molecules in membraneless organelles and the surrounding environment in a highly dynamic and reversible manner, and is widely involved in the assembly of regulatory signal networks for biological events and the formation of a new mechanism for functional molecular order.

[0008] This study, published in Nature Communications, identifies for the first time that RIPK1 undergoes a phase transition, characterized by liquid-liquid phase separation, during programmed necrosis (PNC). The study also demonstrates that PNC is a novel pathway for initiating PNC, independent of classical activation. The study further demonstrates that SUMO1-mediated covalent modification of RIPK1 at lysine 305 is key to promoting RIPK1 "droplet" formation, indicating that SUMOylation is a mechanism regulating RIPK1 phase separation. RIPK1 K305R mutation or competitive SUMOylation of RIPK1 at lysine 305 significantly reduces RIPK1 aggregate formation, restricting RIPK1 PNC liquid-liquid phase separation and thus protecting the host from PNC damage. Given that the present invention discloses that lysine 305 of the RIPK1 protein is a key site for SUMOylation-induced PNC liquid-liquid phase separation, it is readily feasible for those skilled in the art to design polypeptides that inhibit SUMOylation of lysine 305 of the RIPK1 protein using conventional techniques. Therefore, any targeted polypeptide that targets the RIPK1 protein and is used to inhibit the SUMOylation modification of lysine at position 305 of the RIPK1 protein should theoretically fall within the scope of protection of the present invention.

[0009] Furthermore, the present invention designs a RIPK1 protein SUMOylation inhibitory polypeptide based on the principle of competitive inhibition. This peptide can competitively inhibit endogenous RIPK1 protein SUMOylation, thereby limiting RIPK1's ability to undergo liquid-liquid phase separation and reducing programmed cell death. The RIPK1 protein SUMOylation inhibitory polypeptide is a polypeptide that competes for RIPK1 protein SUMOylation at lysine 305.

[0010] Furthermore, the amino acid sequence of the RIPK1 protein SUMOylation modification inhibitory polypeptide is shown in SEQ ID No. 1: EDVKSLKKEYS.

[0011] Furthermore, the cell penetrating peptide is a TAT protein transport peptide.

[0012] Furthermore, the amino acid sequence of the polypeptide that inhibits liquid-liquid phase separation of RIPK1 protein is shown in SEQ ID No. 2: EDVKSLKKEYSGRKKRRQRRRPPQ.

[0013] The present invention also provides use of a preparation for inhibiting the liquid-liquid phase separation of RIPK1 protein in the preparation of a programmed cell necrosis inhibitor.

[0014] Furthermore, the preparation for inhibiting the liquid-liquid phase separation of RIPK1 protein is a preparation for site-directed mutation of lysine 305 of RIPK1, so that lysine is mutated to arginine (K305R).

[0015] Furthermore, the liquid-liquid phase separation inhibitory preparation for RIPK1 protein is any of the above-mentioned polypeptides for inhibiting liquid-liquid phase separation of RIPK1 protein.

[0016] Furthermore, the inhibitor inhibits the liquid-liquid phase separation of RIPK1 protein through the polypeptide, thereby reducing the level of programmed cell necrosis.

[0017] Furthermore, the polypeptide competitively inhibits the SUMOylation modification of RIPK1 to inhibit the liquid-liquid phase separation of the RIPK1 protein.

[0018] The polypeptides of the present invention that inhibit RIPK1 protein liquid-liquid phase separation can inhibit programmed cell death and can be used to prevent and treat diseases caused by programmed cell death. Therefore, the present invention also provides a RIPK1 protein K305R site-directed mutagenesis reagent or the use of any of the polypeptides that inhibit RIPK1 protein liquid-liquid phase separation in the preparation of a medicament for preventing or treating diseases caused by programmed cell death.

[0019] Furthermore, diseases caused by programmed cell necrosis include but are not limited to autoimmune diseases and neurodegenerative diseases.

[0020] The present invention provides a drug for treating diseases caused by programmed cell necrosis, comprising a RIPK1 protein K305R site-directed mutagenesis reagent or any of the polypeptides that inhibit the liquid-liquid phase separation of the RIPK1 protein.

[0021] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a polypeptide that inhibits the liquid-liquid phase separation of RIPK1 protein. The polypeptide is a fusion polypeptide of a cell-penetrating peptide and a RIPK1 protein SUMOylation-inhibiting polypeptide. The RIPK1 protein SUMOylation-inhibiting polypeptide is a targeted polypeptide that targets the RIPK1 protein and is used to inhibit the SUMOylation modification of lysine at position 305 of the RIPK1 protein. The polypeptide that inhibits the liquid-liquid phase separation of RIPK1 protein can inhibit the SUMOylation modification of endogenous RIPK1 protein in cells, thereby limiting the liquid-liquid phase separation of RIPK1 protein, effectively relieving the phase transition of RIPK1 protein, and significantly reducing RIPK1-mediated programmed cell necrosis without changing the RIPK1 kinase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of RIPK1 and K305 amino acid site.

[0025] Figure 2 Figure 3. Sanger sequencing results of the vector constructed for the RIPK1 K305 site mutation.

[0026] Figure 3 The figure shows the results of immunoprecipitation technology combined with immunoblotting analysis of RIPK1 mutants.

[0027] Figure 4 The figure shows the results of immunoblotting analysis of the kinase activity of RIPK1 K305 site mutants.

[0028] Figure 5 Constructed gel image of RIPK1 knockout human cervical cancer cells HeLa.

[0029] Figure 6 This is a fluorescent image showing the disaggregation and fusion of droplets formed by RIPK1 during programmed cell necrosis.

[0030] Figure 7 Image of droplets formed by RIPK1 lacking SUMOylation modification (K305R mutant).

[0031] Figure 8 A gel image was constructed for RIPK1 knockout human colon cancer cell line HT-29.

[0032] Figure 9 Gel images were constructed for human colon cancer cells HT-29 inducibly expressing RIPK1 (WT and K305R mutant).

[0033] Figure 10 The cell death rate in the group treated with RIPK1(K305R) lacking SUMOylation modification.

[0034] Figure 11Schematic diagram of the construction of the cell-penetrating peptide CPP and RIPK1 fusion peptide CPP-K and CPP-R as a control.

[0035] Figure 12 This is the mass spectrometry peak identification diagram of the fusion polypeptide CPP-K (P30929).

[0036] Figure 13 This is the mass spectrometry peak identification diagram of the fusion polypeptide CPP-R (P31346).

[0037] Figure 14 Fluorescence images showing the effects of CPP-K and CPP-R treatment on the formation of RIPK1 droplets.

[0038] Figure 15 This is a statistical graph showing the effects of CPP-K and CPP-R treatment on programmed cell necrosis in human colon cancer cell HT-29. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0041] The RIPK1 protein number is: Q13546.

[0042] Example 1 Identification of RIPK1 SUMOylation Sites and Basic Sequences

[0043] Identify the sites and base sequences where RIPK1 SUMOylation occurs. The specific methods and results are as follows:

[0044] RIPK1 (294-316) amino acid sequence Figure 1 As shown. RIPK1 mutation vector was constructed using site-directed mutagenesis technology, and the vector Sanger sequencing results were shown as follows. Figure 2 As shown in Figure 3, lysine at position 305 was mutated to arginine (K305R). The results of immunoprecipitation combined with immunoblotting analysis are shown in Figure 3. Figure 3 As shown, the RIPK1 mutant (K305R) lacks SUMO1-mediated covalent modification, that is, the RIPK1 mutant (K305R) lacks SUMOylation modification, indicating that lysine K305 at position 305 in RIPK1 undergoes SUMOylation modification.

[0045] Example 2 demonstrates that SUMOylation promotes RIPK1 liquid-liquid phase separation and its mediated programmed cell necrosis

[0046] Overexpression of wild-type RIPK1 or K305R mutant RIPK1 and treatment with the RIPK1 kinase activity inhibitor Nec-1, such as Figure 4 As shown in Figure 3, the kinase activity of the RIPK1 mutant K305R was not significantly different from that of wild-type RIPK1, indicating that the K305R mutation did not affect the kinase activity of RIPK1. Figure 5 As shown, RIPK1 was knocked out in human cervical cancer cells HeLa. The fluorescent plasmid expressing RIPK1 was transfected into human cervical cancer cells HeLa with RIPK1 knocked out, and a cell programmed necrosis model was established using TNFα (20ng / ml), BV6 (1μM) and zVAD (20μM). Green fluorescence was observed after 6 hours. Figure 6 As shown, RIPK1 forms droplets, and the droplets undergo disaggregation and fusion.

[0047] Fluorescent plasmids expressing RIPK1 and RIPK1 mutants were transfected into human cervical cancer cells HeLa with RIPK1 knockout. TNFα (20 ng / ml), BV6 (1 μM) and zVAD (20 μM) were used to establish a cell programmed necrosis model. Green fluorescence was observed after 6 hours. Figure 7 As shown in Figure 3, the droplets formed by RIPK1 lacking SUMOylation modification (K305R) are smaller. Figure 8 As shown, RIPK1 was knocked out in human colon cancer cells HT-29. Wild-type RIPK1 or K305R mutant RIPK1 with Tet-on system was stably transferred into human colon cancer cells HT-29 with RIPK1 knocked out. The cells were treated with Dox and collected after induction with different concentrations of Dox. The expression level of RIPK1 protein was detected by immunoblotting analysis using RIPK1-specific antibody, as shown in Figure 2. Figure 9 As shown in Figure 2, as the concentration of Dox increases, the level of RIPK1 protein increases. The above-mentioned induced expression cells were treated with TNFα (10 ng / ml), BV6 (1 μM), and zVAD (20 μM). After 16 hours, the number of living cells was detected by measuring the intracellular ATP content, as shown in Figure 2. Figure 10 As shown, the cell death rate in the SUMOylated RIPK1(K305R)-deficient group was significantly reduced.

[0048] Based on Examples 1 and 2, the present invention studies the phase changes of RIPK1 during programmed necrosis, which leads to liquid-liquid phase separation. It confirms that RIPK1 liquid-liquid phase separation is a novel pathway for initiating programmed necrosis, independent of the classical activation pathway. It also demonstrates that RIPK1 lysine K305 at position 305 undergoes SUMOylation and that SUMOylation promotes RIPK1 liquid-liquid phase separation and mediated cellular programmed necrosis. This indicates that SUMOylation at position 305 of RIPK1, mediated by SUMO1, is key to promoting the formation of RIPK1 "droplets."

[0049] Example 3 Design of RIPK1 protein SUMOylation inhibitory peptide CPP-K and its effect on RIPK1 liquid-liquid phase separation

[0050] (1) Using the principle of competitive inhibition, we designed a peptide CPP-K that inhibits RIPK1 protein SUMOylation.

[0051] Given that the site of RIPK1 SUMOylation modification is K305, and the short peptide motif of SUMOylation modification is usually about 12 amino acids near the modification site. Therefore, the RIPK1 (299-309) short peptide was designed as a "bait" for SUMOylation modification, and its amino acid sequence is as shown in SEQ ID No.1: EDVKSLKKEYS, which uses the principle of competitive inhibition to reduce the SUMOylation modification of endogenous RIPK1. CPPtat is a transport peptide of HIV-1 virus TAT ​​protein, which can carry in vitro synthesized polypeptides into cells. Figure 11 As shown in Figure 2, a fusion peptide CPP-K of CPPtat and RIPK1 (299-309) and a fusion peptide CPP-R as a control were constructed. After HPLC purification and ESI mass spectrometry identification, the fusion peptide CPP-K results were as follows: Figure 12 The amino acid sequence of CPP-K is shown in SEQ ID No. 2: EDVKSLKKEYSGRKKRRQRRRPPQ, and the results of the control fusion polypeptide CPP-R are shown in Figure 13 shown.

[0052] (2) Using cell-penetrating peptides to deliver in vitro synthesized CPP-K peptides into cells, restricting RIPK1 from undergoing liquid-liquid phase separation

[0053] CPP-K and control peptide CPP-R were added to the culture medium of human cervical cancer cells HeLa transfected with GFP-RIPK1 at a final concentration of 25 μM. After incubation for 4 hours, TNFα (20 ng / ml), BV6 (1 μM) and zVAD (20 μM) were added to establish a cell programmed necrosis model. Green fluorescence was observed after 6 hours. Figure 14As shown, the RIPK1 droplets in the CPP-K treatment group were smaller, but the control peptide CPP-R had no effect on the RIPK1 droplets. CPP-K and the control peptide CPP-R were added to the culture medium of human colon cancer cells HT-29 at a final concentration of 25 μM. After incubation for 4 hours, TNFα (50 ng / ml), BV6 (1 μM) and zVAD (20 μM) were added to establish a cell programmed necrosis model. After 8 hours, the number of living cells was detected by measuring the intracellular ATP content. Figure 15 As shown, the cell death rate in the CPP-K treatment group was significantly reduced, but no effect of the control peptide CPP-R on the cell death rate was observed.

[0054] Since the only difference between CPP-K and CPP-R is that CPP-R cannot undergo SUMOylation modification (carries the K305R mutation), the above results indicate that CPP-K reduces cell programmed necrosis by competing for RIPK1 SUMOylation to limit the liquid-liquid phase separation function of RIPK1.

Claims

1. A polypeptide for inhibiting liquid-liquid phase separation of RIPK1 protein, characterized in that: The polypeptide is a fusion polypeptide of a cell-penetrating peptide and a RIPK1 protein SUMOylation modification inhibitory polypeptide; the RIPK1 protein SUMOylation modification inhibitory polypeptide is a targeting polypeptide targeting the RIPK1 protein, and is used to inhibit the SUMOylation modification of lysine at site 305 of the RIPK1 protein.

2. The polypeptide for inhibiting liquid-liquid phase separation of RIPK1 protein according to claim 1, characterized in that The amino acid sequence of the RIPK1 protein SUMOylation inhibition polypeptide is shown in SEQ ID No.

1.

3. The polypeptide for inhibiting liquid-liquid phase separation of RIPK1 protein according to claim 1, characterized in that The cell penetrating peptide is a TAT protein transport peptide.

4. The polypeptide for inhibiting liquid-liquid phase separation of RIPK1 protein according to claim 1, characterized in that The amino acid sequence of the polypeptide that inhibits the liquid-liquid phase separation of RIPK1 protein is shown in SEQ ID No.

2.

5. Use of a RIPK1 protein K305R site-directed mutagenesis reagent or a polypeptide for inhibiting RIPK1 protein liquid-liquid phase separation according to any one of claims 1 to 4 in the preparation of a necroptosis inhibitor.

6. The application according to claim 5, characterized in that The inhibitor inhibits the liquid-liquid phase separation of RIPK1 protein through the polypeptide, thereby reducing the level of programmed cell necrosis.

7. The application according to claim 6, characterized in that The polypeptide competitively inhibits the SUMOylation modification of RIPK1 to inhibit the liquid-liquid phase separation of the RIPK1 protein.

8. Use of a RIPK1 protein K305R site-directed mutagenesis reagent or a polypeptide for inhibiting RIPK1 protein liquid-liquid phase separation according to any one of claims 1 to 4 in the preparation of a drug for preventing or treating diseases caused by programmed cell necrosis.

9. A drug for treating a disease caused by programmed cell necrosis, characterized in that: A reagent for site-directed mutagenesis of RIPK1 protein K305R or a polypeptide for inhibiting liquid-liquid phase separation of RIPK1 protein according to any one of claims 1 to 4.

10. The drug according to claim 9, characterized in that It also contains pharmaceutically acceptable excipients.

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