Polypeptide and application thereof

Abstract

The invention discloses a polypeptide and application thereof, and relates to the technical field of biology. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. The polypeptide designed based on the precise binding site of the Gabp alpha and the Ppargamma comprises an aa164-189 amino acid sequence fragment of the Ppargamma, ARG168 and LYS184 sites can be specifically bound with ASP332 and GLN324 sites of the Gabp alpha respectively, a natural binding interface of the Gabp alpha and the Ppargamma can be competitively occupied, specific blocking of the interaction of the Gabp alpha and the Ppargamma is achieved, and the effect of inhibiting the Gabp alpha and the Ppargamma is achieved. Non-specific interference caused by interaction of other proteins is avoided, and a high-specificity tool is provided for targeted regulation of a brown adipose tissue related metabolic process.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a polypeptide and its applications. Background Technology

[0002] Glycolysis is a crucial metabolic process in living organisms, playing an irreplaceable role in the thermogenesis regulation of brown adipose tissue (BAT). As a key thermogenic tissue, the glycolytic efficiency of brown adipose tissue directly impacts energy balance and has a potential link to the development of metabolic disorders such as obesity and diabetes. Studies have identified GA-binding protein α (Gabpα) as a core transcriptional regulator maintaining the glycolytic capacity of brown adipocytes. Gabpα interacts with peroxisome proliferator-activated receptor g (Pparγ) to form the Gabpα-Pparγ complex, which synergistically promotes the transcription of key glycolytic genes (such as enolase 1Eno1), ultimately regulating the glycolytic efficiency and thermogenesis function of brown adipose tissue.

[0003] Current technologies have not yet clarified the specific binding sites of Gabpα and Pparγ, nor have they developed any tool molecules or candidate intervention components that can specifically block their binding. This not only makes it impossible to accurately verify the core role of Gabpα-Pparγ binding in the regulation of glycolysis and thermogenesis in brown adipose tissue, but also results in a lack of direct experimental support for the study of related metabolic mechanisms, thus causing the intervention research on metabolic abnormalities related to brown adipose tissue to still face bottlenecks. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the technical problem solved by this application is: how to specifically block the binding sites of Gabpα and Pparγ.

[0005] In a first aspect, embodiments of this application provide a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0006] In conjunction with the first aspect, in one embodiment, the polypeptide comprises an amino acid sequence fragment of peroxisome proliferator-activated receptor Pparγ, specifically amino acid segments aa164-189.

[0007] In conjunction with the first aspect, in one embodiment, the polypeptide contains an ARG168 site and a LYS184 site, wherein ARG168 binds to the ASP332 site of the GA-binding protein a chain, and LYS184 binds to the GLN324 site of the GA-binding protein a chain.

[0008] In conjunction with the first aspect, in one embodiment, the polypeptide competitively binds to the GA-binding protein a chain with Pparγ.

[0009] Secondly, embodiments of this application provide the application of a polypeptide in inhibiting the binding of the GA-binding protein a chain to the peroxisome proliferator-activated receptor Pparγ.

[0010] Thirdly, embodiments of this application provide an application of a polypeptide in regulating the glycolysis process of brown adipose tissue.

[0011] In conjunction with the third aspect, in one embodiment, the regulation of glycolysis in brown adipose tissue is a thermogenic process.

[0012] Fourthly, embodiments of this application provide the application of a polypeptide in reducing lactate concentration or downregulating lactate dehydrogenase expression.

[0013] Fifthly, embodiments of this application provide an application of a polypeptide in regulating the expression of coupling protein 1.

[0014] Compared with the prior art, the advantages of this application are: The peptide designed based on the precise binding sites of Gabpα and Pparγ contains the aa164-189 amino acid sequence fragment of Pparγ, where the ARG168 and LYS184 sites can specifically bind to the ASP332 and GLN324 sites of Gabpα, respectively. This allows it to competitively occupy the natural binding interface between Gabpα and Pparγ, achieving specific blocking of their interaction and avoiding non-specific interference with other protein interactions. This provides a highly specific tool for targeted regulation of metabolic processes related to brown adipose tissue. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the test results in Embodiment 1 of this application; Figure 2 This is a schematic diagram of the test results in Embodiment 2 of this application; Figure 3 This is a schematic diagram of the test results in Embodiment 3 of this application; Figure 4 This is a schematic diagram of the test results in Embodiment 4 of this application. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0018] The flowchart shown in the attached diagram is for illustrative purposes only and does not necessarily include all content and operations / steps, nor does it necessarily have to be performed in the order described. For example, some operations / steps can be broken down, combined, or partially merged, so the actual execution order may change depending on the actual situation.

[0019] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0020] In a first aspect, embodiments of this application provide a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1; the sequence of SEQ ID NO:1 is: YGRKKRRQRRRRRTIRLKLIYDRCDLNCRIHKKSRNK; The polypeptide is defined as GPCBi.

[0021] The polypeptide contains the aa164-189 amino acid sequence fragment of the peroxisome proliferator-activated receptor Pparγ, which contains the ARG168 site and the LYS184 site. The ARG168 site binds to the ASP332 site of the GA-binding protein a chain (Gabpα), and the LYS184 site binds to the GLN324 site of the GA-binding protein a chain (Gabpα).

[0022] This peptide, designed based on the precise binding sites of Gabpα and Pparγ, contains the aa164-189 amino acid sequence fragment of Pparγ. The ARG168 and LYS184 sites can specifically bind to the ASP332 and GLN324 sites of Gabpα, respectively. It can competitively occupy the natural binding interface between Gabpα and Pparγ, achieving specific blocking of their interaction and avoiding non-specific interference with other protein interactions. This provides a highly specific tool for targeted regulation of metabolic processes related to brown adipose tissue.

[0023] In one embodiment, GPCBi is obtained according to the peptide solid-phase synthesis method designed by Merrifield, extending sequentially from the carboxyl terminus to the amino terminus according to the sequence of SEQ ID NO:1 until the full length of GPCBi is synthesized; the specific method for obtaining GPCBi includes: S1. Determine the materials required for the condensation reaction: Fmoc-Asn-OH, TBTU, NMM, and DMF; S2. After the carboxyl group of Lys is tightly bound to the insoluble polymer resin, the amino protecting group adsorbed on the first amino acid of the solid resin is removed, and a condensation reaction is initiated to generate an amide bond. The temperature of the condensation reaction is 35℃ and the reaction time is 40min. S3. Perform washing, deprotection, end sealing and washing processes in sequence; S4. Repeat steps S2 and S3 to extend the peptide chain amino by amino group until the complete peptide chain of GPCBi is obtained. S5. After removing GPCBi from the resin, and then purifying and lyophilizing it, the final polypeptide product, GPCBi, is obtained.

[0024] Secondly, embodiments of this application provide the application of a polypeptide in inhibiting the binding of the GA-binding protein a chain to the peroxisome proliferator-activated receptor Pparγ.

[0025] In this embodiment, GPCBi specifically binds to the ASP332 and GLN324 sites of Gabpα through its ARG168 and LYS184 sites, competitively blocking the natural interaction between Gabpα and Pparγ. It can be used as a specific tool molecule to verify the function of Gabpα-Pparγ interaction in cellular physiological processes, and can also be used as a candidate intervention molecule to regulate pathological processes that depend on Gabpα-Pparγ interaction.

[0026] Thirdly, embodiments of this application provide an application of a polypeptide in regulating the glycolysis process of brown adipose tissue.

[0027] In one embodiment, the glycolysis process in brown adipose tissue is regulated as a thermogenic process.

[0028] In this embodiment, since the Gabpα-Pparγ complex is the core of regulating the transcription of key genes in brown adipose tissue glycolysis, GPCBi can inhibit the expression of key glycolysis genes (such as Eno1) by blocking the interaction between the two, thereby downregulating the glycolysis efficiency of brown adipose tissue. Moreover, the regulation of glycolysis in brown adipose tissue by GPCBi is mainly manifested in the inhibition of thermogenesis. Therefore, it can be used to regulate the thermogenesis function of brown adipose tissue and provide a tool for intervening in diseases related to abnormal thermogenesis.

[0029] Fourthly, embodiments of this application provide the application of a polypeptide in reducing lactate concentration or downregulating lactate dehydrogenase expression.

[0030] In this embodiment, lactic acid is one of the end products of glycolysis, and its concentration is directly related to the efficiency of glycolysis. GPCBi can reduce the production of lactic acid by inhibiting the glycolysis process in brown adipose tissue, thereby reducing the concentration of lactic acid in tissues or blood. At the same time, lactate dehydrogenase (LDH) is a key enzyme that catalyzes the conversion of pyruvate to lactate, and its expression level is closely related to glycolysis activity. GPCBi can downregulate the expression of lactate dehydrogenase by inhibiting the transcription of key genes in glycolysis, thereby further reducing lactic acid production. This application has potential value for intervening in lactate accumulation-related diseases (such as lactic acidosis).

[0031] Fifthly, embodiments of this application provide an application of a polypeptide in regulating the expression of coupling protein 1.

[0032] In this embodiment, uncoupling protein 1 (UCP1) is a core molecule for thermogenesis in brown adipose tissue, and its expression level is positively correlated with thermogenesis efficiency. GPCBi inhibits glycolysis by blocking Gabpα-Pparγ interaction, thereby reducing the energy supply required for thermogenesis and feedback downregulating the expression of UCP1. This application can be used to regulate thermogenesis activity in brown adipose tissue and provide a tool for intervening in metabolic disorders related to excessive or insufficient thermogenesis.

[0033] The functionality of GPCBi is verified through experimental testing, as shown in the following example: Example 1 To verify the binding affinity of Pparγ and Gabpα, Myc-Pparγ (Myc-labeled Pparγ) and His-Gabpα (His-labeled Gabpα) were co-transfected into HEK293T cells (interscapular brown adipose mesenchymal stem cells extracted from 8-week-old wild-type C57BL / 6J mice). Gabpα was immunoprecipitated using anti-His antibody, and Western blot analysis was performed, referring to... Figure 1 The Western blot results shown in Figure a indicate that Gabpα and Pparγ have a significant interaction. Differentiated interscapular brown adipose mesenchymal stem cells (iBAT-SVF) were fixed and subjected to immunohistochemical experiments. The primary antibody was rabbit anti-Gabpα or mouse anti-Pparγ, and the secondary antibody was a green or red fluorescently conjugated secondary antibody. Finally, nuclear staining (blue) was performed using DAPI. The cells were then observed using a Leica DM i8 confocal microscope (scale bar: 10 µm) to obtain... Figure 1 Figure b shows the immunofluorescence analysis results; iBAT-SVF cells were transfected with His-Gabpα and GST-labeled full-length Gabpaγ. The interaction between Gabpα and Pparγ within iBAT-SVF cells was examined. Figure 1 As shown in Figure c, the full-length Gabpα protein labeled with GST in iBAT-SVF cells can immunoprecipitate Pparγ, indicating a direct interaction between these two proteins in i-brown adipose tissue SVF cells.

[0034] like Figure 1 The surface plasmon resonance (SPR) sensing image shown in Figure d shows that the response signal intensity between Gabpα and Pparγ increases with increasing Gabpα concentration, and the binding constant KD is 5.34 nM, indicating a strong binding affinity between the two proteins.

[0035] Example 2 To determine the binding sites of Gabpα and Pparγ, the molecular structures of these two proteins were visualized using a protein database (PDB). The visualization results are shown below. Figure 2 As shown in Figure a; the binding sites of Gabpα and Pparγ were predicted using the HDOCKserver platform, as shown in Figure a. Figure 2 As shown in Figure b, the predicted binding sites are such that five amino acids in Gabpα form hydrogen bonds with six amino acids in Pparγ. Specifically, the GLN324 site of Gabpα binds to the LYS184 site of GPCBi. The calculated protein docking binding energy is -248.52 kcalmol-1. Furthermore, all five binding residues in Gabpα are located in the C-terminal region of Gabpα (aa252-454).

[0036] Build as Figure 2 Figure c shows a C-terminal truncated mutant of His-labeled Gabpa; iBAT-SVF cells were co-transfected with His-labeled full-length Gabpa or mutant Gabpa and Myc-labeled Pparγ, and immunoprecipitation and Western blot analysis were performed to compare the effect of Gabpa C-terminal truncated mutation on its binding to Pparγ. The results are as follows. Figure 2 As shown in Figure d, the immunoprecipitation experiment confirms that the binding site required for interaction with Pparγ is indeed located at the C-terminus of Gabpα.

[0037] Example 3 To investigate the intracellular blocking effect of the peptide GPCBi provided in this invention, an immunoprecipitation assay was performed using HEK293T cells. The binding of Gabpα at the GLN324 site to the LYS184 site of GPCBi was as follows: Figure 3As shown in Figure a, Figure a illustrates the simulated binding of the ARG168 site of GPCBi with the ASP332 site of Gabpα. HEK293T cells were co-transfected with Myc-Pparγ and His-Gabpα and treated with GPCBi. Immunoprecipitation and Western blot analysis were performed to compare the effects of GPCBi treatment on the binding affinity of Gabpα and Pparγ. Figure 3 As shown in Figure b, the binding ability of Gabpα and Pparγ is weakened after cells are treated with GPCBi. like Figure 3 The surface plasmon resonance (SPR) sensing image shown in Figure c shows that as the concentration of GPCBi increases, the surface plasmon resonance reaction between Gabpα and GPCBi becomes stronger. That is, GPCBi is shown to bind directly to Gabpα in a dose-dependent manner, with a binding constant KD value of 7.03 nM. like Figure 3 The surface plasmon resonance (SPR) sensing image shown in Figure d shows that the surface plasmon resonance reaction between Gabpa and Pparg weakens with increasing GPCBi concentration. That is, in the absence of GPCBi, Gabpα exhibits a high affinity for Pparγ, while GPCBi can dose-dependently weaken this interaction. Differentiated iBAT-SVF cells were treated with FITC (green)-labeled GPCBi, followed by nuclear staining with DAPI (blue). Nuclei were obtained using a Leica DM i8 confocal microscope (scale bar: 10 µm). Figure 3 As shown in Figure e, the fluorescence image reveals that FITC-labeled GPCBi is also concentrated in the nucleus of brown adipose tissue SVF cells.

[0038] Example 4 Characterization experiments of GPCBi in mice were conducted. GPCBi (5.5 mg / kg BAT intravenously) was injected into the brown adipose tissue of mice for 7 days. The mice were weighed, and various tissues (including BAT, ingWAT, epiWAT, and liver) were extracted and weighed. The effects of GPCBi injection on BAT, ingWAT, epiWAT, and liver in mice were compared. The results are as follows: Figure 4 As shown in the α-AC plot, the BAT mass of mice treated with GPCBi decreased, while the remaining parameters did not change significantly. Following GPCBi injection, BAT tissue was extracted, fixed, and subjected to H&E fat staining and UCP1 immunohistochemistry to quantify the area of ​​adipocytes and the relative area of ​​UCP1. The effects of GPCBi injection on fat content and UCP1 expression in mouse BAT were compared. The results are as follows: Figure 4As shown in the middle df figure, these mice showed reduced lipid accumulation and decreased expression of coupling protein 1 in BAT. After GPCBi injection, fasting blood glucose levels in mice were measured to compare the effects of GPCBi injection on fasting blood glucose in mice. The results are as follows: Figure 4 As shown in Figure g, the fasting blood glucose level of mice was significantly increased after GPCBi injection. Following GPCBi injection, mice were placed in metabolic cages (at ambient temperature), and their oxygen consumption and heat production were monitored using a Comprehensive Laboratory Animal Monitoring System (CLAMS). The effect of GPCBi injection on oxygen consumption in mice at room temperature was compared, and the results are as follows: Figure 4 As shown in the figure, oxygen consumption and heat production are significantly reduced at room temperature; Mice were placed at 4°C, and their rectal temperature was measured at different time points to compare the effect of GPCBi injection on core body temperature regulation in mice under cold conditions. The results are as follows: Figure 4 As shown in the middle figure, the cold tolerance of GPCBi mice is reduced. After GPCBi injection, mice were placed at 4°C, and their survival time was observed. The results are as follows: Figure 4 As shown in the figure, the mortality rate increased significantly at 4°C.

[0039] It should be noted that the sequence numbers of the embodiments in this application are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0040] The terms "comprising" and "having," and any variations thereof, in the specification, claims, and accompanying drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus. The terms "first," "second," and "third," etc., are used to distinguish different objects, etc., and do not indicate a sequence, nor do they limit "first," "second," and "third" to different types.

[0041] In the description of the embodiments of this application, terms such as "exemplary," "for example," or "for instance" are used to indicate examples, illustrations, or explanations. Any embodiment or design described as "exemplary," "for example," or "for instance" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of terms such as "exemplary," "for example," or "for instance" is intended to present the relevant concepts in a concrete manner.

[0042] In the description of the embodiments of this application, unless otherwise stated, " / " means "or". For example, A / B can mean A or B. The "and / or" in the text is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. In addition, in the description of the embodiments of this application, "multiple" means two or more.

[0043] In some processes described in the embodiments of this application, multiple operations or steps are included in a specific order. However, it should be understood that these operations or steps may not be executed in the order they appear in the embodiments of this application, or they may be executed in parallel. The sequence number of the operation is only used to distinguish different operations, and the sequence number itself does not represent any execution order. In addition, these processes may include more or fewer operations, and these operations or steps may be executed sequentially or in parallel, and these operations or steps may be combined.

[0044] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) as described above, and includes several instructions to cause a terminal device to execute the methods described in the various embodiments of this application.

[0045] The above are merely specific embodiments of this application, but the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application. Therefore, the protection scope of this application should be determined by the scope of the claims.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide contains the aa164-189 amino acid sequence fragment of the peroxisome proliferator-activated receptor Pparγ.

3. A polypeptide as described in claim 2, characterized in that, The polypeptide contains ARG168 and LYS184 sites, wherein ARG168 binds to the ASP332 site of the GA-binding protein a chain, and LYS184 binds to the GLN324 site of the GA-binding protein a chain.

4. A polypeptide as described in claim 3, characterized in that, The polypeptide competitively binds to the GA-binding protein a chain with Pparγ.

5. The use of a polypeptide according to any one of claims 1-2 in inhibiting the binding of the GA-binding protein α chain to the peroxisome proliferator-activated receptor Pparγ.

6. The application of a polypeptide according to any one of claims 1-2 in regulating the glycolysis process of brown adipose tissue.

7. The application as described in claim 6, characterized in that, The process of regulating glycolysis in brown adipose tissue is a thermogenic process.

8. The use of a polypeptide according to any one of claims 1-2 in reducing lactate concentration or downregulating lactate dehydrogenase expression.

9. The use of a polypeptide according to any one of claims 1-2 in downregulating the expression of coupling protein 1.

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