Novel micropeptide MP29 for regulating energy metabolism and use thereof

Abstract

The present application relates to a novel micropeptide MP29 and a use thereof and relates to a use of the micropeptide in the preparation of a reagent or drug for preventing, treating, or alleviating diseases related to abnormal mitochondrial energy metabolism in cells, wherein the diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, aging, photoaging, fatty liver disease, liver fibrosis, liver cirrhosis, liver cancer, diabetic nephropathy, cardiovascular diseases such as heart failure, etc. By means of endogenous overexpression or exogenous synthesis, the reducing equivalents NADH in the tricarboxylic acid cycle are up-regulated to promote intracellular ATP production, thereby significantly enhancing the proliferation of high-energy-demanding cardiac cells and brain tissue cells, or suppressing oxidative damage and apoptosis of cells in Alzheimer's disease models and Parkinsonism, or alleviating hypertrophy and aging of myocardial cells in heart failure models, or ameliorating the photodamage of cells in photoaging models. These results indicate that the micropeptide MP29 has an application value in preventing or treating diseases related to abnormal mitochondrial energy metabolism.

Description

A novel micropeptide MP29 for regulating energy metabolism and its applications

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 2024110153632, filed with the China National Intellectual Property Administration on July 26, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention belongs to the field of biomedical technology, specifically relating to a novel micropeptide MP29 and its applications. Background Technology

[0004] Alzheimer's disease (AD) is a neurodegenerative disease and a leading cause of dementia in the elderly. It ultimately leads to communication and reasoning difficulties, mood swings, progressive memory loss, and even the loss of daily living abilities. Recent studies have found a close correlation between energy metabolism disorders and the development of AD. Alterations in brain tissue energy metabolism pathways, energy-supplying substances, and metabolic patterns of different types of nerve cells are important characteristics of energy metabolism disorders in the brain tissue of AD patients. The mechanisms involve insulin resistance, glucose transport disorders, mitochondrial dysfunction, and lactate shuttle imbalance, among others. Energy metabolism plays a crucial role in the pathogenesis of AD. Currently, there are no drugs available to slow, prevent, or treat the further progression of AD.

[0005] Parkinson's syndrome (PD) is one of the most common neurodegenerative diseases, associated with the progressive loss of dopaminergic neurons in the substantia nigra, including various motor symptoms (such as bradykinesia, rigidity, and resting tremor) and non-motor symptoms (such as cognitive impairment, constipation, fatigue, sleep disturbances, and depression). A key molecular mechanism in the pathogenesis of Parkinson's disease is oxidative stress in the mitochondria of dopaminergic neurons in the substantia nigra and apoptosis caused by neuroinflammation.

[0006] Heart failure (HF) is a disease caused by impaired cardiac pumping function, resulting in the heart's inability to meet the body's basal metabolic needs. Main symptoms include shortness of breath, limited activity, and fluid retention. Disorders of myocardial energy metabolism are one of the pathophysiological causes of HF. Increased myocardial load, myocardial cell damage, cardiac remodeling and fibrosis, activation of the neuroendocrine system, inflammatory responses, and oxidative stress all contribute to the occurrence and development of HF.

[0007] Besides being influenced by age, photoaging of the skin is also directly related to sun exposure. Skin aging caused by sun exposure is called photoaging, and its clinical manifestations include wrinkles, skin laxity, telangiectasia, and uneven skin pigmentation. Accumulated ultraviolet (UV) radiation can cause skin aging and cancer. The main effects of acute and chronic UV exposure are DNA damage, inflammatory responses, and immunosuppression caused by reactive oxygen species (ROS) produced by UV radiation. The dynamic balance and regulation of mitochondrial ATP synthesis and ROS production determine the survival, development, aging, disease, and death of organisms.

[0008] Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder, typically characterized by motor impairment, cognitive decline, and psychiatric disturbances. Many studies speculate that HD is associated with Huntington protein (HTT), a protein ubiquitous in the brain and peripheral tissues. The primary cause is likely a mutation in the mutated Huntington protein (mHTT) gene, caused by an amplification of the cytosine-adenine-guanine repeat sequence. When mHTT forms insoluble aggregates and interacts with other proteins, it leads to an energy imbalance in the body, thereby affecting neuronal function. Mitochondria, as the body's energy producers, can accelerate the development of HD when they malfunction (Mitochondrial dynamics and quality control in Huntington's disease, Neurobiology of Disease, 2016, 90: 51-57; PINK1-induced mitophagy promotes neuroprotection in Huntington's disease, Cell Death & Disease, 2015, 6(1): e1617; Synaptic mitochondria in synaptic transmission and organization of vesicle pools in health and disease, Frontiers in Synaptic Neuroscience, 2010, 2: 139).

[0009] Schizophrenia (SCZ) is a severe mental illness characterized by hallucinations, delusions, and disordered speech, affecting hundreds of millions of people worldwide. Its lifetime prevalence is approximately 1%, and it has a high degree of heritability. The pathogenesis of SCZ is not yet fully understood, but mitochondrial quality control may be a key factor. Mitochondria are crucial organelles within cells, responsible for ATP production, metabolism, regulation of cell signal transduction and apoptosis, and playing a vital role in maintaining cellular survival and normal function. Alterations in the expression of mitochondrial-related genes are associated with the pathogenesis of SCZ. Post-mortem brain tissue studies of SCZ patients have shown that many genes related to mitochondrial quality control processes are dysregulated, with decreased expression of genes involved in mitochondrial respiration and ATP production (Diverse roles of mtDNA in schizophrenia: implications in its pathophysiology and as biomarker for cognitive impairment, Prog Biophys Mol Biol, 2020, 155:36-41). Meanwhile, some scholars have pointed out that genes involved in mitochondrial fusion and division (Mfn1, Mfn2, DRP1, G72) are significantly dysregulated in the brains of SCZ patients (The role of mitochondria in mood disorders: from physiology to pathophysiology and to treatment, Front Psychiatry, 2021, 12:546801).

[0010] Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by excessive fat deposition in hepatocytes, excluding alcohol and other clearly defined liver injury factors. It includes simple fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), NASH-related liver fibrosis, cirrhosis, and hepatocellular carcinoma. Current treatment strategies for NAFLD are not very effective, making in-depth research into its pathogenesis crucial. The widely accepted pathogenesis is the "multiple-hit" theory, which states that insulin resistance, mitochondrial dysfunction, inflammatory activation, dietary factors, and genetic factors all contribute to the progression of NAFLD. Among these, hepatic mitochondrial dysfunction plays a significant role, including mitochondrial morphological changes, mitochondrial DNA damage, fatty acid metabolism disorders and energy metabolism abnormalities, oxidative stress, lipid peroxidation, and abnormal mitophagy (Mitochondrial dys-function and signaling in chronic liver diseases, Gastroenterology, 2018, 155(3):629-647). Therefore, research targeting hepatic mitochondria has become a new and important breakthrough in the prevention and treatment of NAFLD.

[0011] Diabetic nephropathy (DN) is a microvascular complication associated with diabetes. Under prolonged hyperglycemic conditions, renal cells experience oxidative stress and inflammation. Renal interstitial fibrosis (RIF), as a pathological process, involves changes in the structure and function of the renal tubules and interstitium, leading to the gradual loss of kidney function—the ultimate result of DN development. Patient prognosis is closely related to the severity of RIF. Mitochondrial damage can cause cellular energy metabolism disorders and oxidative stress. Since the number of mitochondria in the renal interstitium is far greater than in other tissues, early lesions are often characterized by mitochondrial damage (Macrophages: Versatile players in renal inflammation and fibrosis, Nat Rev Nephrbl, 2019, 15(3): 144-158; Mitochondrial disease and endocrine dysfunction, Nat Rev Endocrinol, 2017, 13(2): 92-104). Therefore, early detection and intervention of mitochondrial damage are crucial for delaying the development of RIF.

[0012] Vascular aging is a crucial pathological basis for the aging of various organs and systems in the human body, significantly impacting the development of common cardiovascular diseases such as hypertension, atherosclerosis, myocardial ischemia-reperfusion injury, and coronary heart disease. The pathological mechanisms of vascular aging mainly involve cellular senescence, oxidative stress, and mitochondrial dysfunction. Mitochondrial dysfunction is characterized by abnormal mitochondrial morphology, decreased membrane potential, increased ROS production and opening of the mitochondrial permeability transition pore (mPTP), and ATP deficiency. This induces oxidative stress and inflammatory responses, while simultaneously exacerbating vascular endothelial damage, promoting endothelial cell senescence, inducing apoptosis, and accelerating vascular aging (New insights into vascular aging: Emerging role of mitochondria function, Biomedecine & Pharmacotherapies, 2022, 156:113954; Current understanding of the pivotal role of mitochondrial dynamics in cardiovascular diseases and senescence, Frontiers in Cardiovascular Medicine, 2022, 9:905072). In aging blood vessels, the mitochondrial electron transport chain malfunctions, leading to increased mitochondrial ROS production, reduced expression of antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD) and glutathione (GSH), and decreased NO bioavailability, thus affecting vascular dilation function and permeability.

[0013] It should be noted that the methods described in this section are not necessarily methods that had been previously conceived or adopted. Unless otherwise specified, no method described in this section should be assumed to be prior art simply because it is included in this section. Similarly, unless otherwise specified, the issues mentioned in this section should not be considered to be accepted in any prior art. Summary of the Invention

[0014] To address the aforementioned technical problems, this application provides a polypeptide and its mutants, which can be used to regulate abnormal mitochondrial metabolism, thereby exhibiting good preventive, alleviating, or therapeutic effects on various diseases, such as Alzheimer's disease, Parkinson's syndrome, Huntington's disease, schizophrenia, aging, photoaging, fatty liver, liver fibrosis, cirrhosis, liver cancer, diabetic nephropathy, and cardiovascular diseases such as heart failure. Therefore, the polypeptide and its mutants provided in this application have promising application prospects and clinical value.

[0015] According to one embodiment of this application, the polypeptide has an amino acid sequence as described in any one of (i)-(iv): (i) the amino acid sequence shown in SEQ ID NO:1; (ii) at least one amino acid sequence obtained by deleting, substituting, or inserting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the amino acid sequence shown in SEQ ID NO:1; (iii) at least one amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1; (iv) at least one sequence containing the amino acid sequence TLT.

[0016] According to one embodiment of this application, a polynucleotide is also provided, said polynucleotide encoding the polypeptide described in this application.

[0017] According to one embodiment of this application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in this application.

[0018] According to one embodiment of this application, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the polypeptide, the polynucleotide, or the recombinant vector described in this application.

[0019] According to one embodiment of this application, the use of the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application in the preparation of a medicament for detecting, preventing, alleviating, or treating a disease is also provided.

[0020] According to one embodiment of this application, the use of the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application in the preparation of a medicament for regulating mitochondrial metabolism or improving mitochondrial metabolic abnormalities is also provided.

[0021] According to one embodiment of this application, a method for detecting, preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application.

[0022] According to one embodiment of this application, a method for regulating mitochondrial metabolism or improving mitochondrial metabolic abnormalities is also provided, the method comprising administering to a subject in need the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application.

[0023] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0024] The accompanying drawings exemplify embodiments and form part of the specification, serving together with the textual description to explain exemplary implementations of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0025] Figure 1 shows the elemental diagram of the plvx-mCMV-zsGreen-Puro plasmid used in Example 1.

[0026] Figure 2 shows the overexpression of MP29 in N2a and HL-1 cells as detected by qPCR and Western blot in Example 1.

[0027] Figure 3 shows the results of KEGG enrichment analysis of RNA-seq sequencing results in Example 2.

[0028] Figure 4 shows the effect of overexpression of micropeptide MP29 and exogenous donor peptide MP29 on the enzyme activity of α-ketoglutarate dehydrogenase complex in N2a and HL-1 cells, as detected by the enzyme-linked bioinformatics kit in Example 2.

[0029] Figure 5 shows the effects of overexpression of the micropeptide MP29 on NADH / NAD+, ATP, and cell proliferation in N2a and HL-1 cells, as detected by the Promega kit in Example 2.

[0030] Figure 6 shows the effects of exogenous administration of micropeptide MP29 on NADH / NAD+, ATP, and cell proliferation in N2a and HL-1 cells, as detected by the Promega kit in Example 3.

[0031] Figure 7 shows the effect of exogenously administered micropeptide MP29 on the proliferation activity and ROS content of N2a cells, as detected by CCK-8 and flow cytometry in Example 4.

[0032] Figure 8 shows the effect of exogenously administered micropeptide MP29 on the expression of Bax and Bcl-2 proteins in N2a cells, as detected by Western blot in Example 4.

[0033] Figure 9 shows the effect of exogenously administered micropeptide MP29 on the proliferative activity of neuroblastoma cells as detected by CCK-8 in Example 4.

[0034] Figure 10 shows the effect of exogenous administration of micropeptide MP29 on the inhibition of renal podocyte proliferation induced by high glucose, as detected by CCK-8 in Example 5.

[0035] Figure 11 shows the effect of exogenously administered micropeptide MP29 on lipid droplets in AML12 cells as detected by Oil Red staining in Example 6.

[0036] Figure 12 shows the effect of exogenous administration of micropeptide MP29 on the cell viability of the photoaged cell model as detected by CCK-8 in Example 7.

[0037] Figure 13 shows the effect of exogenous administration of micropeptide MP29 on ROS content in a photoaged cell model, as detected by flow cytometry in Example 7.

[0038] Figure 14 shows the effect of exogenous administration of micropeptide MP29 on the antioxidant enzyme activity of photoaged cell model cells, as detected by the kit in Example 7.

[0039] Figure 15 shows the skin moisture content and elasticity of the photoaging model mice after drug administration, as detected in Example 8.

[0040] Figure 16 shows the epidermal condition of the photoaging model mice after drug administration, as detected by HE staining in Example 8.

[0041] Figure 17 shows the dermal condition of the photoaging model mice after drug administration, as detected by MASSON staining in Example 8.

[0042] Figure 18 shows the SOD content of photoaging model mice after drug administration, as detected by the kit in Example 8.

[0043] Figure 19 shows the echocardiograms of heart failure model mice after one week of continuous drug administration, as well as the quantitative analysis results of ejection fraction (EF) and fractional shortening (FS) in Example 9.

[0044] Figure 20 shows the echocardiograms of heart failure model mice after one month of continuous drug administration, as well as the quantitative analysis results of ejection fraction (EF) and fractional shortening (FS) in Example 9.

[0045] Figure 21 shows the HE staining results of the heart chambers of a heart failure model mouse after one month of continuous administration in Example 9.

[0046] Figure 22 shows the ATP content and OGDHc enzyme activity of heart failure model mice after administration of the kit in Example 9, as well as the expression of related proteins in different groups as detected by Western blot.

[0047] Figure 23 shows the distribution of exogenous MP29 in mice as detected by radiolabeling in Example 10.

[0048] Figure 24 is a schematic diagram of the binding of the micropeptide MP29 and OGDH in the molecular docking simulation of Example 11. In the figure, Figure A shows the secondary structure of the micropeptide MP29 predicted using PEPFOLD; Figure B shows the homology modeling of the OGDH protein using SWISS-MODEL; and Figure C shows the rigid docking of the micropeptide MP29 and the OGDH protein structure using HDOCK.

[0049] Figure 25 shows the effects of different concentrations of micropeptide MP29 and its truncated peptides (MP29-13, MP29-46, MP29-79, MP29-1012, MP29-1618, MP29-1921, MP29-2224, MP29-2527, MP29-2829) on the proliferation activity of N2a cells as detected by CCK-8 in Example 12.

[0050] Figure 26 is a summary bar chart of the proliferative activity of different concentrations of micropeptide MP29 and its truncated peptides (MP29-13, MP29-46, MP29-79, MP29-1012, MP29-1618, MP29-1921, MP29-2224, MP29-2527, MP29-2829) on N2a cells as detected by CCK-8 in Example 12.

[0051] Figure 27 shows the binding of the micropeptide MP29 and its truncated peptides MP29-1012 and MP29-1315 to the interacting protein OGDH, as detected by the MST method in Example 12.

[0052] Figure 28 shows the effects of different concentrations of the micropeptide MP29 and its substitution mutants (MP29-1A, MP29-8A, MP29-10A, MP29-28A, MP29-29A) on the proliferation activity of N2a cells as detected by CCK-8 in Example 13.

[0053] Figure 29A shows the binding of the micropeptide MP29 and its substitution mutants (MP29-1A, MP29-8A) to the interacting protein OGDH, as detected by the MST method in Example 13. Figure 29B shows the binding of the micropeptide MP29 substitution mutants (MP29-10A, MP29-28A, MP29-29A) to the interacting protein OGDH, as detected by the MST method in Example 13.

[0054] Figure 30 shows the effects of different concentrations of the micropeptide MP29 and its substitution mutants (MP29-13A, MP29-14A, MP29-15A) on the proliferation activity of N2a cells as detected by CCK-8 in Example 13.

[0055] Figure 31 is a summary bar chart of the CCK-8 assay used in Example 13 to detect the proliferation activity of the micropeptide MP29 and its substituted mutants (MP29-13A, MP29-14A, MP29-15A) on N2a cells at the same concentration.

[0056] Figure 32A shows the binding of the micropeptide MP29 and its mutant MP29-13A to the interacting protein OGDH, as detected by the MST method in Example 13. Figure 32B shows the binding of the micropeptide MP29 mutants MP29-14A and MP29-15A to the interacting protein OGDH, as detected by the MST method in Example 13.

[0057] Figure 33 shows the effects of exogenous administration of micropeptide MP29 and its substituted mutant MP29-13A on OGDHc enzyme activity, α-KG, succinate, NADH / NAD+, and ATP in N2a cells, as detected by the kit in Example 13. Detailed Implementation

[0058] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0059] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0060] Unless otherwise stated or contradicted by the context, the terms or expressions used herein should be read in the context of the document and as understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0061] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0062] In this application, the terms "polypeptide" and "peptide" are used interchangeably, referring to a polymer of amino acids of any length. Therefore, polypeptides, oligopeptides, micropeptides, proteins, antibodies, and enzymes are all included in the definition of polypeptide.

[0063] The terms “nucleic acid” and “polynucleotide” used in this application are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues.

[0064] It should be noted that, in the context of this application, upstream refers to the 5' end of the nucleic acid or the N-terminus of the polypeptide, and downstream refers to the 3' end of the nucleic acid or the C-terminus of the polypeptide. The direction from upstream to downstream is from the 5' end to the 3' end or from the N-terminus to the C-terminus.

[0065] The term "vector" as used in this application refers to a self-replicating DNA molecule that transfers a foreign target gene into a host organism, and is often in the form of a circular double-stranded DNA molecule. Typical vectors include plasmids, viruses, bacteriophages, kinases, and mini-chromosomes. Among these, plasmids are the most common form of vector, referring to circular double-stranded DNA that can accept foreign nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0066] The terms "expression vector" and "recombinant vector" used in this application are interchangeable and refer to a vector containing a foreign gene, and also containing regulatory elements that are expressed in a specified host organism. Introducing the expression vector into a suitable host organism enables it to express the inserted target gene (e.g., the nucleotide sequence encoding a micropeptide provided in this application).

[0067] The terms "exogenous" and "heterogeneous" used in this application are used interchangeably and refer to sources different from the native (original) organism, such as organisms derived from another species. The terms "heterogeneous gene" or "exogenous gene" used in this application refer to genes that do not naturally exist in the host organism and are introduced into the host organism through gene transfer.

[0068] The terms "relief" and "treatment" as used in this application, and their synonyms, refer to the improvement of a disease, symptom, and / or condition. "Relief" and "treatment" can be an improvement in at least one measurable physical parameter, which is not necessarily identifiable by the patient. "Relief" and "treatment" can also be the physical (e.g., stabilizing identifiable symptoms), physiological (e.g., stabilizing physical parameters), or both, suppression of the progression of a disease, symptom, and / or condition. "Relief" and "treatment" can also be the slowing of or reversal of the progression of a disease, symptom, and / or condition.

[0069] The term “prevention” as used in this application and its synonyms refer to delaying the onset of a particular disease, condition and / or symptom or related symptoms of such disease, condition and / or symptom or reducing the risk of acquiring such disease, condition and / or symptom.

[0070] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below.

[0071] Micropeptide MP29 and its mutants

[0072] This application provides a polypeptide having an amino acid sequence as described in any one of (i)-(iv): (i) the amino acid sequence shown in SEQ ID NO:1; (ii) at least one amino acid sequence obtained by deleting, substituting, or inserting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the amino acid sequence shown in SEQ ID NO:1; (iii) at least one amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1; (iv) at least one sequence containing the amino acid sequence TLT.

[0073] In mutants obtained from the amino acid sequence shown in SEQ ID NO:1, the mutation can be a deletion mutation. In some embodiments, the deleted sites include positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 of the amino acid sequence shown in SEQ ID NO:1. At least one of the amino acids at positions 7, 28, and / or 29, for example, the deleted site can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 of these amino acids. In some specific embodiments of this application, polypeptides obtained by deleting any three of the amino acids at positions 1 to 12 and positions 14 to 29 in the amino acid sequence shown in SEQ ID NO:1 all have regulatory activity on mitochondrial metabolism. Therefore, those skilled in the art can expect that polypeptides obtained by mutating multiple sites (e.g., multiple sites at positions 1 to 12 and positions 14 to 29) based on the amino acid sequence shown in SEQ ID NO:1 can also achieve the regulatory effect on mitochondrial metabolism.

[0074] In some preferred embodiments, the deleted site includes any three of the following amino acids from the amino acid sequence shown in SEQ ID NO:1: position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, and / or position 29.

[0075] In some preferred embodiments, the missing sites include any three adjacent sites from the amino acid sequences shown in SEQ ID NO:1, specifically positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and / or 29.

[0076] In some more preferred embodiments, the deleted site is selected from at least one group of: (1) amino acids at positions 1, 2, and 3 of the amino acid sequence shown in SEQ ID NO:1; (2) amino acids at positions 4, 5, and 6 of the amino acid sequence shown in SEQ ID NO:1; (3) amino acids at positions 7, 8, and 9 of the amino acid sequence shown in SEQ ID NO:1; (4) amino acids at positions 10, 11, and 12 of the amino acid sequence shown in SEQ ID NO:1; (5) amino acids at positions 16, 17, and 18 of the amino acid sequence shown in SEQ ID NO:1; (6) amino acids at positions 19, 20, and 21 of the amino acid sequence shown in SEQ ID NO:1; (7) amino acids at positions 22, 23, and 24 of the amino acid sequence shown in SEQ ID NO:1; (8) amino acids at positions 25, 26, and 27 of the amino acid sequence shown in SEQ ID NO:1; and / or (9) amino acids at positions 1, 2, 2, and 24 of the amino acid sequence shown in SEQ ID NO:1; and / or (9) amino acids at positions 25, 26, and 27 of the amino acid sequence shown in SEQ ID NO:1. The 28th and 29th amino acids in the amino acid sequence shown in NO:1.

[0077] In the mutant obtained from the amino acid sequence shown in SEQ ID NO:1, the mutation can be a substitution mutation. In some embodiments, the substitution site includes at least one of the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and / or 29 of the amino acid sequence shown in SEQ ID NO:1.

[0078] In some preferred embodiments, the substituted sites include at least one of the amino acids at positions 1, 8, 10, 14, 15, 28, and / or 29 of the amino acid sequence shown in SEQ ID NO:1.

[0079] In some more preferred embodiments, the substituted site is selected from at least one of the following: (1) the first amino acid in the amino acid sequence shown in SEQ ID NO:1 is mutated from M to A; (2) the eighth amino acid in the amino acid sequence shown in SEQ ID NO:1 is mutated from Q to A; (3) the tenth amino acid in the amino acid sequence shown in SEQ ID NO:1 is mutated from T to A; (4) the fourteenth amino acid in the amino acid sequence shown in SEQ ID NO:1 is mutated from L to A; (5) the fifteenth amino acid in the amino acid sequence shown in SEQ ID NO:1 is mutated from T to A; (6) the twenty-eighth amino acid in the amino acid sequence shown in SEQ ID NO:1 is mutated from T to A; and / or (7) the twenty-ninth amino acid in the amino acid sequence shown in SEQ ID NO:1 is mutated from T to A.

[0080] In some more preferred embodiments, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO:1-5,7-14,16-19.

[0081] Polynucleotides, recombinant vectors

[0082] According to one embodiment of this application, a polynucleotide is also provided, said polynucleotide encoding the polypeptide described in this application.

[0083] According to one embodiment of this application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in this application.

[0084] In some embodiments, the polynucleotide or the recombinant vector further includes a promoter. The promoter can be any suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by a host cell expressing a nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates polypeptide expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to those of the host cell.

[0085] In some embodiments, the polynucleotide or the recombinant vector further includes a transcription termination sequence. The transcription termination sequence is a sequence that can be recognized by the host cell to terminate transcription. In some embodiments, the transcription termination sequence is operatively attached to the 3' end of a nucleic acid sequence encoding a protein or polypeptide. Any terminator that can function in a selected host cell can be used in this application.

[0086] Pharmaceutical Composition

[0087] According to one embodiment of this application, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the polypeptide, the polynucleotide, or the recombinant vector described in this application.

[0088] In some embodiments, the pharmaceutical composition further includes a pharmaceutically or physiologically acceptable carrier. The carrier may be any compatible, physiologically acceptable, non-toxic substance suitable for delivering the polypeptide, polynucleotide, or recombinant vector provided in this application into a mammal (e.g., a human).

[0089] "Pharmaceutically acceptable carrier" refers to a carrier, diluent, or adjuvant used in the formulation or administration of the polypeptide, polynucleotide, or recombinant carrier provided in this application, which is not an essential active ingredient and does not cause excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art.

[0090] "Physiologically acceptable carrier" refers to a carrier, diluent, or adjuvant that does not cause significant irritation to an organism and does not eliminate the pharmaceutical activity and properties of the peptide, polynucleotide, or recombinant carrier provided in this application. Suitable physiologically acceptable carriers are also well known to those skilled in the art.

[0091] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients. In some embodiments, the excipients include at least one selected from solubilizers, disintegrants, wetting agents, stabilizers, thickeners, diluents, buffers, and flavoring agents.

[0092] In some non-limiting embodiments, the carrier and / or excipients used in the pharmaceutical compositions of this application may comprise, for example, liquid, gel or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending agents, dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[0093] Uses and methods

[0094] According to one embodiment of this application, the use of the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application in the preparation of a medicament for the prevention, relief, or treatment of a disease is also provided.

[0095] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application.

[0096] In some implementations, the disease includes diseases related to abnormal mitochondrial energy metabolism. Currently, many diseases are known to be associated with abnormal mitochondrial energy metabolism, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, aging, photoaging, fatty liver, liver fibrosis, cirrhosis, liver cancer, diabetic nephropathy, and cardiovascular diseases such as heart failure (Mitochondrial dynamics and quality control in Huntington's disease, Neurobiology of Disease, 2016, 90:51-57; PINK1-induced mitophagy promotes neuroprotection in Huntington's disease, Cell Death & Disease, 2015, 6(1):e1617; Synaptic mitochondria in synaptic transmission and organization of vesicle pools in health and disease, Frontiers in Synaptic Neuroscience, 2010, 2:139; Diverse roles of mtDNA in schizophrenia: implications in its pathophysiology and as biomarker for cognitive impairment, Prog Biophys Mol). Biol,2020,155:36-41; Mitochondrial dys-function and signaling in chronic liver diseases,Gastroenterology,2018,155(3):629-647; Macrophages:Versatile players in renal inflammation and fibrosis,Nat Rev Nephrbl,2019,15(3):144-158; Mitochondrial disease and endocrine dysfunction,Nat Rev Endocrinol,2017,13(2):92-104;New insights into vascular aging: Emerging role of mitochondria function, Biomedecine & Pharmacotherapies, 2022, 156:113954; Current understanding of the pivotal role of mitochondrial dynamics in cardiovascular diseases and senescence, Frontiers in Cardiovascular Medicine, 2022, 9:905072), these disclosures are all incorporated herein by reference in their entirety. In some specific embodiments of this application, the peptides, polynucleotides, and recombinant vectors described in this application have shown good activity in regulating abnormal mitochondrial energy metabolism. Therefore, those skilled in the art can reasonably expect, based on the disclosures of this application, that the peptides, polynucleotides, and recombinant vectors described in this application have therapeutic effects on the aforementioned diseases related to abnormal mitochondrial energy metabolism.

[0097] In some embodiments, the diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, aging, photoaging, fatty liver, liver fibrosis, cirrhosis, liver cancer, diabetic nephropathy, heart failure, and other cardiovascular diseases.

[0098] In some embodiments, the detection, prevention, mitigation or treatment of disease includes at least one of the following: (1) promoting the activity of enzymes involved in the synthesis of mitochondrial energy metabolism complexes; (2) upregulating the reducing equivalent NADH / NAD+ in mitochondrial energy metabolism; (3) promoting intracellular ATP production; (4) promoting cell proliferation; and / or (5) reducing intracellular oxidative stress levels.

[0099] According to one embodiment of this application, the use of the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application in the preparation of a medicament for regulating mitochondrial metabolism or improving mitochondrial metabolic abnormalities is also provided.

[0100] According to one embodiment of this application, a method for regulating mitochondrial metabolism or improving mitochondrial metabolic abnormalities is also provided, the method comprising administering to a subject in need the polypeptide, polynucleotide, recombinant vector, or pharmaceutical composition described in this application.

[0101] In some embodiments, the regulation of mitochondrial metabolism or improvement of mitochondrial metabolic abnormalities includes at least one of the following: (1) promoting the activity of enzymes related to the synthesis of mitochondrial energy metabolism complexes; (2) upregulating the reducing equivalent NADH / NAD+ in mitochondrial energy metabolism; (3) promoting intracellular ATP production; (4) promoting cell proliferation; and / or (5) reducing intracellular oxidative stress levels.

[0102] In the methods described in this application, the dosage of the polypeptide, polynucleotide, recombinant vector, or composition provided in this application may depend on several factors, including the severity and responsiveness of symptoms, the route of administration, the duration of treatment (from days to months to years), and the time to symptom improvement. Those skilled in the art can adjust the dosage regimen to provide a therapeutic response based on the patient's specific circumstances. For example, a single dose may be administered, several separate doses may be administered over a predetermined time period, or the dose may be reduced or increased as indicated by the treatment outcome. The dosage specification is determined by the specific therapeutic effect to be achieved. The dosage value may also vary depending on the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the treating clinician.

[0103] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory), and all embodiments formed by such combinations are considered as part of the disclosure of this application.

[0104] The exemplary embodiments of this application will now be described with reference to the accompanying drawings, including various details of the embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0105] Example

[0106] Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product instructions shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0107] Example 1: Construction of MP29 overexpressing cell lines

[0108] First, the plvx-mCMV-zsGreen-Puro plasmid (constructed by Universal Biotechnology) was constructed. The element map of the plasmid is shown in Figure 1, where the nucleotide sequence of the element "MP29 gene sequence" is shown in SEQ ID NO:20. HEK-293T cells (Shanghai Institute of Life Sciences, Chinese Academy of Sciences Cell Resource Center) were selected for lentiviral packaging and infection of target cells N2a and HL-1 (both from Shanghai Chenying Biotechnology Co., Ltd.) to construct a cell line overexpressing MP29. The experimental procedure is briefly described below:

[0109] HEK-293T was passaged into 100mm culture dishes. When the density reached 60-70%, lentivirus packaging was performed. Two 1.5mL EP tubes were prepared. In tube 1, 500μL of DMEM basal medium, 10μg of expression vector, 7.5μg of psPAX2 (purchased from General Biotechnology (Anhui) Co., Ltd.), and 2.5μg of pMD2.G (purchased from General Biotechnology (Anhui) Co., Ltd.) were added. In tube 2, 500μL of DMEM basal medium and 72μL of... Using EZTrans transfection reagent (AC04L091, Shanghai Liji Biotechnology Co., Ltd.), add the virus from tube 2 to tube 1 and incubate for 10 min. Add the transfection complex along the tube wall and gently shake to mix. Replace with fresh culture medium after 12 h. Collect the supernatant of the culture medium at 48 h and 72 h after transfection. Centrifuge the virus supernatant collected twice at 1000 rpm for 5 min to remove cell debris. Filter the supernatant through a 0.45 μm filter into an ultracentrifuge tube. Add virus concentrate (AC04L441, Shanghai Liji Biotechnology Co., Ltd.) at a virus solution:virus concentrate ratio of 4:1. Incubate overnight at 4℃. Centrifuge the virus solution at 4℃ and 4000g for 15 min, discard the supernatant to obtain the virus.

[0110] Resuspend the virus in 1 mL of culture medium, add 3 μL of polybrene (TA003, General Biotechnology (Anhui) Co., Ltd.), gently mix, and then infect N2a and HL-1 cells. After 9 h of virus infection, replace with fresh DMEM / F12 complete culture medium. After 72 h of infection, begin drug screening for the resistance gene to construct a stable cell line overexpressing the micropeptide MP29. The positive rate of overexpressing cells was detected by fluorescence microscopy and flow cytometry, and the overexpression efficiency was verified by qPCR and Western blotting.

[0111] Total RNA was extracted from MP29 overexpressing cells and control cells using Trizol reagent (Tiangen Biotech). The concentration and purity of the extracted RNA were quantified using a Nano Drop ND-1000 nucleic acid quantification instrument, and agarose gel electrophoresis was used to ensure RNA integrity. The extracted total RNA was reverse transcribed into cDNA using the Abm 5×All-In-One RT MasterMix kit, and real-time quantitative PCR was performed using the Abm Blas Taq 2×qPCR MasterMix. Primer information used is shown in Table 1. The specificity of the reaction was determined based on the melting curve, and the relative gene expression level was calculated based on the Ct value.

[0112] Total protein was extracted from MP29-overexpressing cells and control cells using Beyotime's Western & IP lysis buffer, and then analyzed by Western blot. The antibodies used for Western blot were Anti-α-Tubulin Mouse pAb (Hangzhou Xianzhi Biotechnology Co., Ltd.) and Anti-α-MP29 Mouse pAb (ordered from Abimate Biopharmaceutical (Shanghai) Co., Ltd.).

[0113] The results are shown in Figure 2. The qPCR and Western blot results showed that the expression level of the micropeptide MP29 in the overexpressing cells plvx-MP29 (the coordinate axes and lanes in Figure 2 are all labeled "MP29") was significantly higher than that in the control cells (the coordinate axes and lanes in Figure 2 are all labeled "ctrl"), indicating that the MP29 overexpressing cell line was successfully constructed.

[0114] Table 1 Primer sequences used in qPCR

[0115] Example 2: Effects of MP29 overexpression on mitochondrial energy metabolism in nerve and cardiomyocytes

[0116] 2.1 Effects of MP29 overexpression on the activity of enzymes involved in mitochondrial energy metabolism synthesis

[0117] RNA-seq sequencing was performed on the stable cell line overexpressing the micropeptide MP29 obtained in Example 1 and its control cell line, and KEGG enrichment analysis was performed on the differentially expressed genes. The results are shown in Figure 3. The results showed that the overexpression of the micropeptide MP29 had the most significant effect on metabolic pathways; BioCyc is a collection of metabolic pathway databases, and BioCyc enrichment analysis showed that the overexpression of the micropeptide MP29 had the most significant effect on mitochondrial energy metabolism in metabolic pathways.

[0118] Dissolve or aliquot each component according to the kit instructions (Shanghai Enzyme-Link Biotechnology Co., Ltd.). First, perform sample pretreatment, taking 5 × 10⁻⁶ samples.6 N2a and HL-1 cells overexpressing MP29 and their control cells (obtained in Example 2) were homogenized on ice for 5 min using a glass homogenizer. After centrifugation to obtain the precipitate, the appropriate reagents were added, and the cells were sonicated for 3 s at 20% power, with 10 s intervals, repeated 30 times in an ice bath. Then, the cells were proceeded to the next step of activity assay. The UV spectrophotometer was preheated for at least 30 min, the wavelength was set to 340 nm, and the zeroing was performed using distilled water. The working solution was incubated at 37°C for 5 min. The working solution was added to a quartz cuvette, mixed, and the absorbance value A1 was recorded immediately after 20 s and the absorbance value A2 after 2 min 20 s. ΔA = A2 - A1 was calculated.

[0119] The results are shown in Figure 4, indicating that the micropeptide MP29 can significantly increase the activity of enzymes related to mitochondrial energy metabolism synthesis in cardiomyocytes and nerve cells.

[0120] 2.2 Effect of MP29 overexpression on reducing equivalent NADH / NAD+

[0121] The NADH / NAD+ assay was performed according to the kit instructions (Promega, USA), and is briefly described below: First, a standard curve was prepared. Cells were digested and collected, washed twice with PBS, and then counted. 4000 N2a cells overexpressing MP29 and 4000 control cells (obtained in Example 2) were collected, along with 2000 HL-1 cells overexpressing MP29 and 2000 control cells (obtained in Example 2). The cell suspension was diluted to 50 μL and added to an opaque white plate. 50 μL of a basic alkaline solution containing 1% DTAB was added to lyse the cells, and the mixture was gently shaken for 10 min. 100 μL of the lysed cell sample was divided into two aliquots and treated differently: 25 μL of 0.4N HCl was added to the acid-treated wells, and the wells were heated at 60°C for 15 min; the alkaline-treated wells were heated at 60°C for 15 min. After equilibration to room temperature, 25 μL of Trizma Base solution was added to the acid-treated wells, and 50 μL of HCl / Trizma Base solution was added to the alkaline-treated wells. Base buffer; the above-treated sample was divided into two equal parts, 50 μL per well, and 50 μL of working solution was added to each well. The mixture was gently shaken to lyse the cells and incubated at room temperature for 30 min; the bioluminescence signal was measured using a multi-functional microplate reader and the data were recorded.

[0122] The results are shown in Figures A and B in Figure 5. Overexpression of MP29 upregulated the reducing equivalent NADH / NAD+ in mitochondrial energy metabolism in nerve cells and cardiomyocytes.

[0123] 2.3 Effects of MP29 overexpression on ATP production

[0124] The ATP assay was performed according to the kit instructions (Shanghai Beyotime Biotechnology Co., Ltd.), briefly described as follows: First, prepare a standard curve, taking 1×10⁻⁶...6 Add 200 μL of ATP lysis buffer to each cell, pipette and shake the plate several times to ensure complete cell lysis, centrifuge at 12000×g for 5 min at 4℃, and transfer the supernatant to a new centrifuge tube; prepare the working solution: each sample requires 20 μL of luciferase assay reagent and 80 μL of diluent. Add the working solution to a transparent white plate in advance and let it stand at room temperature for 3-5 min to eliminate background signal; add 10-100 μL of sample or standard to every 100 μL of working solution, and detect the bioluminescent signal using a multi-functional microplate reader after 2 min.

[0125] The results are shown in Figures C and D of Figure 5. Overexpression of MP29 can promote ATP production in nerve cells and cardiomyocytes.

[0126] 2.4 Effects of MP29 overexpression on cell proliferation

[0127] Cell proliferation detection is based on cell viability levels. The method is briefly described as follows: Cells are seeded into 96-well plates. After different drug administration operations, CCK-8 reagent is added at a ratio of 1:100, and the cells are incubated at 37°C for 1-3 hours. The readings are taken at a wavelength of 450 nm using an ELISA reader.

[0128] The results are shown in Figures E and F in Figure 5. Overexpression of MP29 can promote the proliferation of nerve cells and cardiomyocytes in a dose-dependent manner.

[0129] In summary, the results in Figure 5 confirm that overexpression of MP29 upregulates the reducing equivalent NADH / NAD+ in mitochondrial energy metabolism in nerve cells and cardiomyocytes, promotes ATP production, and is beneficial to cell proliferation.

[0130] The results of this embodiment indicate that the micropeptide MP29 can improve mitochondrial energy metabolism in nerve cells and cardiomyocytes, and has the potential to be applied to the treatment of diseases with abnormal mitochondrial energy metabolism, such as diseases related to abnormal mitochondrial energy metabolism in the nervous system (Alzheimer's disease, Parkinson's syndrome, Huntington's disease, schizophrenia, etc.) and diseases related to abnormal mitochondrial energy metabolism in the cardiovascular system (heart failure, etc.). It may also have therapeutic effects on other diseases related to abnormal mitochondrial energy metabolism (aging, photoaging, fatty liver, liver fibrosis, cirrhosis, diabetic nephropathy, etc.).

[0131] Example 3: Effects of exogenous MP29 administration on mitochondrial energy metabolism in nerve and cardiomyocytes

[0132] Wild-type N2a and HL-1 cells (both cell types were purchased from Shanghai Chenying Biotechnology Co., Ltd.) were treated with exogenous 0 nM, 10 nM, and 1000 nM MP29 (synthesized by chemical methods by Changzhou Kanglong Biotechnology Co., Ltd.) for 48 h, and then tested using the same detection method as in Example 2.

[0133] As shown in Figure 6, the addition of synthetic micropeptide MP29 to the culture medium and the overexpression of micropeptide MP29 in cells achieved the same biological effects: upregulation of the reducing equivalent NADH / NAD+ in mitochondrial energy metabolism, promotion of ATP production, and benefit to cell proliferation. Therefore, both synthetic and endogenously expressed micropeptide MP29 can increase the activity of energy metabolism in mitochondria and have the potential for application in the treatment of diseases with abnormal mitochondrial energy metabolism.

[0134] In summary, the results of Examples 2-3 indicate that endogenous expression or exogenous administration of micropeptide MP29 in nerve cells and cardiomyocytes can significantly affect mitochondrial energy metabolism, including significantly increasing the activity of enzymes related to mitochondrial energy metabolism synthesis complexes, upregulating the reducing equivalent NADH / NAD+ in mitochondrial energy metabolism, promoting ATP production, and promoting cell proliferation. This suggests that micropeptide MP29 may have good therapeutic effects on various diseases related to abnormal mitochondrial energy metabolism in the nervous or cardiovascular systems, including but not limited to Alzheimer's disease, Parkinson's syndrome, Huntington's disease, schizophrenia, and heart failure.

[0135] Example 4: Effects of exogenous MP29 administration on neurological diseases

[0136] 4.1 Effects of exogenous MP29 administration on ROS content in nerve cells

[0137] N2a cells (purchased from Shanghai Chenying Biotechnology Co., Ltd.) induced by 0.5 mM H2O2 were used as an Alzheimer's disease (AD) cell model. Cells were co-incubated with 10 nM micropeptide MP29 for 24 h. Cell viability was assessed by CCK-8 assay, and changes in intracellular reactive oxygen species (ROS, Shanghai Beyotime Biotechnology Co., Ltd.) levels were detected by flow cytometry. The results are shown in Figure 7. Exogenous administration of micropeptide MP29 can alleviate the inhibitory effect of oxidative damage on nerve cell proliferation and reduce the ROS levels caused by oxidative damage.

[0138] 4.2 Effects of exogenous MP29 administration on the expression of apoptosis-related proteins in neural cells

[0139] After cell collection, probe loading was performed by diluting DCFH-DA with serum-free culture medium at a ratio of 1:50,000 to a final concentration of 10 μmol / L. Cells were then collected and suspended in the diluted DCFH-DA at a concentration of 5 × 10⁶ cells / L.5 Cells were incubated at 37°C for 20 minutes at a density of 1000 μL. The cells were inverted and mixed every 3-5 minutes to ensure thorough contact between the probe and cells. Cells were washed three times with serum-free cell culture medium to remove any unextracted DCFH-DA. Cells were collected and analyzed by flow cytometry. Western blotting was used to detect apoptosis-related proteins Bcl-2 and Bax (antibody purchased from Abogen (Shanghai) Trading Co., Ltd.). An increased Bax / Bcl-2 ratio indicated apoptosis. The results are shown in Figure 8, demonstrating that exogenous administration of the micropeptide MP29 alleviated neuronal apoptosis.

[0140] 4.3 Exogenous administration of MP29 alleviates the inhibition of nerve cell proliferation by MPTP.

[0141] In addition, SH-SY5Y human neuroblastoma cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were induced with 1 μM MPTP (purchased from MedChemExpress) as a commonly used cell model for Parkinson's syndrome (PD). Exogenous treatment with 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM MP29 for 24 h was performed, and cell viability was detected by CCK-8 assay. The results are shown in Figure 9. Exogenous administration of the micropeptide MP29 alleviated the inhibitory effect of MPTP on nerve cell proliferation.

[0142] In summary, the results in Figures 7, 8, and 9 further confirm that the micropeptide MP29 has potential therapeutic effects on diseases related to abnormal mitochondrial energy metabolism in the nervous system, such as Alzheimer's disease, Parkinson's syndrome, Huntington's disease, and schizophrenia.

[0143] Example 5: Exogenous administration of MP29 alleviates the inhibition of renal podocyte proliferation caused by high glucose.

[0144] MPC5 mouse renal podocytes induced by 30 μM high glucose (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) are a commonly used cell model for diabetic nephropathy. They were treated with 1 nM, 10 nM, 100 nM, 1 μM and 10 μM MP29 for 48 h. The therapeutic effect of MP29 on the model cells was detected by CCK-8 assay.

[0145] The results are shown in Figure 10. Exogenous administration of micropeptide MP29 can treat the inhibition of renal podocyte proliferation caused by high glucose, restore the proliferative capacity of MPC5 renal podocytes, and improve the energy supply of MPC5 cells, thereby improving diabetic nephropathy. These results indicate that micropeptide MP29 has potential therapeutic effects on diabetic nephropathy and other diseases.

[0146] Example 6: Effect of exogenous MP29 administration on intracytoplasmic lipid droplets of hepatocytes

[0147] Early-stage fatty liver disease is characterized primarily by fat accumulation in the liver. However, persistent inflammation and oxidative stress can activate hepatic stellate cells, leading to collagen deposition and ultimately resulting in liver fibrosis, cirrhosis, and even liver cancer. Therefore, this embodiment evaluates the therapeutic activity of exogenous MP29 against the aforementioned diseases by detecting the lipid droplet content within hepatocytes.

[0148] Inducing mouse hepatocytes AML12 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) with 0.5 mM sodium oleate (OA) and 0.25 mM sodium palmitate (PA) for 24 h is the most commonly used method for establishing a fatty liver cell model.

[0149] Specifically, the cells were treated with exogenous MP29 at concentrations of 10 nM, 100 nM, and 1000 nM for 48 h, and the changes in lipid droplet content in the cytoplasm were observed by Oil Red staining.

[0150] The specific method for oil red staining is as follows: fix with 4% paraformaldehyde for 20 min, stain with oil red for 30 min, wash twice with PBS, and then observe under a microscope.

[0151] The results are shown in Figure 11. Compared with the control group, the model group induced by OA and PA became darker, indicating successful modeling. Meanwhile, the color of the model group became lighter after exogenous administration of micropeptide MP29, indicating a decrease in the content of lipid droplets in the cytoplasm of hepatocytes, confirming its potential therapeutic effect on fatty liver and its possible causes such as liver fibrosis, cirrhosis, and liver cancer.

[0152] Example 7: Effects of exogenous MP29 administration on human dermal fibroblasts

[0153] 7.1 Effects of exogenous MP29 on skin cell proliferation

[0154] HDF cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) cultured to the logarithmic growth phase were digested with trypsin and collected. They were resuspended in fresh serum-containing medium and counted using a cell counter at a concentration of 1 × 10⁻⁶. 5 Cells were seeded at a ratio of 100 μL per cell in 96-well plates, and the cell suspension was added to each well at a rate of 100 μL. The plates were then incubated overnight at 37°C in a CO2 incubator.

[0155] After removing the 96-well plate, place it in a clean bench and expose it to ultraviolet light at 40 mJ / cm². 2HDF cells were treated with a radiation dose (UV irradiation meter, Shenzhen Baorui Instrument Co., Ltd.) or 100 μM H2O2 for 24 h, followed by co-incubation with different concentrations of micropeptide MP29 (1 nM, 10 nM, 100 nM, 1 μM, 10 μM) for 48 h. Under light-protected conditions, 10 μL of CCK-8 solution was added to each well, and after incubation at 37°C for approximately 1.5 h, the OD450 value of each well was measured using a microplate reader. The calculation formula is as follows: Cell viability (%) = OD450 of blank treatment group / OD450 of sample treatment group × 100%.

[0156] The results are shown in Figure 12. Exogenous administration of different concentrations of the micropeptide MP29 can enhance the vitality of damaged cells in both modeling methods and reduce oxidative damage and aging of skin cells caused by ultraviolet radiation or H2O2. This demonstrates that MP29 possesses anti-aging activity.

[0157] 7.2 Effects of exogenous MP29 administration on ROS content in skin cells

[0158] ROS (such as H2O2, superoxide anion O2) - Hydroxyl radicals (OH, etc.) have strong oxidizing properties and can react with probes to generate detectable signals, which can be detected using ROS kits (Beyotime Biotechnology, No.: S0033S).

[0159] Drug administration and grouping: Cells were divided into blank group (no UV irradiation), control group (no UV irradiation, no drug administration, DCFH-DA probe), model group (with UV irradiation, no drug administration, DCFH-DA probe), and drug administration group (no UV irradiation, given 1 μM micropeptide MP29, DCFH-DA probe).

[0160] Solution preparation: Prepare the DCFH-DA fluorescent probe solution according to the kit, and dilute the DCFH-DA stock solution with serum-free culture medium at a ratio of 1:3000.

[0161] Sample processing: Remove the 6-well plate after 48 hours of drug treatment, discard the culture medium, digest with trypsin, collect the samples according to the group into 1.5 mL EP tubes, centrifuge at 1500 rpm for 5 min, and wash twice with PBS.

[0162] Fluorescence incubation: Discard the PBS in the EP tube, add 200 μL of diluted DC FH-DA probe solution to each centrifuge tube, and incubate at 37°C in the dark for 30 min, gently inverting the EP tube every 3-5 min.

[0163] ROS detection: Remove the EP tube, centrifuge at 1500 rpm for 5 min, discard the probe solution, wash the cells 1-2 times with PBS, resuspend the cells in 0.2-0.3 mL of PBS, filter the cells through a cell strainer, and perform flow cytometry detection.

[0164] The results are shown in Figure 13. Exogenous administration of MP29 can reduce the ROS level of senescent cells, indicating that MP29 has significant anti-aging activity.

[0165] 7.3 Effects of exogenous MP29 administration on the activity of antioxidant enzymes in skin cells

[0166] Environmental oxidants and ultraviolet radiation primarily stimulate skin cells to produce reactive oxygen species (ROS), thereby inducing cell damage, mitochondrial dysfunction, and ultimately aging. Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), are core components of the endogenous antioxidant defense system, synergistically blocking oxidative stress responses and thus protecting skin cells.

[0167] UV-induced damage regimen: Control group (no UV irradiation, no drug administration), Model group (UV irradiation, no drug administration), Positive agent group (UV irradiation, administered 1 μM palmitoyl pentapeptide-3, Shanghai Lanyan Cosmetics Co., Ltd.), Drug administration group (UV irradiation, administered 1 μM MP29), where UV intensity was 40 mJ / cm². 2 The radiation dose.

[0168] H2O2-induced damage protocol: blank group (no H2O2 treatment, no drug administration), model group (H2O2 treatment, no drug administration), positive control group (H2O2 treatment, administered 1 μM palmitoyl pentapeptide-3, Shanghai Lanyan Cosmetics Co., Ltd.), and drug administration group (H2O2 treatment, administered 1 μM MP29). H2O2 was administered at a dose of 100 μM.

[0169] Sample pretreatment: Take out the 6-well plate 48 hours after drug administration, digest it and collect it into 1.5 mL EP tubes according to the group, add 0.15 mL PBS buffer, insert the probe of the sonicator below the liquid surface, sonicate at 300 W once every 3-5 seconds, repeat 5 times with a 30-second interval, and place the whole plate on ice to lyse the cells.

[0170] SOD was measured according to the kit instructions, and the absorbance value was measured at 550 nm. The result was calculated according to the instructions (T-SOD test kit (hydroxylamine method), Nanjing Jiancheng A001-1-2); In addition, CAT was measured according to the kit instructions (CAT determination kit (visible light method) (ammonium molybdate method), Nanjing Jiancheng A007-1-1).

[0171] The results are shown in Figure 14. In both modeling methods, exogenous administration of MP29 increased the activities of SOD and CAT, and the improvement effect was better than that in the positive control group. This indicates that the micropeptide MP29 has an enhancing effect on the activity of cellular antioxidant enzymes, demonstrating that MP29 has significant anti-aging activity, superior to the positive control group.

[0172] The above results indicate that micropeptide MP29 has potential therapeutic effects on diseases related to abnormal mitochondrial energy metabolism, such as aging and photoaging.

[0173] Example 8: Effects of exogenous MP29 administration on mouse skin

[0174] 1. Establishment of a mouse model of skin aging

[0175] This study used 5-week-old SPF-grade female ICR mice (purchased from Spiford (Suzhou) Biotechnology Co., Ltd.). All animals underwent one week of acclimatization in an SPF-grade animal laboratory. Mice were randomly divided into 5 groups (n = 5 mice / group): blank group (saline), model group (UV + saline), positive control group (UV + pentapeptide, Shanghai Lanyan Cosmetics Co., Ltd.), and MP29 administration group (UV + 10 μM MP29, UV + 1 mM MP29). Mice were anesthetized using an inhalation anesthesia machine according to their respective groups. The limbs and tail of the mice were then secured with tape to a plastic board (25cm × 15cm). Next, the plastic board holding the mice was inhaled at a total dose of 400 mJ / cm². 2 Irradiation was performed for 10 minutes daily, with the same intensity of ultraviolet radiation (UV irradiation meter, Shenzhen Baorui Instrument Co., Ltd.) for 21 consecutive days. The back of the mouse was selected as the shaving area (2cm×2cm). The mouse was fixed in a stable position and a manual razor was used to ensure that no hair residue and no epidermal damage were left on the skin after shaving. The mice in each group were shaved every five days, and the UV modeling and drug administration were performed 24 hours after the shaving.

[0176] 2. Animal grouping and administration

[0177] After each day's modeling is completed, allow the mouse to stand for 30 minutes before applying the drug. The drug administration protocol is shown in Table 2 below. Dissolve the drug in physiological saline. Apply the drug to the irradiated skin (2cm × 2cm) on the back of the mouse using a cotton swab and wait for complete absorption (5-10 minutes).

[0178] Table 2 Animal grouping and administration

[0179] 3. Measurement of skin moisture content and elasticity

[0180] One day before the mice were euthanized, the water content and elasticity of the skin on the back of each group of mice were measured using a multifunctional skin analyzer (Henan Miao Electronic Technology Co., Ltd.). The results were displayed directly on the instrument as a numerical content (%). The back area of ​​the mice was randomly selected for testing, and the test was repeated three times. The final results were statistically analyzed, and the results are shown in Figure 15.

[0181] The results showed that, compared with the Model group, the high-dose MP29 group mice exhibited significantly increased skin hydration and elasticity (p<0.05). This indicates that MP29 has a restorative effect on the decline in skin hydration and elasticity in aging mice, and the high-dose MP29 was more effective than the positive control drug, suggesting that MP29 has a good effect in the treatment of aging.

[0182] 4. HE and MASSON pathological analysis

[0183] Skin sections (0.5cm × 0.5cm) were randomly excised from the backs of mice. Five randomly selected samples (per group) were fixed in 10% formaldehyde solution and stained with hematoxylin and eosin (HE) and Masson's stain. Skin sections were observed using an inverted microscope. Four clear fields of view were randomly selected at 400x magnification, and one clear field of view was randomly selected at 200x magnification for photographing. Pathological scoring was performed based on the selected fields of view. HE and Masson's staining sections were obtained from a third-party institution. The HE and Masson's staining scoring criteria were as follows: 1 point (very mild), 2 points (mild), 3 points (moderate), 4 points (severe), and 0 points (virtually normal), based on the severity of the lesion. The mean score (mean ± SD) for each group of animals was calculated.

[0184] The results are shown in Figure 16 and Table 3. HE staining results and scores showed that, compared with the Model group, the thickening of the epidermal spinous layer was improved, the inflammatory response (inflammatory cell infiltration, skin tissue necrosis) was weakened, and the number of hair follicles increased. Furthermore, the high-dose group showed effects comparable to the positive control group. This indicates that the micropeptide MP29 has a repairing effect on epidermal structural damage and can improve the inflammatory response in photoaging model mice.

[0185] Table 3 HE staining results and scores

[0186] Note: One-way ANOVA, *P<0.05; **P<0.01; ***P<0.001 compared with the Model group.

[0187] The results are shown in Figure 17 and Table 4. Masson staining results and scores showed that, compared with the Model group, the high-dose group had a decreased degree of dermal collagen fiber proliferation, and the improvement effect was better than that of the positive drug group. This indicates that the micropeptide MP29 has a repair effect on dermal collagen damage in aging model mice.

[0188] Table 4. Masson staining results and scores

[0189] Note: One-Way ANOVA, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001, compared with the Model group.

[0190] 5. SOD content determination

[0191] Twenty-four hours after the final UV irradiation, mice were euthanized, and the shaved skin from the back of the mice was collected, weighed, and minced with ophthalmic scissors. Pre-cooled PBS buffer (tissue weight: PBS buffer weight = 1:9) was added, along with grinding beads, and the mixture was placed in a grinder and ground at 4°C for 10 minutes. Finally, it was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected for later use. The absorbance was measured at 550 nm according to the kit instructions, and the results were calculated according to the instructions (Total Superoxide Dismutase (T-SOD) Assay Kit (Hydroxyamine Method), Nanjing Jiancheng A001-1-2).

[0192] The results are shown in Figure 18. The micropeptide MP29 can significantly enhance the SOD activity of aging model mice, and the improvement effect is better than that of the positive drug group.

[0193] In summary, the micropeptide MP29 can enhance SOD and participate in the regulation of antioxidant enzymes and lipid peroxidation to improve the oxidative stress response of the skin, thereby demonstrating a repairing effect on damage in both the dermis and epidermis of an aging mouse model, while also reducing inflammatory responses. This suggests that the micropeptide MP29 has a good therapeutic effect on aging, photoaging, and other related diseases.

[0194] Example 9: Therapeutic effect of exogenous MP29 on a mouse model of acute heart failure

[0195] An animal model of acute heart failure was established using isoproterenol (ISO) to investigate the effects of MP29 on heart failure.

[0196] Forty-two 6-week-old male C57BL / 6J mice were housed in an SPF animal facility and acclimatized for 7 days before the start of the experiment. The facility was maintained at 24±2.0℃ and 55±10% humidity, with a standard diurnal cycle. The mice had free access to drinking water and a standard diet. The mice were randomly divided into 7 groups of 6 mice each, with 36 mice serving as the model group. These mice received subcutaneous injections of ISO for two consecutive days (85 mg / kg on the first day and 340 mg / kg on the second day).

[0197] Peptide administration: Mice were divided into four groups based on administration route and dosage, as detailed in Table 5 below. The peptide was administered concurrently with ISO, with the peptide given every 24 hours. The final administration time was determined based on changes in echocardiogram and blood BNP (a marker protein for heart failure) levels (measured after one week and one month of continuous administration). The administration volume was adjusted according to mouse body weight, calculated at 2.5 μL / g.

[0198] Table 5 Animal grouping and administration

[0199] The experimental endpoint indicators included: echocardiography, HE staining to examine myocardial status, and Western blot to detect the expression of key proteins.

[0200] The cardiac function of Vevo mice is typically assessed using the Vevo 2100 high-resolution small animal ultrasound imaging system. The following are the general operating procedures for cardiac function testing:

[0201] 1. Preparation

[0202] (1) Instrument preparation: Ensure that the Vevo 2100 system is powered on and completes its self-test.

[0203] (2) Anesthetize mice: Use an appropriate anesthetic (such as isoflurane) to generalize the mice and maintain the anesthesia state by ventilator.

[0204] (3) Restraining mice: After anesthesia, mice are fixed supine on a constant temperature heating plate to maintain body temperature.

[0205] (4) Shave: Shave the hair off the mouse’s chest so that the probe can make better contact with the skin.

[0206] 2. Operating Procedures

[0207] (1) Select a probe: Select a high-frequency probe suitable for mouse heart detection (usually 30MHz).

[0208] (2) Apply coupling agent: Apply coupling agent evenly to the probe.

[0209] (3) Place the probe: Place the probe on the left side of the mouse sternum to perform a short-axis view scan of the heart.

[0210] 3. Acquire the image:

[0211] (1) Adjust the position and angle of the probe to find a clear four-chamber view of the heart.

[0212] (2) Adjust parameters such as depth, gain, and time gain compensation (TGC) to optimize image quality.

[0213] 4. Cardiac function testing:

[0214] (1) M-mode ultrasound: M-mode ultrasound images are acquired in the middle of the ventricle to measure the thickness of the interventricular septum and the posterior wall of the left ventricle.

[0215] (2) Two-dimensional ultrasound: to observe the morphology and movement of each chamber of the heart.

[0216] (3) Pulse wave Doppler: Measure the blood flow velocity and direction of the mitral and aortic valves.

[0217] (4) Color Doppler: Observe the direction and speed of blood flow in the heart chambers.

[0218] (5) Tissue Doppler: to assess the speed of myocardial movement.

[0219] 5. Measurement of cardiac function parameters:

[0220] Using the measurement tools in the Vevo software, cardiac function parameters were measured in the short-axis view of the ventricle, including: ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic volume (EDV), left ventricular end-systolic volume (ESV), cardiac output (CO), and heart rate (HR).

[0221] Figure 19 shows that after one week of continuous administration, compared with the model group, the EF and FS values ​​of different doses of exogenous MP29 administration groups were improved, and the retrograde effect was better than that of the positive drug group.

[0222] Figure 20 shows that after one month of continuous administration, compared with the model group, the EF and FS values ​​of different doses of exogenous MP29 were improved, and the high-dose group (G6: 4.5 mg / kg / day, tail vein injection) showed better reversion effect than the positive drug group.

[0223] Figure 21 shows that after one month of continuous administration, compared with the model group, the enlargement of the heart chambers in mice was inhibited, the intercellular spaces were reduced, and the medium- and high-dose exogenous MP29 administration group was more effective than the positive drug group.

[0224] Figure 22 shows that the micropeptide MP29 promotes ATP production in the heart tissue of heart failure mice, with the high-dose group showing a better promoting effect than the positive control group. MP29 can increase the activity of OGDHc enzyme in the heart tissue of heart failure mice, with the high-dose group (G6: 4.5 mg / kg / day, tail vein injection) showing a better effect than the positive control group. Western blot results showed that the BNP protein level was lower in the treatment group compared to the model group. The decrease in BNP protein indicates that MP29 has inhibitory activity against heart failure.

[0225] In summary, exogenous MP29 demonstrates superior therapeutic efficacy against heart failure compared to the positive control drug trimetazidine. It enhances OGDHc enzyme activity, promotes ATP production in cardiomyocytes within the heart failure model, and alleviates cardiomyocyte hypertrophy and senescence. Trimetazidine has been used for many years in the treatment of chronic heart failure and other diseases, thus proving the potential therapeutic efficacy of exogenous MP29 for cardiovascular diseases such as acute heart failure, chronic heart failure, and coronary artery disease.

[0226] Example 10: Distribution of exogenous MP29 in mice

[0227] 1. Radioactive labeling

[0228] Peptide synthesis: Peptide synthesis and chelating agent coupling were performed using solid-phase synthesis, followed by purification in semi-preparative liquid phase and lyophilization to obtain the sample;

[0229] Isotope selection: commonly used in PET imaging. 68 Ga(t 1 / 2 =68min), 64 Cu(t 1 / 2 =12.7h);

[0230] Labeling of coordination reaction (with) 68 (Taking Ga as an example): Take DOTA-protein solution (0.1-1 mg / mL) and... 68 Mix GaCl3 (activity 10-20 MBq); reaction conditions are pH 4.0-5.0 (sodium acetate buffer), heating at 95℃ for 10-15 min.

[0231] Purification: Free ions were removed by NAP-5 column chromatography. 68 Ga 3+ Collect protein peaks;

[0232] Radioactive TLC verification: Develop silica gel plate (developing solvent: 50mM DTPA solution), and detect the labeling rate with a gamma counter (must be >95%).

[0233] 2. In vivo injection and imaging in mice

[0234] C57BL / 6 or BALB / c nude mice (depending on experimental requirements) were selected and anesthetized by inhalation of 2% isoflurane, maintaining a body temperature of 37°C. Radiolabeled exogenous MP29 (volume <200 μL, activity 1-5 MBq) was injected via the tail vein. At four time points (0.5h, 1h, 2h, and 4h), the tissue distribution of radiolabeled exogenous MP29 was scanned by PET / CT, and quantitative analysis was performed.

[0235] The results are shown in Figure 23. At 0.5h, 1h, 2h, and 4h, exogenous MP29 was distributed in the heart, liver, kidney, lung, and brain of mice. These results indicate that exogenous administration of MP29 can achieve good drug distribution in mice, and therefore holds promise for alleviating and treating diseases related to various organs such as the heart, liver, kidney, lung, and brain.

[0236] Example 11: Molecular docking simulates the binding of MP29 to protein OGDH

[0237] The secondary structure of the micropeptide MP29 was predicted using PEPFOLD, and then the OGDH protein was homologated using SWISS-MODEL. Finally, the structure of the micropeptide MP29 and the OGDH protein was rigidly docked using HDOCK. The results are shown in Figure 24.

[0238] The results indicate that OGDH is a direct target of micropeptide MP29. Therefore, those skilled in the art can anticipate that micropeptide MP29 can be used to treat a variety of diseases related to OGDH, including but not limited to neurodevelopmental disorders, neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), inflammation, oxidative stress, etc.

[0239] Example 12: Activity assay of MP29 truncated mutant

[0240] The micropeptide MP29 was truncated and mutated using an exogenous synthesis method (sequence shown in Table 6 below, synthesized by Changzhou Kanglong Biotechnology Co., Ltd.). In N2a cell lines, cells were treated with gradient concentrations of peptide (0 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) for 48 hours. Cell proliferation was detected using the CCK-8 assay, and the results are shown in Figures 25 and 26. Subsequently, the micropeptide MP29 was validated using MST, and the results are shown in Figure 27.

[0241] The MST experiment first identifies the target molecule: OGDH (experimental group) or BSA (negative control), which carries a fluorescent signal and has a concentration lower than the estimated Kd value. Preparation method: Dilute the protein solution to 1 μM, add 200 μL of an equal volume of CY5 fluorescent labeling solution (CY5 can label cysteine ​​residues in the protein), incubate at room temperature for 90 min, and pass through a molecular sieve column. After these steps, the protein solution is diluted approximately 10 times, i.e., the concentration is approximately 100 nM.

[0242] Then, the Ligand molecule, MP29, was identified. The highest concentration should be 20 times higher than the estimated Kd value, and the buffer solutions used for dilution gradients should be kept consistent. Preparation method: Dilute the chemically synthesized peptide MP29 to a 200 μM stock solution using PBS.

[0243] Preliminary experiment: Take the sieved Target molecular solution with a standard capillary tube; detect whether it can produce a suitable fluorescence intensity (NT.115 fluorescence range of 200-2000), sample homogeneity, whether it adsorbs onto the inner wall of the capillary tube, and whether the sample aggregates.

[0244] Formal Experiment: Prepare 16 PCR tubes. Add 20 μL of high-concentration MP29 stock solution to tube 1, and 10 μL of PBS solution to tubes 2-16. Take 10 μL of MP29 stock solution from tube 1 and add it to tube 2. Mix well, then take 10 μL and add it to tube 3. Repeat the above operation until tube 16. Finally, discard 10 μL from tube 16. Add 10 μL of labeled Target molecule solution to each tube. Mix thoroughly, then use a capillary tube to aspirate the sample, avoiding the generation of air bubbles, and perform the PCR test.

[0245] Data analysis: Check the uniformity of fluorescence intensity and whether adsorption or aggregation occurs; use MO.Affinity Anlysis software to analyze the detection results, fit the binding affinity constant Kd value, and control the signal-to-noise ratio to be greater than 5 to ensure the reliability of the results.

[0246] The results in Figures 25 and 26 show that the truncated peptide MP29-1315 has little effect on cell proliferation activity. The other nine mutants obtained after truncating mutation (MP29-13, MP29-46, MP29-79, MP29-1012, MP29-1618, MP29-1921, MP29-2224, MP29-2527, MP29-2829) can all effectively promote cell proliferation. The above results confirm that mutants obtained by truncating amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 of the MP29 amino acid sequence all maintained the regulatory activity of MP29 on cell proliferation; however, the mutant MP29-1315 obtained by truncating amino acids at positions 13, 14, and 15 reduced the original cell proliferation regulatory activity of MP29.

[0247] In Figure 27, the MST experiment results show that both the micropeptide MP29 and the truncated peptide MP29-1012 can bind to the interacting protein OGDH, but the truncated peptide MP29-1315 does not bind to the interacting protein OGDH.

[0248] In summary, the results in Figures 25, 26, and 27 confirm that the binding site of MP29 to OGDH may be at position 13, 14, or 15.

[0249] Table 6. Sequences of micropeptides and their truncated peptides

[0250] Example 13: Activity assay of MP19 substitution mutant

[0251] The micropeptide MP29 was mutated using an exogenous synthesis method (sequence shown in Table 7 below, synthesized by Changzhou Kanglong Biotechnology Co., Ltd.). In N2a cell lines, cells were treated with gradient concentrations of peptides (0 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) for 48 hours. Cell proliferation was detected using the CCK-8 assay, and the results are shown in Figure 28. Subsequently, the micropeptide MP29 was validated using MST, and the results are shown in Figures 29A and 29B.

[0252] Table 7 Sequences of micropeptides and their substituted mutant peptides

[0253] As shown in Figures 28, 29A, 29B, and Table 8, the five mutants obtained after substitution mutation (MP29-1A, MP29-8A, MP29-10A, MP29-28A, and MP29-29A) all effectively promoted cell proliferation and exhibited binding activity with the interacting protein OGDH. Furthermore, the mutant peptide MP29-8A showed a stronger binding affinity to OGDH than the micropeptide MP29. These results indicate that mutations at sites 1, 8, 10, 28, and 29 do not affect the binding of MP29 to OGDH or its proliferative activity. Therefore, it is evident that these five sites can be mutated with different amino acids without affecting the activity of MP29.

[0254] Table 8 Affinity results of the substituted mutant peptides

[0255] Meanwhile, based on the results of Example 12, the binding site of MP29 to OGDH may be amino acid at position 13, 14, or 15, so this example further verifies this.

[0256] Figures 30, 31, 32A, and 32B show that the other two mutants (MP29-14A and MP29-15A) obtained after truncation mutation can effectively promote cell proliferation and have binding activity with the interacting protein OGDH. These results confirm that mutants obtained by substituting amino acids 1, 8, 10, 14, 15, 28, and 29 of the MP29 amino acid sequence can maintain the regulatory activity of MP29 on cell proliferation and its binding activity with the interacting protein OGDH.

[0257] Figures 30, 31, 32A, and 32B also show that the mutant MP29-13A, obtained by substituting the 13th amino acid, reduces the original cell proliferation regulatory activity of MP29 and its binding activity with the interacting protein OGDH. This result further confirms that the key binding site between MP29 and OGDH is the 13th amino acid.

[0258] To further verify that the binding site of MP29 to OGDH is at amino acid position 13, Figure 33 shows:

[0259] (1) The mutant peptide MP29-13A upregulates OGDHc enzyme activity (tested by an OGDHc enzyme activity assay kit, which was purchased from Shanghai Enzyme Link Biotechnology Co., Ltd.) less effectively than the micro peptide MP29.

[0260] (2) The micropeptide MP29 can accelerate the conversion of α-KG to succinic acid (tested by α-ketoglutarate ELISA kit and succinic acid detection kit, both kits were purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.), while the mutant peptide MP29-13A slows down the conversion of α-KG to succinic acid.

[0261] (3) The mutant peptide MP29-13A does not increase the intracellular reducing equivalent NADH; the mutant peptide MP29-13A reduces the production of intracellular ATP.

[0262] Based on the results of Examples 12-13, it can be concluded that substitution mutations or truncation mutations at any site other than the 13th amino acid of MP29 can maintain the regulatory activity on cell proliferation and the binding activity with the interacting protein OGDH.

[0263] It should be noted that the above are merely preferred embodiments of this application and are not intended to limit the application. Various modifications and variations are possible for those skilled in the art. Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents of the above embodiments may exist or be unforeseeable to the applicant or other those skilled in the art. Therefore, the appended claims and any possible amendments to the claims are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents. Importantly, as technology evolves, many elements described herein can be replaced by equivalent elements appearing after this application.

Claims

1. A polypeptide, characterized in that, The polypeptide has an amino acid sequence of any one of (i)-(iv) below: (i) the amino acid sequence set forth in SEQ ID NO: 1; (ii) at least one of an amino acid sequence obtained by deletion, substitution or insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (iii) at least one of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:

1. (iv) at least one of a sequence containing the amino acid sequence TLT.

2. The polypeptide of claim 1, wherein the site of deletion comprises at least one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, and / or 29th amino acid in the amino acid sequence set forth in SEQ ID NO:

1.

3. The polypeptide of claim 2, wherein the site of deletion is selected from at least one of the following groups: (1) the 1st, 2nd, and 3rd amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (2) the 4th, 5th, and 6th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (3) the 7th, 8th, and 9th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (4) the 10th, 11th, and 12th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (5) the 16th, 17th, and 18th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (6) the 19th, 20th, and 21st amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (7) the 22nd, 23rd, and 24th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (8) the 25th, 26th, and 27th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; and / or (9) the 28th and 29th amino acids in the amino acid sequence set forth in SEQ ID NO:

1.

4. The polypeptide of claim 1, wherein the substituted position comprises at least one of the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and / or 29 of the amino acid sequence set forth in SEQ ID NO:

1. Preferably, the substituted position comprises at least one of the amino acids at positions 1, 8, 10, 14, 15, 28, and / or 29 of the amino acid sequence set forth in SEQ ID NO:

1.

5. The polypeptide of claim 4, wherein the substituted position is selected from at least one of the following group: (1) the amino acid at position 1 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from M to A; (2) the amino acid at position 8 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from Q to A; (3) the amino acid at position 10 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from T to A; (4) the amino acid at position 14 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from L to A; (5) the amino acid at position 15 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from T to A; (6) the amino acid at position 28 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from T to A; and / or (7) the amino acid at position 29 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from T to A.

6. The polypeptide of any one of claims 1-5, having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-5, 7-14, 16-19.

7. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-6. The polynucleotide encodes the polypeptide of any one of claims 1-6.

8. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 7.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, or the recombinant vector of claim 8.

10. Use of the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing, alleviating, or treating a disease.

11. The use of claim 10, wherein the disease comprises a disease associated with abnormal mitochondrial energy metabolism.

12. The use of claim 10, wherein the disease comprises Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, schizophrenia, aging, photoaging, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, diabetic nephropathy, or a cardiovascular disease. Preferably, the cardiovascular disease comprises heart failure.

13. Use of the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 in the manufacture of a medicament for modulating mitochondrial metabolism or ameliorating mitochondrial metabolic abnormality.

14. The use of claim 13, wherein the modulating mitochondrial metabolism or ameliorating mitochondrial metabolic abnormality comprises at least one of the following group: (1) promoting mitochondrial energy metabolism synthesis-related complex enzyme activity; (2) up-regulating reduced equivalent NADH / NAD+in mitochondrial energy metabolism; (3) promoting intracellular ATP generation; (4) promoting cell proliferation; and / or (5) reducing intracellular oxidative stress level.

15. A method of preventing, alleviating or treating a disease, characterized in that, The method comprises administering to a subject in need thereof the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9.

16. The method of claim 15, wherein the disease comprises a mitochondrial energy metabolism abnormality-related disease.

17. The method of claim 15, wherein the disease comprises Alzheimer's disease, Parkinson's syndrome, Huntington's disease, schizophrenia, aging, photoaging, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, diabetic nephropathy, or cardiovascular disease; Preferably, the cardiovascular disease comprises heart failure.

18. A method of modulating or ameliorating mitochondrial metabolism abnormality, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-17. The method comprises administering to a subject in need thereof the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9.

19. The method of claim 18, wherein the modulating mitochondrial metabolism or ameliorating mitochondrial metabolic abnormality comprises at least one of the following group: (1) promoting mitochondrial energy metabolism synthesis-related complex enzyme activity; (2) up-regulating reduced equivalent NADH / NAD+in mitochondrial energy metabolism; (3) promoting intracellular ATP generation; (4) promoting cell proliferation; and / or (5) reducing intracellular oxidative stress level.

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