Separated polypeptide for inducing mitochondrial calcium overload and application thereof

Abstract

The invention discloses a separated polypeptide for inducing mitochondrial calcium overload and application of the separated polypeptide. The separated polypeptide has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The separated polypeptide disclosed by the invention can be combined with NCLX protein, and degradation of the NCLX protein can be promoted through m-AAA protease, so that calcium ions can be prevented from being excreted, and calcium overload in mitochondria is avoided. In addition, the separated polypeptide is obviously superior to the existing calcium release inhibitor CGP37157 in the aspect of inducing mitochondrial calcium overload.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to the isolation of a polypeptide for inducing mitochondrial calcium overload and its application. Background Technology

[0002] mitochondria contain calcium ions (Ca) within the cell. 2 Calcium (NCL) plays a crucial role in the regulation of mitochondrial Na+. Calcium homeostasis has a significant impact on cellular energy metabolism, apoptosis, and signal transduction. NCLX, as a mitochondrial Na+... + / Ca 2+ Exchange proteins are the main calcium ion efflux channels on the inner mitochondrial membrane, responsible for transporting calcium ions out of the mitochondrial membrane. 2+ Na is transported out of the mitochondria and simultaneously transported in. + The regulation of its function is crucial for maintaining mitochondrial calcium homeostasis.

[0003] Current NCLX inhibitors can inhibit intramitochondrial calcium... 2 + outflow, but some inhibitors are not specific to intramitochondrial Ca. 2 + Regulation, and may also inhibit Ca2+ on the cell membrane 2 The effect of efflux is limited. Therefore, there is an urgent need to develop a new type of calcium overload reagent. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a polypeptide for inducing mitochondrial calcium overload and its application.

[0005] This application is based on the following discoveries of the inventors:

[0006] Existing calcium release inhibitors, such as CGP37157, can inhibit intramitochondrial calcium release. 2 + outflow, but can also inhibit Ca on the cell membrane 2 The outflow of + affects the intramitochondrial Ca2+. 2 The regulation of calcium overload is nonspecific and has limited effects. Therefore, it is of great significance to develop a novel, specific, efficient, or biosafe calcium overload agent.

[0007] However, the inventors of this application unexpectedly discovered a novel Kuafu peptide translated from zebrafish long non-coding RNA (lncRNA). This peptide, composed of 65 amino acids, is located in the inner mitochondrial membrane and significantly induces mitochondrial calcium overload by binding to and promoting the degradation of NCLX protein via the m-AAA (ATPases Associated with various cellular activities) protease, thereby inhibiting mitochondrial calcium ion efflux. Furthermore, experiments showed that the Kuafu peptide of this application is more effective than the existing calcium release inhibitor CGP37157 in inducing calcium overload, and due to its short peptide chain length and stable molecular structure, it exhibits better biocompatibility. Therefore, the Kuafu peptide of this application provides a more specific new tool for studying mitochondrial calcium homeostasis and its related mechanisms, helping to reveal more cellular physiological processes. Based on the calcium overload-inducing properties of the Kuafu peptide, it can also be used to further develop novel anti-tumor drugs, providing new avenues for improving clinical treatment efficacy and reducing side effects.

[0008] Based on this, in a first aspect of this application, an isolated polypeptide is proposed. According to an embodiment of this application, the isolated polypeptide includes a functional region having an amino acid sequence as shown in SEQ ID NO:1.

[0009] In a second aspect of this application, an isolated nucleic acid is provided. According to embodiments of this application, the isolated nucleic acid is used to encode the isolated polypeptide described in the first aspect.

[0010] In a third aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the isolated nucleic acid described in the second aspect; or the expression vector expresses the isolated polypeptide described in the first aspect.

[0011] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell comprises: carrying the isolated nucleic acid described in the second aspect or the expression vector described in the third aspect; or expressing the isolated polypeptide described in the first aspect.

[0012] In a fifth aspect of this application, the use of the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of NCLX protein inhibitors and mitochondrial calcium overload agents is proposed.

[0013] In a sixth aspect of this application, an NCLX protein inhibitor or mitochondrial calcium overload agent is proposed. According to embodiments of this application, the NCLX protein inhibitor or mitochondrial calcium overload agent comprises: the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect.

[0014] In a seventh aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises: the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the NCLX protein inhibitor or mitochondrial calcium overload agent described in the sixth aspect.

[0015] In an eighth aspect of this application, the use of the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the NCLX protein inhibitor or mitochondrial calcium overload agent described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect in the preparation of antibodies, pharmaceuticals, or reagents is provided.

[0016] In a ninth aspect of this application, a method is provided for inhibiting NCLX protein activity and / or inducing mitochondrial calcium overload in vitro. According to embodiments of this application, the method includes: overexpressing the isolated polypeptide described in the first aspect in cells; wherein the cells contain mitochondria and the NCLX protein.

[0017] In a tenth aspect of this application, a method for detecting NCLX protein is provided. According to an embodiment of this application, the method includes: contacting the isolated polypeptide described in the first aspect with a sample to be tested to form an immune complex.

[0018] Beneficial effects:

[0019] The Kuafu peptide of this application can specifically target the inner mitochondrial membrane, promoting the degradation of NCLX protein and thus significantly inducing mitochondrial calcium overload. Compared with existing calcium release inhibitors, the Kuafu peptide has significant advantages in terms of specificity, efficacy, and biosafety.

[0020] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0021] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0022] Figure 1In Example 1 of this application, antibodies are used to recognize in vitro overexpressed Kuafu peptides (i.e., isolated polypeptides).

[0023] Figure 2 The image shows the results of using MitoProt to predict mitochondrial signal peptides (A) and TMpred to predict transmembrane domains (B) in Example 2 of this application.

[0024] Figure 3 This is a diagram showing the results of Kuafu peptide localization in the inner mitochondrial membrane in Example 2 of this application;

[0025] Figure 4 This is a diagram showing the results of Kuafu peptide promoting NCLX degradation by enhancing the binding of NCLX and m-AAA in Example 3 of this application;

[0026] Figure 5 This is a diagram showing the results of Kuafu peptide-induced mitochondrial calcium overload in Example 4 of this application. Detailed Implementation

[0027] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0028] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0029] This application details

[0030] Definitions and General Terms

[0031] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0032] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0033] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0034] The isolated polypeptide described in this application can be prepared by biosynthesis or by solid-phase peptide synthesis. Based on the nucleotide sequence shown in this application, those skilled in the art can readily obtain the encoded nucleic acid of this application using various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc., and specific methods can be found in J. Sambrook, *Molecular Cloning: A Laboratory Manual*. As one embodiment of this application, the encoded nucleic acid sequence of this application can be constructed by segmenting and synthesizing the nucleotide sequence followed by overlap extension PCR.

[0035] In this document, the term "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of the isolated peptide containing that amino acid sequence. Such modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated peptides of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0036] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, containing a nucleotide sequence that expresses a target protein, and capable of being transferred to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0037] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a recipient cell (or host cell) is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., expression vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0038] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by homogenizing and adequately combining an active, isolated peptide, isolated nucleic acid, or expression carrier with a liquid carrier, a finely fragmented solid carrier, or both.

[0039] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients is also within the scope of consideration for this application, except for any range of incompatibilities with the isolated polypeptide, isolated nucleic acid, or expression vector of this application, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition.

[0040] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The isolated polypeptides, isolated nucleic acids, expression vectors, NCLX protein inhibitors, mitochondrial calcium overload agents, or pharmaceutical compositions of this application may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are anticipated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration. Preferably, the compositions of this application are administered via intravenous or subcutaneous injection.

[0041] In this document, the term "treatment" refers to the use of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a disease in individuals susceptible to the disease but not yet diagnosed with it; (b) inhibition of disease, such as blocking disease progression; or (c) alleviation of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers isolated peptides, isolated nucleic acids, expression vectors, or drugs containing them to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administering drugs containing the isolated peptides, isolated nucleic acids, or expression vectors described herein to an individual in need.

[0042] This application provides a detailed description of the isolated peptides used to induce mitochondrial calcium overload and their applications.

[0043] This application proposes a polypeptide for inducing mitochondrial calcium overload and its applications, which will be described in detail below.

[0044] isolated peptides

[0045] In a first aspect of this application, an isolated polypeptide is provided. According to an embodiment of this application, the isolated polypeptide includes a functional region having an amino acid sequence as shown in SEQ ID NO:1.

[0046] The isolated peptide of this application can bind to NCLX protein and AFG3L2 E575Q, and can be used to detect NCLX protein and AFG3L2 E575Q. The isolated peptide can also serve as an antigen for preparing antibodies to detect the isolated peptide. Furthermore, the binding of the isolated peptide to NCLX protein can promote the degradation of NCLX protein through the m-AAA (ATPases Associated with various cellular activities) protease, thereby preventing calcium ion efflux and leading to mitochondrial calcium overload. Moreover, the isolated peptide is significantly superior to the existing calcium release inhibitor CGP37157 in inducing mitochondrial calcium overload.

[0047] Since mitochondrial calcium homeostasis plays a crucial role in maintaining cellular function and life activities, the Kuafu peptide described in this application provides a more specific and novel tool for studying mitochondrial calcium homeostasis and its related mechanisms, helping to elucidate more cellular physiological processes. Based on the calcium overload-inducing properties of the Kuafu peptide, novel drugs can be further developed, potentially providing new avenues for treating diseases such as tumors.

[0048] VAGEVQAPVHDRSVNQIAMMLAIMGLSLSYYSAKQMTEKVRQEPAI (SEQ ID NO: 1).

[0049] In one optional embodiment of this application, the functional region includes, from the N-terminus to the C-terminus, the mitochondrial intermembrane region, the transmembrane region, and the mitochondrial matrix region.

[0050] In an optional embodiment of this application, the mitochondrial intermembrane region has an amino acid sequence as shown in SEQ ID NO:6.

[0051] VAGEVQAPVHDR (SEQ ID NO:6).

[0052] In an optional embodiment of this application, the transmembrane region has an amino acid sequence as shown in SEQ ID NO:7.

[0053] SVNQIAMMLAIMGLSLSYYSA (SEQ ID NO:7).

[0054] In one optional embodiment of this application, the C-terminus of the transmembrane region is connected to the N-terminus of the mitochondrial matrix region.

[0055] In an optional embodiment of this application, the mitochondrial matrix region has an amino acid sequence as shown in SEQ ID NO:8.

[0056] KQMTEKVRQEPAI (SEQ ID NO:8).

[0057] According to embodiments of this application, the isolated polypeptide further includes a signal peptide.

[0058] In one optional embodiment of this application, the C-terminus of the signal peptide is connected to the N-terminus of the cytoplasmic region.

[0059] In one alternative embodiment of this application, the signal peptide is used to localize the functional region to the mitochondria, preferably to the inner mitochondrial membrane.

[0060] In an optional embodiment of this application, the signal peptide has an amino acid sequence as shown in SEQ ID NO:5.

[0061] MIIRLGRLLTPGYFRLLQRQ (SEQ ID NO: 5).

[0062] According to embodiments of this application, the isolated polypeptide has an amino acid sequence as shown in SEQ ID NO:2.

[0063] MIIRLGRLLTPGYFRLLQRQVAGEVQAPVHDRSVNQIAMMLAIMGLSLSYYSAKQMTEKVRQEPAI (SEQ ID NO: 2).

[0064] The isolated peptides of this application can specifically locate on the inner mitochondrial membrane and bind to NCLX protein and AFG3L2E575Q. These isolated peptides can be used to detect NCLX protein and AFG3L2E575Q. Furthermore, the isolated peptides can also be used as antigens for antibody screening.

[0065] isolated nucleic acids

[0066] In a second aspect of this application, an isolated nucleic acid is provided. According to embodiments of this application, the isolated nucleic acid is used to encode the isolated polypeptide described in the first aspect. The isolated nucleic acid of this application can encode the isolated polypeptide of the first aspect.

[0067] According to an embodiment of this application, the isolated nucleic acid is DNA.

[0068] It should be noted that, for the isolated nucleic acids mentioned herein, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0069] According to embodiments of this application, the isolated nucleic acid has a nucleotide sequence as shown in SEQ ID NO:3 or 4.

[0070] GTAGCAGGAGAAGTCCAGGCCCCGGTGCATGACCGCTCCGTCAACCAGATCGCCATGATGCTG GCCATCATGGGTCTCAGCCTGTCCTATTACAGCGCCAAACAGATGACAGAGAAAGTTCGCCAAGAA CCTGCGATATGA (SEQ ID NO: 3).

[0071] ATGATCATCAGACTGGGCAGACTGACTCCTGGATACTTCCGTCTTTTGCAGCGGCAGGTAGCAGGAGAAGTCCAGGCCCCGGTGCATGACCGCTCCGTCAACCAGATCGCCATGATGCTGGCCATCATGGGTCTCAGCCTGTCCTATTACAGCGCCAAACAGATGACAGAGAAAGTTCGCCAAGAACCTGCGATATGA (SEQ ID NO: 4).

[0072] expression carrier

[0073] In a third aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the isolated nucleic acid described in the second aspect; or the expression vector expresses the isolated polypeptide described in the first aspect. When the isolated nucleic acid is ligated to the expression vector, the isolated nucleic acid can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the isolated nucleic acid. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the isolated nucleic acid and the control elements only need to be operably linked.

[0074] In this article, "operable ligation" refers to ligating a foreign gene into an expression vector so that the control elements within the expression vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages.

[0075] According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, the expression of the aforementioned isolated polypeptide can be effectively realized under the mediation of the regulatory system, thereby realizing the large-scale in vitro acquisition of the isolated polypeptide.

[0076] According to embodiments of this application, the expression vector is selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0077] According to embodiments of this application, the vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this application is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0078] In one optional embodiment of this application, the expression vector is a plasmid expression vector or a viral expression vector.

[0079] Recombinant cells

[0080] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell comprises: carrying the isolated nucleic acid described in the second aspect or the expression vector described in the third aspect; or expressing the isolated polypeptide described in the first aspect. Using this recombinant cell, under suitable conditions, the aforementioned isolated polypeptide can be effectively expressed intracellularly.

[0081] According to some specific embodiments of this application, the recombinant cells can efficiently and in large quantities separate polypeptides under suitable conditions.

[0082] It should be noted that "suitable conditions" refers to conditions suitable for the expression of the isolated peptides described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the isolated peptides include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the isolated peptides based on the specific environment of their laboratory.

[0083] According to embodiments of this application, the recombinant cells are obtained by introducing the expression vector described in the third aspect into host cells.

[0084] It should be noted that the recombinant cells described in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0085] According to embodiments of this application, the recombinant cells are eukaryotic cells, preferably mammalian cells.

[0086] Applications in the preparation of NCLX protein inhibitors and mitochondrial calcium overload agents

[0087] In a fifth aspect of this application, the use of the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of NCLX protein inhibitors and mitochondrial calcium overload agents is proposed.

[0088] NCLX protein inhibitors or mitochondrial calcium overload agents

[0089] In a sixth aspect of this application, an NCLX protein inhibitor or mitochondrial calcium overload agent is proposed. According to embodiments of this application, the NCLX protein inhibitor or mitochondrial calcium overload agent comprises: the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect.

[0090] Pharmaceutical Composition

[0091] In a seventh aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises: the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the NCLX protein inhibitor or mitochondrial calcium overload agent described in the sixth aspect.

[0092] According to embodiments of this application, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0093] In one alternative embodiment of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients conventional in the pharmaceutical field, such as diluents, buffers, osmotic pressure regulators, pH regulators, protectants, solutions (such as water), etc.

[0094] In one alternative embodiment of this application, suitable pharmaceutically acceptable excipients are well known in the art. Pharmaceutical compositions comprising such excipients can be formulated using known conventional methods.

[0095] In some alternative embodiments, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0096] The pharmaceutical compositions of this application can be administered via any acceptable method of administration. The pharmaceutical compositions of this application can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, as currently known or readily apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this application are formulated to ensure that the bioactive components contained therein are bioavailable after administration to a patient.

[0097] The clinical dosage of the pharmaceutical composition of this application can be determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including the patient's physique, weight, body surface area, age, the drug to be administered, sex, time and route of administration, general health, and other concurrently administered medications. The pharmaceutical composition of this application can be administered topically or systemically. Preferably, it can be administered intravenously or subcutaneously. The pharmaceutical composition of this application can also be administered directly to the target site, for example, by targeted delivery to internal or external target sites.

[0098] Uses in the preparation of drugs or reagents

[0099] In an eighth aspect of this application, the use of the isolated polypeptide described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the NCLX protein inhibitor or mitochondrial calcium overload agent described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect in the preparation of antibodies, pharmaceuticals, or reagents is provided.

[0100] According to embodiments of this application, the drug is used to prevent and / or treat diseases related to mitochondrial calcium ion regulation.

[0101] According to embodiments of this application, the reagent is used to inhibit NCLX protein activity and / or induce mitochondrial calcium overload.

[0102] method

[0103] In a ninth aspect of this application, a method is provided for inhibiting NCLX protein activity and / or inducing mitochondrial calcium overload in vitro. According to embodiments of this application, the method includes: overexpressing the isolated polypeptide described in the first aspect in cells; wherein the cells contain mitochondria and the NCLX protein. This method is particularly suitable for in vitro scientific research.

[0104] According to embodiments of this application, the overexpression of the isolated polypeptide described in the first aspect in cells is achieved by carrying the isolated nucleic acid described in the second aspect or the expression vector described in the third aspect.

[0105] In a tenth aspect of this application, a method for detecting NCLX protein is provided. According to an embodiment of this application, the method includes: contacting the isolated polypeptide described in the first aspect with a sample to be tested to form an immune complex.

[0106] According to embodiments of this application, the presence or content of NCLX protein in the sample to be tested is determined based on the signal of the immune complex.

[0107] It should be noted that the "sample to be tested" mentioned above can be any sample, and its specific type is not significant, all of which are within the scope of protection of this application. In one optional example, the sample to be tested contains NCLX protein.

[0108] In some alternative embodiments of this application, the signal includes a fluorescence signal.

[0109] In the eleventh aspect of this application, a method for preventing and / or treating a disease is proposed. According to embodiments of this application, the method comprises administering to a subject a pharmaceutically acceptable dose of the isolated polypeptide of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect, the recombinant cells of the fourth aspect, the NCLX protein inhibitor or mitochondrial calcium overload agent of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0110] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0111] The effective amount of the drug in this application may vary depending on the administration method and the severity of the disease to be treated. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0112] The isolated peptides, isolated nucleic acids, expression vectors, recombinant cells, NCLX protein inhibitors, mitochondrial calcium overload agents, or pharmaceutical compositions of this application may be incorporated into suitable pharmaceuticals, which may be prepared in various forms, such as liquids. Various routes of administration of the isolated peptides, isolated nucleic acids, expression vectors, recombinant cells, NCLX protein inhibitors, mitochondrial calcium overload agents, or pharmaceutical compositions of this application are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, and oral administration; however, this application is not limited to these exemplified routes of administration.

[0113] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0114] Example 1: Synthesis and Detection of Kuafu Small Peptides

[0115] Method 1: Amino acid synthesis (used for the preparation of polyclonal antibodies)

[0116] Chemical synthesis of small peptides: Kuafu small peptides (abbreviated as small peptides) were synthesized using solid-phase peptide synthesis technology based on the amino acid sequence shown in SEQ ID NO:2.

[0117] Purification of small peptides: The synthesized small peptides were purified by high performance liquid chromatography (HPLC) to obtain high-purity Kuafu small peptides.

[0118] Validation of purified small peptides: Mass spectrometry and amino acid analysis were used to verify the molecular weight and sequence accuracy of the purified small peptides. The results showed that the small peptide synthesis was successful.

[0119] Furthermore, polyclonal antibodies were prepared using the small peptides synthesized above.

[0120] The preparation method is as follows:

[0121] 1. Animal immunization

[0122] Immunize New Zealand white rabbits with the obtained Kuafu small peptide antigen via injection as follows (routine immunization 4 times): The injection time and dosage are as follows:

[0123]

[0124] 2. Antibody titer testing

[0125] One week after the 3rd / 4th immunization, blood samples should be collected for testing. The antiserum titer after the 3rd / 4th immunization should be measured using an indirect ELISA method, and the antiserum titer should be >1:32000. If the titer does not meet the requirement, 1-2 more immunizations should be administered.

[0126] 3. Final bloodletting: After confirming that the antibody titer has reached the expected level, final bloodletting is performed to collect serum containing a high concentration of antibodies.

[0127] 4. Antibody affinity purification: Specific antibodies are separated from serum using Protein A affinity chromatography, and then purified and preserved.

[0128] Method 2: Obtaining small peptides from long non-coding RNA translation

[0129] The origin of the Kuafu peptide: This peptide is translated from a long non-coding RNA (nucleotide sequence as shown in SEQ ID NO:9), and the specific ORF sequence is shown in SEQ ID NO:4.

[0130] (SEQ ID NO:9)。

[0131] Construction of overexpression vector: A eukaryotic expression vector containing a nucleotide sequence encoding a small peptide (amino acid sequence as shown in SEQ ID NO:2) was constructed using molecular cloning technology. The vector is named pCS2-Kuafu.

[0132] Cell culture: HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. When the cell density reached about 90%, transfection was performed.

[0133] PEI-based overexpression vector transfection: Before transfection, the culture dish was filled with preheated serum-free DMEM medium, and then an HBS-DNA-PEI mixture was prepared. The HBS buffer formulation was 20 mM HEPES (Sigma, H3375), 50 mM NaCl, and pH 7.4. The pCS2-Kuafu vector was transfected into HeLa cells using PEI.

[0134] Eukaryotic transcription and translation: The target Kuafu peptide is synthesized through the transcription and translation mechanism of HeLa eukaryotic cells.

[0135] Western Blot analysis: 36 hours after transfection, cells were collected, total mitochondrial protein was extracted, and the expression of small peptides was detected using the Kuafu small peptide polyclonal antibody obtained in Method 1 of this embodiment. The detection results are as follows: Figure 1 As shown.

[0136] Example 2: Kuafu small peptides are localized in the inner mitochondrial membrane

[0137] MitoProt and TMpred predict mitochondrial signaling peptides and transmembrane domains using the following steps:

[0138] 1. Mitochondrial localization and transmembrane domain prediction analyses were performed on the amino acid sequence of the Kuafu peptide (amino acid sequence as shown in SEQ ID NO:2). The prediction results for MitoProt and TMpred are shown in [link to MitoProt and TMpred predictions]. Figure 2 The results showed that the N-terminal amino acid sequence of Kuafu peptide, from position 1 to 19, is a mitochondrial targeting sequence, while the N-terminal amino acid region from position 32 to 52 is highly hydrophobic, suggesting that the peptide may be located on the mitochondrial membrane.

[0139] 2. To further verify the localization of the Kuafu peptide to mitochondria, the pCS2-Kuafu-eGFP expression vector and the mito-DsRed expression vector were constructed using conventional methods in this field. These two vectors were co-transfected into HeLa cells, and the mitochondrial localization of the peptide was verified using confocal microscopy. The results are as follows: Figure 3 As shown in Figure A, confocal microscopy revealed that the Kuafu peptide (green) was completely co-localized with the mitochondrial marker protein mito-DsRed (red). This further confirms that the Kuafu peptide is located in mitochondria.

[0140] 3. Verify whether positions 1-19 of the Kuafu peptide are mitochondrial signal peptides.

[0141] The expression vector pCS2-Kuafu-ΔMSP-eGFP (i.e., Kuafu-ΔSP, which deletes amino acids 1-19 from the N-terminus of the small peptide) and pCS2-Kuafu-MSP-eGFP (i.e., Kuafu-SP), containing only the signal peptide (amino acid sequence as shown in SEQ ID NO:7), were constructed using conventional methods in the art. See details [link to documentation]. Figure 3 B.

[0142] Then, HeLa cells were co-transfected with the pCS2-Kuafu-ΔMSP-eGFP and mito-DsRed expression vectors to overexpress Kuafu-ΔMSP-eGFP and the mitochondrial marker protein Mito-DsRed in HeLa cells. Live-cell imaging was then performed, and the results are shown below. Figure 3 C. The results showed that Kuafu-ΔMSP-eGFP (green) and Mito-DsRed (red) could not co-localize.

[0143] Furthermore, pCS2-Kuafu-ΔMSP-eGFP was co-transfected with the endoplasmic reticulum marker protein Sec61b-mCherry (red), followed by live-cell imaging detection. The results are shown in [link to results]. Figure 3 D. The results showed that Kuafu-ΔMSP-eGFP (green) was completely localized in the endoplasmic reticulum.

[0144] Furthermore, HeLa cells were co-transfected with the pCS2-Kuafu-MSP-eGFP and mito-DsRed expression vectors to overexpress Kuafu-MSP-eGFP and the mitochondrial marker protein Mito-DsRed in HeLa cells. Live-cell imaging was then performed, and the results are shown below. Figure 3 E. The results showed that Kuafu-MSP-eGFP (green), which contains only signal peptides at positions 1-19, can also co-localize with Mito-DsRed (red).

[0145] Therefore, the above results indicate that amino acids 1-19 of this Kuafu peptide are mitochondrial signal peptides, which is essential for Kuafu to be localized to mitochondria.

[0146] 4. Verification of the localization of Kuafu peptide to the inner mitochondrial membrane.

[0147] The pCS2-Kuafu-eGFP expression vector and the mito-DsRed expression vector were constructed using conventional methods in this field. Cos7 cells were co-transfected with both pCS2-Kuafu-eGFP and mito-DsRed expression vectors to obtain Cos7 cells overexpressing Kuafu-eGFP and Mito-DsRed. The localization of Kuafu peptides in mitochondria was observed using super-resolution SIM confocal imaging. Results are detailed below. Figure 3 F. The results showed that Kuafu peptides (green) were distributed in a crest pattern and were interspersed with mitochondrial matrix protein Mito-DsRed (red).

[0148] Furthermore, the pCS2-Kuafu-Flag expression vector was constructed using conventional methods in the art, and Kuafu-Flag was transfected into HeLa cells. After cell homogenization, mitochondria were extracted using conventional methods in the art. Then, the cells were treated with proteinase K in the presence and absence of digitonin or Triton X-100. Proteinase K treatment digested proteins exposed on the outer mitochondrial membrane, digitonin treatment disrupted the outer mitochondrial membrane structure, and Triton X-100 treatment disrupted the inner mitochondrial membrane structure. Immunoblot analysis was then performed using Flag antibody (Invitrogen, M20008), Fundc1 (mitochondrial outer membrane protein, provided by Professor Chen Quan's laboratory), Tim23 (mitochondrial inner membrane protein; BD, 612278) antibody, and Hsp60 (mitochondrial matrix protein; Cell Signaling Technology, 4870) antibody. The results are as follows: Figure 3 As shown in G. The results show that the Kuafu peptide is located in the same region as the mitochondrial inner membrane protein Tim23, within the mitochondrial inner membrane.

[0149] pass Figure 3 F and Figure 3 The results from G confirm the mitochondrial inner membrane localization of the Kuafu peptide.

[0150] 5. Verify whether positions 32-52 of the Kuafu peptide are mitochondrial transmembrane domains.

[0151] A truncated pCS2-Kuafu-ΔTM-eGFP expression vector (with the amino acid sequence of the truncated Kuafu-ΔTM vector deleted from the transmembrane domain shown in SEQ ID NO:10) and a pCS2-mito-DsRed expression vector were constructed. Both pCS2-Kuafu-ΔTM-eGFP and pCS2-mito-DsRed expression vectors were co-transfected into HeLa cells to overexpress Kuafu-ΔTM-eGFP and the mitochondrial marker protein Mito-DsRed. Live-cell imaging was then performed, and the results are shown below. Figure 3 H. Imaging results showed that Kuafu-ΔTM-eGFP (green) and Mito-DsRed (red) still colocalized.

[0152] MIIRLGRLLTPGYFRLLQRQVAGEVQAPVHDRKQMTEKVRQEPAI (SEQ ID NO: 10).

[0153] Furthermore, the pCS2-Kuafu-ΔTM-Flag expression vector was constructed using conventional methods in the art. Kuafu-ΔTM-Flag was transfected into HeLa cells. After cell homogenization, mitochondria were extracted using conventional methods in the art, and treated with proteinase K in the presence and absence of didigitonin or Triton X-100. Immunoblot analysis was performed using Flag antibody, Tom20 (mitochondrial outer membrane protein; BD, 611223) antibody, Tim23 (mitochondrial inner membrane protein) antibody, and Hsp60 (mitochondrial matrix protein) antibody. The results are as follows: Figure 3 As shown in Figure I, the results indicate that Kuafu-ΔTM is localized in the mitochondrial matrix and not in the inner mitochondrial membrane. Therefore, it can be demonstrated that the amino acid region at positions 32-52 of the N-terminus of the Kuafu peptide is a transmembrane domain.

[0154] Example 3: Kuafu small peptides promote the degradation of NCLX protein by m-AAA protease

[0155] 1. The pCS2-Kuafu-HA expression vector was constructed using conventional methods in this field. Different doses of Kuafu-HA were overexpressed in 293T cells. Mitochondria were extracted 36 hours after transfection and analyzed with NCLX antibody. (Abcam,ab83551) Immunoblot analysis was performed; results are shown below. Figure 4 A. The results showed that NCLX protein expression decreased significantly with increasing Kuafu expression.

[0156] Therefore, it can be concluded that the Kuafu peptide of this application can promote the degradation of NCLX.

[0157] 2. Mitochondria originated from archaea, and the quality control and degradation of proteins within them are similar to those in prokaryotes. m-AAA protease and i-AAA protease are two important AAA proteases (ATPases associated with various cellular activities) within mitochondria. i-AAA protease is located in the mitochondrial inner membrane protein hydrolysis domain facing the intermembrane space, composed of the YME1L1 subunit, forming a hexameric complex; m-AAA protease is located in the mitochondrial inner membrane but its hydrolysis domain faces the matrix side, composed of the AFG3L2 and SPG7 subunits, forming a heterohexameric complex. To investigate which AAA protease degrades NCLX located in the mitochondrial inner membrane, the following experiment was conducted in this example:

[0158] Overexpression vectors of pCS2-YME1L1-Myc, pCS2-AFG3L2-Myc, and pCS2-SPG7-Myc were constructed using conventional methods in the art. These vectors were transfected into 293T cells at different doses. Thirty-six hours after transfection, total cellular protein was extracted using conventional methods in the art, and Western blotting analysis was performed using the NCLX antibody. Results are shown below. Figure 4 B. Figure 4 Figures C and D show that the NCLX protein expression level did not change significantly with the gradient overexpression of YME1L1-Myc. Figure 4 B); however, with the increase of AFG3L2-Myc and SPG7-Myc expression levels, the NCLX protein expression level decreased significantly ( Figure 4 C and Figure 4 D). These results indicate that NCLX is degraded via m-AAA.

[0159] The amino acid sequence of YME1L1 is shown in SEQ ID NO:11:

[0160] MFSLSSTVQPQVTVPLSHLINAFHTPKNTSVSLSGVSVSQNQHRDVVPEHEAPSSECMFSDFLTKLNIVSIGKGKIFEGYRSMFMEPAKRMKKSLDTTDNWHIRPEPFSLSIPPSLNLRDLGLSELKIGQIDQLVENLLPGFCKGKNISSHWHTSHVSAQSFFENKYGNLDIFSTLRSSCLYRHHSRALQSICSDLQYWPVFIQSRGFKTLKSRTRRLQSTSERLAETQNIAPSFVKGFLLRDRGSDVESLDKLMKTKNIPEAHQDAFKTGFAEGFLKAQALTQKTNDSLRRTRLILFVLLLFGIYGLLKNPFLSVRFRTTTGLDSAVDPVQMKNVTFEHVKGVEEAKQELQEVVEFLKNPQKFTILGGKLPKGILLVGPPGTGKTLLARAVAGEADVPFYYASGSEFDEMFVGVGASRIRNLFREAKANAPCVIFIDELDSVGGKRIESPMHPYSRQTINQLLAEMDGFKPNEGVIIIGATNFPEALDNALIRPGRFDMQVTVPRPDVKGRTEILKWYLNKIKFDQSVDPEIIARGTVGFSGAELENLVNQAALKAAVDGKEMVTMKELEFSKDKILMGPERRSVEIDNKNKTITAYHESGHAIIAYYTKDAMPINKATIMPRGPTLGHVSLLPENDRWNETRAQLLAQMDVSMGGRVAEELIFGTDHITTGASSDFDNATKIAKRMVTKFGMSEKLGVMTYSDTGKLSPETQSAIEQEIRILLRDSYERAKHILKTHAKEHKNLAEALLTYETLDAKEIQIVLEGKKLEVR(SEQ ID NO:11);

[0161] The amino acid sequence of AFG3L2 is shown in SEQ ID NO:12:

[0162] MAHRCLRLWGRGGCWPRGLQQLLVPGGVGPGEQPCLRTLYRFVTTQARASRNSLLTDIIAAYQRFCSRPPKGFEKYFPNGKNGKKASEPKEVMGEKKESKPAATTRSSGGGGGGGGKRGGKKDDSHWWSRFQKGDIPWDDKDFRMFFLWTALFWGGVMFYLLLKRSGREITWKDFVNNYLSKGVVDRLEVVNKRFVRVTFTPGKTPVDGQYVWFNIGSVDTFERNLETLQQELGIEGENRVPVVYIAESDGSFLLSMLPTVLIIAFLLYTIRRGPAGIGRTGRGMGGLFSVGETTAKVLKDEIDVKFKDVAGCEEAKLEIMEFVNFLKNPKQYQDLGAKIPKGAILTGPPGTGKTLLAKATAGEANVPFITVSGSEFLEMFVGVGPARVRDLFALARKNAPCILFIDEIDAVGRKRGRGNFGGQSEQENTLNQLLVEMDGFNTTTNVVILAGTNRPDILDPALLRPGRFDRQIFIGPPDIKGRASIFKVHLRPLKLDSTLEKDKLARKLASLTPGFSGADVANVCNEAALIAARHLSDSINQKHFEQAIERVIGGLEKKTQVLQPEEKKTVAYHEAGHAVAGWYLEHADPLLKVSIIPRGKGLGYAQYLPKEQYLYTKEQLLDRMCMTLGGRVSEEIFFGRITTGAQDDLRKVTQSAYAQIVQFGMNEKVGQISFDLPRQGDMVLEKPYSEATARLIDDEVRILINDAYKRTVALLTEKKADVEKVALLLLEKEVLDKNDMVELLGPRPFAEKSTYEEFVEGTGSLDEDTSLPEGLKDWNKEREKEKEEPPGEKVAN (SEQ ID NO:12);

[0163] The amino acid sequence of SPG7 is shown in SEQ ID NO:13:

[0164] MAVLLLLLRALRRGPGPGPRPLWGPGPAWSPGFPARPGRGRPYMASRPPGDLAEAGGRALQSLQLRLLTPTFEGINGLLLKQHLVQNPVRLWQLLGGTFYFNTSRLKQKNKEKDKSKGKAPEEDEEERRRRERDDQMYRERLRTLLVIAVVMSLLNALSTSGGSISWNDFVHEMLAKGEVQRVQVVPESDVVEVYLHPGAVVFGRPRLALMYRMQVANIDKFEEKLRAAEDELNIEAKDRIPVSYKRTGFFGNALYSVGMTAVGLAILWYVFRLAGMTGREGGFSAFNQLKMARFTIVDGKMGKGVSFKDVAGMHEAKLEVREFVDYLKSPERFLQLGAKVPKGALLLGPPGCGKTLLAKAVATEAQVPFLAMAGPEFVEVIGGLGAARVRSLFKEARARAPCIVYIDEIDAVGKKRSTTMSGFSNTEEEQTLNQLLVEMDGMGTTDHVIVLASTNRADILDGALMRPGRLDRHVFIDLPTLQERREIFEQHLKSLKLTQSSTFYSQRLAELTPGFSGADIANICNEAALHAAREGHTSVHTLNFEYAVERVLAGTAKKSKILSKEEQKVVAFHESGHALVGWMLEHTEAVMKVSITPRTNAALGFAQMLPRDQHLFTKEQLFERMCMALGGRASEALSFNEVTSGAQDDLRKVTRIAYSMVKQFGMAPGIGPISFPEAQEGLMGIGRRPFSQGLQQMMDHEARLLVAKAYRHTEKVLQDNLDKLQALANALLEKEVINYEDIEALIGPPPHGPKKMIAPQRWIDAQREKQDLGEEETEETQQPPLGGEEPTWPK(SEQ ID NO:13).

[0165] 3. The Kuafu peptide can promote the binding of NCLX and m-AAA

[0166] 1) To further investigate how the Kuafu peptide participates in regulating the degradation of NCLX protein via m-AAA, an expression vector of the m-AAA negative dominant mutant AFG3L2 E575Q-Myc was constructed using conventional methods in this field (studies have shown that the 575th amino acid of mutated AFG3L2 can affect its proteolytic activity without affecting its binding to protein substrates). Then, AFG3L2E575Q-Myc was transfected into 293T cells at different doses, and Western blot analysis was performed using an NCLX antibody. The results are shown in [link to results]. Figure 4 E. The results showed that NCLX protein expression was significantly increased, confirming that AFG3L2 E575Q is indeed a mutant with negative dominant function.

[0167] 2) Expression vectors pCS2-Kuafu-Flag, pCS2-NCLX-Myc, and pCS2-AFG3L2E575Q-Myc were constructed using conventional methods in this field. These vectors were then transfected into 293T cells. Thirty-six hours after transfection, cells were collected and lysed. Kuafu-Flag and NCLX-Myc or AFG3L2E575Q-Myc were then subjected to co-immunoprecipitation (Co-IP), and the results are shown below. Figure 4 F and Figure 4 G. The results showed that the Kuafu peptide could bind to NCLX ( Figure 4 F), can also be combined with AFG3L2 E575Q ( Figure 4 G).

[0168] 3) Referring to step 2) above, perform Co-IP on NCLX-Flag and AFG3L2 E575Q-Myc, as well as Kuafu-Flag and AFG3L2E575Q-Myc, respectively. See the results below. Figure 4 H. The results showed that NCLX and AFG3L2 interacted weakly, while Kuafu and AFG3L2 interacted significantly. This result suggests that the Kuafu peptide may promote NCLX protein degradation by enhancing the interaction between NCLX and AFG3L2.

[0169] 4) Referring to step 2) above, the interaction between NCLX and AFG3L2 was further examined in the absence and presence of Kuafu peptide. The results are shown below. Figure 4I. The results showed that in the absence of Kuafu peptide, the interaction between NCLX and AFG3L2 was weak, while in the presence of Kuafu peptide, the interaction between NCLX and AFG3L2 was significantly enhanced. This result indicates that Kuafu peptide can promote NCLX protein degradation by enhancing the interaction between NCLX and the m-AAA protease AFG3L2.

[0170] Example 4: Kuafu small peptide induces mitochondrial calcium overload

[0171] To investigate the role of Kuafu peptides in mitochondrial homeostasis regulation, 100 μM ATP was used as an agonist to induce the release of calcium from the endoplasmic reticulum calcium stores. 2+ Ca2+ is absorbed into the cytoplasm and mitochondria. 2+ Concentration was indicated by the fluorescence intensity of mito-GCaMP6s. HeLa cells transfected with mito-DsRed served as the control group, and Kuafu was overexpressed at a 3:1 ratio. The experimental group was treated with CGP-37157, an inhibitor of the mitochondrial calcium release channel NCLX. Mitochondrial Ca concentration was measured in an external calcium-free system. 2+ The concentration of Ca in mitochondria changes over time. 2+ Concentration changes over time as follows Figure 5 A and Figure 5 As shown in B. Then, mito-DsRed positive cells were selected for the experiment, and 100 μM ATP was added to the culture medium at approximately 60 seconds. Typical images were captured at five time points: 30s, 100s, 180s, 360s, and 720s. Higher heat indicates higher mitochondrial calcium levels. 2+ The higher the concentration, the better. Figure 5 A and Figure 5 As can be clearly seen in B, the mitochondrial Ca in the control group is lower. 2+ The concentration briefly increased to a peak and then rapidly decreased. In contrast, mitochondrial Ca2+ concentrations were significantly higher in the Kuafu and CGP-37157 overexpression groups. 2+ The concentration rises to its peak and then declines slowly.

[0172] Furthermore, the mitochondrial Ca2+ levels per unit time before and after reaching peak values ​​were analyzed. 2+ Concentration changes represent mitochondrial uptake rate and mitochondrial release rate, respectively; results are shown in [reference needed]. Figure 5 C and Figure 5 D. Analysis results showed that, compared with the control group, the mitochondrial uptake of Ca in the Kuafu overexpression experimental group was significantly higher. 2+ The rate was not affected, but like the mitochondrial calcium release inhibitor CGP37157, it slowed down the release of calcium from mitochondria. 2+The rate of change was observed, and Kuafu peptide exhibited a larger area under the curve compared to CGP37157. Therefore, this indicates that Kuafu peptide can inhibit mitochondrial calcium release and induce mitochondrial calcium overload, with a more significant effect than CGP37157.

[0173] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0174] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An isolated polypeptide, characterized in that, The isolated polypeptide includes a functional region having an amino acid sequence as shown in SEQ ID NO:

1.

2. The isolated polypeptide according to claim 1, characterized in that, The isolated polypeptide further includes a signal peptide; Optionally, the C-terminus of the signal peptide is connected to the N-terminus of the functional region; Optionally, the isolated polypeptide has an amino acid sequence as shown in SEQ ID NO:

2.

3. An isolated nucleic acid, characterized in that, The isolated nucleic acid is used to encode the isolated polypeptide according to any one of claims 1 to 2; Optionally, the isolated nucleic acid has a nucleotide sequence as shown in SEQ ID NO:3 or 4.

4. An expression carrier, characterized in that, The expression vector carries the isolated nucleic acid as described in claim 3; or The expression vector expresses the isolated polypeptides described in any one of 1 to 2; Optionally, the expression vector is selected from eukaryotic expression vectors or prokaryotic expression vectors.

5. A recombinant cell, characterized in that, include: Carrying the isolated nucleic acid as described in claim 3 or the expression vector as described in claim 4; or, Expressing the isolated polypeptide according to any one of claims 1 to 2; Optionally, the recombinant cells are obtained by introducing the expression vector of claim 4 into a host cell.

6. Use of the isolated polypeptide according to any one of claims 1 to 2, the isolated nucleic acid according to claim 3, the expression vector according to claim 4, or the recombinant cell according to claim 5 in the preparation of NCLX protein inhibitors and mitochondrial calcium overload agents.

7. An NCLX protein inhibitor or mitochondrial calcium overload agent, characterized in that, include: The isolated polypeptide according to any one of claims 1 to 2, the isolated nucleic acid according to claim 3, the expression vector according to claim 4, or the recombinant cell according to claim 5.

8. A pharmaceutical composition, characterized in that, include: The isolated polypeptide according to any one of claims 1-2, the isolated nucleic acid according to claim 3, the expression vector according to claim 4, the recombinant cell according to claim 5, or the NCLX protein inhibitor or mitochondrial calcium overload agent according to claim 7; and Optional pharmaceutically acceptable excipients.

9. Use of the isolated polypeptide of any one of claims 1 to 2, the isolated nucleic acid of claim 3, the expression vector of claim 4, the recombinant cell of claim 5, the NCLX protein inhibitor or mitochondrial calcium overload agent of claim 7, or the pharmaceutical composition of claim 8 in the preparation of antibodies, pharmaceuticals or reagents; Optionally, the drug is used to prevent and / or treat diseases related to mitochondrial calcium ion regulation; Optionally, the reagent is used to inhibit NCLX protein activity and / or induce mitochondrial calcium overload.

10. A method for inhibiting NCLX protein activity and / or inducing mitochondrial calcium overload in vitro, characterized in that, include: Overexpression of the isolated polypeptide according to any one of claims 1 to 2 in cells; The cells contain mitochondria and NCLX protein; Optionally, the overexpression of the isolated polypeptide according to any one of claims 1 to 2 in cells is achieved by carrying the isolated nucleic acid according to claim 3 or the expression vector according to claim 4.

11. A method for detecting NCLX protein, characterized in that, include: The isolated polypeptide according to any one of claims 1 to 2 is contacted with the sample to be tested to form an immune complex; Optionally, the presence or content of NCLX protein in the sample to be tested is determined based on the signal of the immune complex.

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