Application of Emc3 protein in protein sedimentation related diseases

Abstract

The invention belongs to the technical field of biomedicine and biology, particularly relates to a medicine capable of treating diseases related to protein sedimentation, and particularly discloses a medicine for treating or improving diseases or symptoms related to protein sedimentation, the medicine contains Emc3 protein, and the amino acid sequence of the Emc3 protein is shown as SEQ ID No: 1; the nucleotide sequence of the Emc3 protein is as shown in SEQ ID No: 2. The Emc3 can be used as a target gene for screening and preparing drugs for improving cardiac protein sedimentation, cardiac myocardial cell hypertrophy, cardiac hypertrophy, cardiac failure and the like, reducing death caused by protein deposition and prolonging survival time.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and biotechnology, and specifically relates to a drug that can treat diseases related to protein deposition. Background Technology

[0002] Proteins are among the most important structural and functional molecules in cells, participating in almost all intracellular life activities, including signal transduction, metabolic regulation, and providing structural support. Protein homeostasis refers to the dynamic equilibrium achieved within the cell in processes such as protein synthesis, folding, repair, and degradation. Maintaining protein homeostasis is crucial for normal cellular function and the entire life process. To achieve this, eukaryotes have evolved various protein quality control (PQC) mechanisms, including heat shock proteins, autophagy, and the ubiquitin-proteasome system. These mechanisms can recognize and degrade abnormal proteins to maintain protein homeostasis. When protein homeostasis is disrupted, it can lead to the accumulation and deposition of abnormal proteins, forming protein deposits that may impair cellular structure and function. For example, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are closely related to abnormal protein deposition. In addition to these neurodegenerative diseases, abnormal protein deposition is also associated with liver diseases (such as liver fibrosis and cirrhosis), cardiovascular diseases (such as myocardial hypertrophy and heart failure), and kidney diseases (such as glomerulonephritis and renal failure).

[0003] Currently, treatment methods for protein homeostasis-related diseases vary depending on the disease type and individual differences, and there are four main treatment approaches, including (1) drug therapy: For specific diseases, doctors may prescribe drugs to alleviate symptoms or slow disease progression. For example, for neurodegenerative diseases such as Alzheimer's and Parkinson's, drugs can be used to improve symptoms and control disease progression. However, drug therapy may have side effects, and for some diseases, there is currently no effective drug treatment; (2) gene therapy: This aims to treat diseases by repairing or replacing abnormal genes. For some hereditary protein deposition-related diseases, gene therapy may provide a potential treatment option. However, currently... Gene therapy is still in the research stage and faces many technical and safety challenges; (3) Protein degradation enhancers: Researchers are exploring some drugs to enhance the ability of cells to degrade proteins, thereby clearing abnormal protein deposits. These drugs can promote the degradation of abnormal proteins by activating the intracellular quality control system. However, the efficacy and safety of these drugs still need further research; (4) Symptomatic treatment: For some protein deposition-related diseases, doctors may adopt symptomatic treatment methods to relieve symptoms and improve the patient's quality of life. For example, for patients with cardiovascular disease, doctors may recommend adjusting diet, exercising, or using drugs to control blood pressure and cardiac load. Although many current research and treatment strategies are mainly focused on the treatment of neurodegenerative diseases, the causes of protein homeostasis imbalance are multifaceted, including the complexity of the disease, the side effects of treatment, and individual differences. The types of diseases it causes are also very wide. Therefore, more strategies and means need to be developed to treat various complex diseases caused by protein homeostasis imbalance.

[0004] The endoplasmic reticulum membrane protein complex (Emc) is a transmembrane protein complex required for protein folding in the endoplasmic reticulum. First identified in yeast, the loss of Emc subunits in yeast leads to the inability of membrane proteins to fold properly into secondary structures, resulting in their accumulation in the endoplasmic reticulum and the production of unfolded protein reactions. There are 10 Emc members in mammals, including membrane proteins Emc1, Emc3, Emc4, Emc5, Emc6, Emc7, and Emc10, and cytoplasmic proteins Emc2, Emc8, and Emc9. Proteomics studies have revealed that Emc is involved in the degradation of endoplasmic reticulum-associated proteins, the formation of endoplasmic reticulum-mitochondrial plugs with mitochondria, and the proper assembly of multi-transmembrane proteins (Chen Kuangyang, Wang Xuanchun, Research progress on endoplasmic reticulum membrane protein complex 10 (Emc10); Fudan Journal). Endoplasmic reticulum membrane protein complex 3 (Emc3), also known as TMEM111, is a subunit of the highly conserved endoplasmic reticulum membrane protein complex Emc protein. Emc3 protein plays a regulatory role in the transport and signal transduction of the morphogenetic factor Wingless (wg). Emc protein was first discovered in yeast and mammals to be essential for maintaining endoplasmic reticulum homeostasis. Studies have shown that the Emc3 subunit of the ER membrane protein complex controls angiogenesis via the FZD4 / WNT signaling axis. This research group constructed Emc3 conditional vascular endothelial cell knockout mice and used mouse retinal vessels as a model to investigate the effects of Emc3 knockout on retinal vessel development. The study found that Emc3 vascular endothelial cell-specific knockout led to abnormal phenotypes such as delayed retinal vessel development, reduced apical cells, and leakage. Further knockout of Emc3 in primary human retinal endothelial cells (HRECs) also resulted in in vitro angiogenesis defects. Using transcriptome sequencing, real-time quantitative PCR (RT-qPCR), and dual-luciferase reporter gene assays, it was found that Emc3 regulates vessel development by controlling the proper folding and expression of the Norrin / β-catenin signaling pathway receptor FZD4. Patent CN115697390A discloses a drug combination for treating tumors, which includes a first membrane component comprising a membrane derived from the inner membrane of bacteria and a component derived from other organisms besides said bacteria, one of the components from other organisms being ER membrane protein complex subunit 3 (Emc3). Summary of the Invention

[0005] This invention verifies the relationship between the expression of Emc3 protein or gene and diseases related to protein sedimentation. Specifically, it demonstrates in cell and animal models that Emc3 protein or gene can affect the sedimentation of protein oligomers in cells and the occurrence and progression of diseases caused by it, and that activating Emc3 function can treat diseases related to protein sedimentation. Based on this, this invention was completed.

[0006] In a first aspect, the present invention provides a medicament for treating or improving protein deposition-related diseases or symptoms, the medicament containing Emc3 protein, the amino acid sequence of which is shown in SEQ ID No:1.

[0007] Furthermore, the amino acid sequence of the Emc3 protein also includes 80%-99% homologous sequences, preferably 80%-85%; more preferably 85%-90%; and more preferably 90%-95%.

[0008] Furthermore, the Emc3 protein also includes its fusion protein, conjugate, nucleic acid encoding its fusion protein or conjugate, and vector expressing the aforementioned nucleic acid molecules.

[0009] Furthermore, the drug may also contain a pharmaceutically acceptable carrier.

[0010] Furthermore, the administration methods of the drug dosage form include, but are not limited to, injectable formulations or oral formulations.

[0011] Furthermore, the drug dosage forms include gels, suspensions, emulsions, polymers, nanoparticles, microspheres, rectal capsules, enemas, oral solutions, and implants, which may optionally be controlled-release and / or sustained-release via dosage forms or devices.

[0012] Furthermore, protein deposition-related diseases or symptoms are preferably those related to the heart.

[0013] Furthermore, the diseases include: rheumatic heart disease, endocarditis, myocarditis, restrictive heart disease, hypertrophic heart disease, dilated cardiomyopathy, and other heart diseases with abnormal protein sedimentation rates.

[0014] In a second aspect, the present invention provides the use of Emc3 protein in the preparation of medicaments for treating or improving protein deposition-related diseases or symptoms, wherein the amino acid sequence of the Emc3 protein is shown in SEQ ID No:1.

[0015] Furthermore, the amino acid sequence of the Emc3 protein also includes 80%-99% homologous sequences, preferably 80%-85%; more preferably 85%-90%; and more preferably 90%-95%.

[0016] Furthermore, the Emc3 protein also includes its fusion protein, conjugate, nucleic acid encoding its fusion protein or conjugate, and vector expressing the aforementioned nucleic acid molecules.

[0017] Furthermore, protein deposition-related diseases or symptoms are preferably those related to the heart.

[0018] Furthermore, the diseases include: rheumatic heart disease, endocarditis, myocarditis, restrictive heart disease, hypertrophic heart disease, dilated cardiomyopathy, and other heart diseases with abnormal protein sedimentation rates.

[0019] Thirdly, the present invention provides the application of Emc3 protein or Emc3 gene in screening drugs for the treatment of protein deposition-related diseases, wherein the Emc3 protein is consistent with that described in the first aspect of the present invention, and the Emc3 gene sequence is shown in SEQ ID No:2. When the nucleotides of Emc3 protein or Emc3 gene are highly expressed, the occurrence and progression of protein deposition-related diseases are inhibited, that is, the drug to be screened can be used to treat protein deposition-related diseases.

[0020] Fourthly, the present invention provides a kit for screening drugs for treating protein deposition-related diseases, the kit containing reagents for detecting Emc3 protein or Emc3 gene expression, wherein when Emc3 protein or Emc3 gene is highly expressed, the occurrence and progression of protein deposition-related diseases are inhibited; the Emc3 protein is consistent with that described in the first aspect of the present invention, and the Emc3 gene sequence is shown in SEQ ID No:2.

[0021] Beneficial effects

[0022] This invention discovers a novel function of the Emc3 protein / gene: knocking out the Emc3 gene significantly reduces autophagy levels in the heart, causes cardiomyocyte hypertrophy, weakens cardiac function, and increases mortality. When the Emc3 gene is overexpressed in cells, it can improve cardiac protein deposition, cardiomyocyte hypertrophy, cardiac hypertrophy, and heart failure, reducing protein deposition-induced death and prolonging survival. Therefore, Emc3 can serve as a target gene for screening and developing drugs that improve cardiac protein deposition, cardiomyocyte hypertrophy, cardiac hypertrophy, and heart failure, reducing protein deposition-induced death and prolonging survival. Attached Figure Description

[0023] Figure 1 Emc3 expression affects CryAB in neonatal rat ventricular myocytes (NRVMs). R120G Induced aggregate content. A. Schematic diagram of experimental design. NRVMs are laid flat on a 2-well glass slide (1 x 10⁻⁶). 5 / well), infected overnight with lentivirus (Emc3 knockout, panels B, C) or adenovirus (Emc3 expression, panel D), and the next day infected with the aggregate reporter factor (CryAB). R120G Adenovirus infection with GFP was performed. Cells were harvested on day 7 for aggregate content analysis. B. Lentiviral-mediated short hairpin (sh) RNA infection reduced Emc3 RNA levels in NRVMs (n=5). C. Decreased Emc3 expression increased CryAB levels in NRVMs. R120G The aggregate content, n=5. The figure shows the repeat region, scale bar: 200μm. D. Adenovirus-mediated Emc3 expression reduces CryAB in NRVMs. R120G The aggregation content, n = 4-6. The diagram shows the repeating region, scale bar: 200 μm. CryAB is expressed as... R120G -GFP adenovirus infection of NRVMs (1X10) 5 Cells were incubated overnight in ( / well) and then infected with an adenovirus expressing Emc3 on day 3 when cell aggregates accumulated. Aggregate levels were measured on day 6. E. Schematic diagram of the experiment investigating Emc3's clearance of protein aggregates. F. In the later stages of the aggregation process, adenovirus-mediated Emc3 expression slowed down CryAB in NRVMs. R120G Accumulation of aggregate content, n=5. Scale bar: 100μm. Error bars represent SEM. Compared with the control group shown, *P<0.05, **P<0.01, ***P<0.001; #P<0.05, ###P<0.001.

[0024] Figure 2 Reducing Emc3 expression decreases autophagy activity. A. Neonatal rat ventricular myocytes (NRVMs) were infected with an adenovirus expressing GFP-LC3, followed by co-infection with a lentivirus (Ctrl) expressing Emc3 or scrambled-shRNA for 48 hours, and then treated with bafloxacin A1 (50 nmol / L) or DMSO (vector) for 4 hours. Scale bar: 100 μm. B. NRVMs were infected with a lentivirus expressing Emc3 or scrambled-shRNA (Ctrl) for 48 hours, followed by treatment with bafloxacin A1 (50 nmol / L) or DMSO (vector) for 4 hours. Western blot analysis was performed on the cell lysates. C. 12-week-old Emc3 cells... flox / + (Emc3 f / + ) and Emc3 + / flox / αMHC-Cre(CM-Emc3 + / - Hearts of mice were analyzed using Western blot analysis of cardiac lysates. D. 12-week-old non-transgenic (Ntg) and CM-Emc3 mice. + / -Mice were injected with reserpine (ip 40 mg / kg) or PBS for 1 hour, and their hearts were harvested for Western blot analysis. CM-Emc3 + / - Mice exhibited systolic dysfunction (E) and left ventricular dilatation (F, G) at 6 months of age. The effects of Emc3 knockout in the heart on mouse survival during development were investigated. Error bars in the BG panel represent SEM. Data were analyzed using one-way ANOVA and Tukey post-hoc analysis. *P<0.05, **P<0.01, ***P<0.001. H, Kaplan-Meier analysis. Log-rank test was used; P<0.0001, n=11-16. BAF: baferomycin. αCre: αMHC-Cre; Emc3 f / + Emc3 flox / + Emc3 flox / + CM-Emc3 + / - Emc3 + / flox / αMHC-Cre; FS: fractional shortening rate; LV: left ventricle; LV Vol(d): diastolic volume of the left ventricle; LV Vol(s): systolic volume of the left ventricle.

[0025] Figure 3 CM-specific expression of Emc3 can reduce the content of aggregates in vivo. A. Schematic diagram of experimental design. B. Emc3 immunostaining shows that CM-specific expression of Emc3 can reduce CryAB. R120G Aggregate content. Heart sections were stained with CryAB antibody (green) and α-actin (red) to identify visible aggregates and CMs. The area of ​​aggregates and CMs was measured, and their ratio was calculated as an indicator of aggregate content, n = 6–8, scale bar: 100 μm. C. Cardiac hypertrophy was quantified using the heart weight to body weight (HW / BW) ratio. D. CM surface area was examined using wheat germ lectin staining, n = 4–8. Scale bar: 100 μm. E. Cardiac contractile function (fractional shortening, FS) was measured, n = 9–17. In the BE panel, error bars represent SEM. Data were analyzed using one-way ANOVA and Tukey post-hoc analysis. *P<0.05, **P<0.01, ***P<0.001, compared with the Ntg group; ##P<0.01, ###P<0.001, between groups. F. Kaplan-Meier survival curves. Log-rank test was used, P < 0.0001, n = 14-21. Ntg: non-GMO; Emc3 / αMCM: CAG-CAT cMyc-Emc3 / αMHC-MerCreMer;Emc3 / CryAB R120G CAG-CAT cMyc-Emc3 / CryAB R120G Emc3 / αMCM / CryABR120G CAG-CAT cMyc-Emc3 / αMHC-MerCreMer / CryAB R120G . Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0027] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0028] The Emc3 gene, acting as a protein degradation enhancer, significantly reduces protein aggregation and deposition when overexpressed in cells. Overexpression of the Emc3 gene in animal tissues can improve diseases related to protein aggregation and deposition. 1) Clearing abnormal protein deposits: Some diseases cause abnormal proteins to deposit within cells, forming aggregates or plaques. Protein degradation enhancers can help clear these abnormal protein deposits, thereby alleviating disease symptoms and progression. In this study, it significantly reduced protein deposition in cells and the heart, improving cardiac structural and functional damage caused by protein deposition. 2) Promoting cellular function recovery: The presence of abnormal protein deposits can interfere with normal cellular function. By enhancing protein degradation, protein degradation enhancers can help cells restore normal function, thereby improving disease symptoms. 3) Delaying disease progression: Some protein deposition-related diseases are characterized by progressive development. Protein degradation enhancers may be able to delay disease progression, reduce the accumulation of abnormal proteins, thereby prolonging patient survival and improving quality of life. 4) Increasing patient survival time: Abnormal accumulation of certain proteins can lead to organ failure and death. In this study, it significantly improved individual death caused by abnormal protein deposition and significantly prolonged the survival time of mice.

[0029] The amino acid sequence of the Emc3 protein described in this article is shown in SEQ ID No:1:

[0030] MAGPELLLDSNIRLWVVLPIVIITFFVGMIRHYVSILLQSDKKLTQEQVSDSQVLIRSRVLR

[0031] ENGKYIPKQSFLTRKYYFNNPEDGFFKKTKRKVVPPSPMTDPTMLTDMMKGNVTNVLPM

[0032] ILIGGWINMTFSGFVTTKVPFPLTLRFKPMLQQGIELLTLDASWVSSASWYFLNVFGLRSIY

[0033] SLILGQDNAADQSRMMQEQMTGAAMAMPADTNKAFKTEWEALELTDHQWALDDVEEELMARDLHFEGMFKKELQTSIF。

[0034] The nucleotide sequence of the Emc3 gene in this article is shown as SEQ ID No:2:

[0035] ATGGCGGGGCCCGAGCTGCTGCTTGACTCCAACATCCGCCTCTGGGTGGTCCTGCCCA

[0036] TCGTTATCATCACTTTCTTCGTGGGCATGATCCGCCACTACGTGTCAATCCTACTGCAGA

[0037] GCGACAAGAAGCTCACCCAGGAACAAGTGTCTGACAGTCAGGTCCTAATTCGAAGCA

[0038] GAGTCCTCAGGGAAAATGGAAAATACATTCCCAAGCAGTCTTTCTTAACACGAAAATA

[0039] TTACTTCAACAACCCAGAGGATGGATTTTTCAAAAAAACAAAAAGGAAGGTTGTGCC

[0040] ACCTTCCCCCATGACAGACCCCACCATGCTCACAGACATGATGAAAGGGAATGTCACA

[0041] AATGTCCTCCCAATGATTCTTATCGGCGGATGGATCAACATGACGTTTTCAGGCTTTGT

[0042] CACAACTAAGGTCCCGTTTCCACTGACACTTCGCTTCAAGCCTATGCTTCAGCAAGGA

[0043] ATAGAGCTGCTCACACTAGACGCATCCTGGGTGAGTTCTGCATCCTGGTACTTCCTCAA

[0044] TGTGTTTGGGCTCCGGAGCATTTACTCTCTAATCCTGGGCCAAGATAACGCCGCCGACC

[0045] AGTCACGAATGATGCAGGAGCAGATGACAGGAGCAGCGATGGCCATGCCTGCAGACA

[0046] CCAACAAAGCTTTCAAGACAGAGTGGGAAGCTTTGGAACTGACAGATCACCAGTGGG

[0047] CGCTCGATGATGTGGAAGAAGAACTCATGGCCAGAGACCTCCACTTTGAAGGCATGTTCAAAAAGGAACTACAGACGTCCATATTCTAA.

[0048] The Emc3 protein mentioned in this article is the product of Emc3 gene expression, wherein the amino acids of Emc3 protein and Emc3 gene are interchangeable. When the Emc3 protein is an amino acid sequence that also includes 80%-99% homologous sequences, fusion proteins, conjugates, nucleic acids encoding its fusion proteins or conjugates, and vectors expressing the aforementioned nucleic acid molecules, its Emc3 gene is the nucleotide sequence corresponding to the amino acid sequence of the Emc3 protein that also includes 80%-99% homologous sequences, fusion proteins, conjugates, nucleic acids encoding its fusion proteins or conjugates, and vectors expressing the aforementioned nucleic acid molecules.

[0049] The CryAB mentioned in this article R120G Mutant proteins are abnormal protein deposits characterized by mutant proteins. The degree of protein deposition can be quantitatively assessed by green fluorescence, and the abundance of oligomers labeled with green fluorescent protein can be used to evaluate and quantify cellular protein aggregation.

[0050] The term "fusion protein" as used herein refers to a protein that contains amino acids other than those encoding the original or natural full-length protein or its subsequences, contains amino acid sequences that replace the amino acid sequences encoding the original or natural full-length protein or its subsequences, contains fewer amino acid sequences than those encoding the original or natural full-length protein or its subsequences, and / or contains amino acid sequences different from those encoding the original or natural full-length protein or its subsequences.

[0051] The terms “nucleic acid” and “nucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and polymers thereof. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or links, said nucleic acids being synthetic, naturally occurring, or non-natural, and said nucleic acids also comprising 80%-99% of their homologous sequence, preferably 80%-85%; more preferably 85%-90%; and most preferably 90%-95%.

[0052] The term "isolated" nucleic acid or polynucleotide as used herein is intended to refer to a nucleic acid molecule, DNA, or RNA removed from its native environment. For example, for the purposes of this invention, a recombinant polynucleotide encoding the Emc3 protein contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or purified (partial or substantial) polynucleotides in solution. Isolated polynucleotides or nucleic acids according to the invention also include said molecules synthesized. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0053] The term "expression vector" as used herein refers to a nucleic acid construct produced through recombination or synthesis using a set of designated nucleic acid elements that allow specific nucleic acids to be transcribed in a host cell. An expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, an expression vector comprises a nucleic acid to be transcribed, operatively linked to a promoter.

[0054] The term "amino acid" as used herein refers to naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those subsequently modified. In this document, amino acids may be represented by their known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their generally accepted single-letter symbols.

[0055] The terms "fusion protein," "protein conjugate," etc., refer to proteins that contain amino acids other than those encoding the original or natural full-length protein or its subsequences, contain amino acid sequences that replace those encoding the original or natural full-length protein or its subsequences, contain fewer amino acid sequences than those encoding the original or natural full-length protein or its subsequences, and / or contain amino acid sequences different from those encoding the original or natural full-length protein or its subsequences. More than one additional domain may be added to the Emc3 protein as described herein, such as an epitope tag or purification tag, or multiple epitope tags or purification tags. Additional domains may be linked, for example, to enhance additional activity, targeting function, or influence on physiological processes.

[0056] Unless otherwise stated, the terms “symptom” and “disease” are used interchangeably in this document.

[0057] Example

[0058] 1. Constructing a cell protein sedimentation model at the cellular level

[0059] Heart tissue from Sprague-Dawley pups, day 1 or 2 after birth, was digested overnight at 4°C with 0.05% trypsin. Other cells in the heart were removed via pre-treatment steps, and cardiomyocytes were purified. The isolated cardiomyocytes were cultured in DMEM (Gibco) containing 10% FBS and 1% penicillin / streptomycin and incubated at 37°C for 24 hours at 5% CO2 humidity. Cells were then cultured in DMEM containing 2% FBS and 1% penicillin / streptomycin (Gibco). The cardiomyocyte density on a 10 cm agar plate was 1.5 × 10⁻⁶ cells / cm². 6 The cardiomyocyte density on a 2-well glass slide (Thermo Fisher Scientific) is 1 × 10⁻⁶ cells / plate. 5 Cells / room. On the second day of cell culture, use cells coated with CryAB. R120G -GFP adenovirus transfection of cultured cells. CryAB R120G -GFP vector, which will express CryAB labeled with green fluorescent protein GFP. R120G Mutant proteins form protein oligomers that precipitate. The abundance of these oligomers, labeled with green fluorescent protein, can be used to assess and quantify cellular protein aggregation. Figure 1 (As shown in A).

[0060] Example 1: Protein sedimentation analysis of Emc3 knockdown at the cellular level

[0061] Primary cardiomyocyte culture was performed as described in section 1. On the first day of cell culture, cells were infected with adenovirus and lentivirus packaged with Emc3 shRNA to reduce Emc3 RNA levels in the cells. On the second day of cell culture, cells were infected with CryAB-coated... R120G-GFP adenovirus transfection of cultured cells resulted in abnormal protein accumulation in the cells. The protein accumulation in the cells was examined on day 7.

[0062] The results showed that Emc3 shRNA significantly reduced Emc3 expression levels in cells (approximately 70% reduction). Cells with knocked-down Emc3 showed a significant increase in green fluorescent protein abundance, with a maximum increase of approximately 70% in protein deposition area. Figure 1 The results showed that inhibiting Emc3 significantly increased the protein deposition phenotype in cells, which can serve as a more severe model of protein deposition disease (increasing the severity by about 70% compared to the conventional model). This model can be used for the study of the mechanisms of severe protein deposition-related diseases and drug development. Emc3 knockdown can be used as a model for the development of therapies aimed at restoring or increasing Emc3 levels, and Emc3 itself can serve as a target for drug development.

[0063] Example 2: Effect of Emc3 overexpression at the cellular level on protein sedimentation in cells

[0064] Emc3 overexpression system: Using Agilent Technologies' adenovirus packaging kit, mouse Emc3 cDNA with or without the cMyc tag was cloned into multiple cloning sites of the pShuttle-cytomegalovirus vector to generate adenoviruses (Ad-Emc3 and Ad-cMyc-Emc3). Empty β-gal reporter adenovirus (Ad-βgal) was used as a control. Upon adenovirus infection, cardiomyocytes were cultured in serum-free DMEM containing an appropriate amount of virus at 37°C for 4 hours. Cells were then cultured in DMEM containing 2% FBS and 1% penicillin / streptomycin (Gibco). Primary cardiomyocyte culture was performed as described in Example 1, using CryAB-coated cells on day 1 of cell culture. R120G -GFP adenovirus transfection of cultured cells caused the abnormal protein to accumulate. On day three, cells were infected with adenovirus containing the Emc3 cDNA sequence to achieve Emc3 overexpression. Cells were collected on day six for protein sedimentation and quantification.

[0065] The results showed that overexpression of Emc3 significantly reduced protein deposition in cells, and this negative correlation was dose-dependent. Figure 1 -D) and time ( Figure 1 -EF) dependence, showing good preventive and therapeutic effects. First, with the increase of Emc3 overexpression, the abnormal protein clearance effect also reaches its optimal level. Figure 1The fluorescence intensity in D is positively correlated with protein precipitation. It can be seen that the higher the Emc3 viral infection level and the higher the Emc3 content, the lower the fluorescence intensity, indicating less abnormal protein precipitation. In the optimal case, it can even eliminate 99% of abnormal protein deposits in the cell. Figure 1 D). Secondly, Emc3 overexpression also showed a good therapeutic effect on abnormal protein sedimentation: Emc3 expression was increased on the third day after protein sedimentation (Ad-Emc3), and the protein sedimentation content was detected for three consecutive days thereafter.

[0066] The results showed that, over time, the deposition of abnormal proteins in the control group cells almost doubled every day, while the increase in the Emc3 overexpression group was significantly slower. Compared to the control group, significantly fewer mutant proteins, CryAB, were observed in the Emc3 overexpression group by the second day. R120G The accumulation of aggregates, and the gradual increase in the difference between the two over time, indicate a significant therapeutic effect. Figure 1 EF).

[0067] Example 3: Decreased expression of Emc3 protein in cardiomyocytes can lead to reduced cardiac function, ventricular structural remodeling, and death in mice.

[0068] To specifically knock out the Emc3 gene in cardiomyocytes (CMs), this experiment used Emc3... flox / flox (Emc3 knockout tool) mice were crossed with αMHC-Cre transgenic mice (cardiomyocyte-specific knockout system) to obtain αMHC-Cre,Emc3 flox / flox Mice (hereinafter referred to as CM-Emc3) - / - Emc3 in cardiac cardiomyocytes was specifically knocked out.

[0069] The results showed that no CM-Emc3 was observed when genotyping was performed 5 days after birth. - / - The pups (in which the Emc3 gene was knocked out) suggest that knockout of the Emc3 gene in cardiomyocytes may lead to mouse death. Further research revealed that CM-Emc3... - / - The pups died within 24 hours of birth. Immunohistochemistry was used to identify CM-Emc3 at E18.5 days. - / - The absence of Emc3 protein in mouse hearts ( Figure 2 AE). CM-Emc3 + / - (Emc3 is knocked out on one strand of the two chromosomes) The Emc3 mRNA level in the heart of heterozygous mice is about 50% of the normal level. Although these mice have normal heart function at 3 months of age, at 6 months of age, CM-Emc3... + / -The mice exhibited decreased cardiac contractile function, accompanied by left ventricular dilation. Figure 2 (FG). By approximately 44 weeks, the mortality rate of mice reached 100%. This indicates that insufficient Emc3 dosage in cardiomyocytes severely affects mouse survival; mice with complete Emc3 knockout died within 24 hours of birth, and mice with half Emc3 knockout (CM-Emc3)... + / - The heterozygous baby also died at 7 months of age. Figure 2 H).

[0070] Example 4: Expression of Emc3 in cardiomyocytes can treat diseases caused by cardiac protein deposition.

[0071] 4.1 Establishment of an animal model of cardiac protein sedimentation (cardiomyocyte-specific overexpression of Emc3)

[0072] Animal experiments were conducted using the FVB / N mouse strain, with mice initially introduced to carry CryAB. R120G Mice carrying CyrAB (their CyrAB gene has a mutation at amino acid 120 where amino acid R is changed to G, leading to abnormal precipitation of CyrAB protein and formation of oligomers). R120G Significant protein oligomer precipitation was observed in the mutant mouse cardiomyocytes, leading to the development of restrictive, hypertrophic, or dilated cardiomyopathy. Secondly, the αMHC-Cre and αMHC-MerCreMer splicing systems are used to coordinate genetic manipulation of the target gene Emc3. The Cre-loxP strategy was used to express Emc3, CAG-CAT... cMyc-Emc3 Tg mice were bred and maintained in an FVB / N background. Finally, crossbreeding was used to obtain mice that all carried CryAB as a background component. R120G The experimental group consisted of Emc3 / αMCM / CryAB mice. R120G :CAG-CAT cMyc-Emc3 / αMHC-MerCreMer / CryAB R120G (Emc3 is overexpressed in the cardiac cardiomyocytes of this mouse). The control group consisted of a blank control group (Ntg: non-transgenic) without genetic manipulation and mice that overexpressed Emc3 but did not have abnormal protein deposition (Emc3 / αMCM: CAG-CAT). cMyc-Emc3 / αMHC-MerCreMer), mice carrying an Emc3 overexpression system but unable to activate it (Emc3 / CryAB) R120G :CAG-CAT cMyc-Emc3 / CryAB R120 CAG-CAT cMyc-Emc3 / CryAB R120G (Emc3 levels in the mouse heart were not affected by the genetic manipulation itself).

[0073] 4.2 Expression of Emc3 in cardiomyocytes can significantly reduce the accumulation of protein oligomer deposits in the heart.

[0074] To determine whether increased Emc3 expression directly affects the accumulation of protein oligomers in mice, CAG-CAT was first... cMyc-Emc3 / αMHC-MerCreMer (abbreviated as Emc3 / αMCM) mice and CryAB R120G Transgenic mice were hybridized to produce the composite transgenic mouse CAG-CAT. cMyc-Emc3 / αMCM / CryAB R120G (abbreviated as Emc3 / αMCM / CryAB) R120G Mice were fed tamoxifen diets starting at 6 weeks of age and continued for 3 weeks to induce specific expression of Emc3 in cardiomyocytes. Subsequently, this experiment examined the Emc3 / αMCM / CryAB ratio at 6 months of age. R120G CryAB in mouse heart R120G The size of protein oligomer precipitates.

[0075] The results showed that, compared with the control group, Emc3 / αMCM / CryAB R120G The protein oligomer deposits in mouse hearts are smaller in size. Figure 3 AB).

[0076] 4.3 Expression of Emc3 in cardiomyocytes can significantly improve the symptoms of myocardial hypertrophy in mice.

[0077] CryAB R120G Protein oligomer deposition can lead to myocardial hypertrophy in mice. To investigate whether Emc3 expression can alleviate this pathological process, this experiment conducted the following study. At 6 months of age, CryAB... R120G Transgenic mice showed an increased heart weight to body weight ratio and developed cardiac hypertrophy; in contrast, Emc3-overexpressing mice had a significantly lower heart weight to body weight ratio. Figure 3 C).

[0078] This experiment also examined the size of cardiomyocytes in mice at 6 months of age. Mouse hearts anesthetized with isoflurane and perfused with 10% formalin were then fixed overnight in 10% formalin at room temperature. A series of graded alcohol dehydration processes were followed, and the tissues were embedded in paraffin blocks for histological processing. Tissue sections (5 μm) were used for immunohistochemistry or immunofluorescence staining. For immunohistochemistry, bright-field images were acquired using a Zeiss Axio Imager A2 microscope equipped with AxioVision software. Immunofluorescence staining was performed using an inverted Nikon A1R confocal microscope equipped with NIS Elements AR 4.13 software. To determine aggregate size, 10 fields of view (20x, 1.5x zoom) were taken for each heart. Aggregate area (anti-CryAB staining, green) and CM area (anti-α-actin staining, red) were determined using NIS Elements AR analysis software (Nikon Instruments), and the average aggregate size was determined proportionally. CM size was determined using wheat germ agglutinin staining. The NIS-Elements software (Nikon) was used to image 10 regions of each heart at 40x magnification, and more than 500 cells were quantified. All samples were blind-identified prior to quantification.

[0079] Data shows Emc3 / αMCM / CryAB R120G Cardiac cells were significantly smaller than those expressing CryAB. R120G Cardiomyocytes, indicating that overexpression of Emc3 can improve CryAB R120G Caused myocardial hypertrophy ( Figure 3 D).

[0080] 4.3 Expression of Emc3 in cardiomyocytes can significantly improve cardiac dysfunction in mice.

[0081] CryAB R120G Protein oligomer deposition can lead to myocardial hypertrophy in mice. To investigate whether Emc3 expression can alleviate this pathological process, the following study was conducted. Mice were anesthetized by inhalation of isoflurane and subjected to two-dimensional guided M-mode echocardiography. Left ventricular function was measured using a VisualSonics Vevo 2100 imaging system and a 40-MHz transducer. The main measurements included, but were not limited to, fractional shortening (FS) and ejection fraction (EF).

[0082] By measuring cardiac function in mice at 6 months of age, the results showed that CryAB... R120G (CryAB R120G and αMCM / CryAB R120G This caused a significant decrease in mouse function, manifested by FS levels being much lower than in blank control mice. Figure 3E). However, overexpression of Emc3 protein improved the symptoms, manifested as Emc3 / αMCM / CryAB. R120G The mice exhibited significantly better cardiac function than the control group, and even showed no difference compared to the blank control group, indicating that Emc3 overexpression completely treated CryAB. R120G Decreased cardiac function ( Figure 3 CE).

[0083] 4.4 Expression of Emc3 in cardiomyocytes significantly increases mouse survival time.

[0084] CryAB R120G Protein oligomer deposition can lead to death in mice. To investigate whether Emc3 expression can alleviate this pathological process, this experiment collected data on mouse mortality. Figure 3 F). All CryAB R120G The mice died 7 months ago (median survival 142 days), but approximately 80% of the Emc3 / αMCM / CryAB mice survived. R120G Mice were still alive at 7 months, and the survival rate exceeded 40% at 9 months (median survival of 223 days). These results indicate that overexpression of Emc3 in cardiomyocytes can improve CryAB. R120G The mice died as a result.

[0085] Therefore, increasing the expression level of Emc3 protein in cardiomyocytes can reduce the accumulation of protein oligomers, alleviate pathological myocardial hypertrophy, protect cardiac function, delay the onset of heart failure, and thus prolong the survival time of mice.

Claims

1. A medicament for treating or improving protein deposition-related diseases or symptoms, said medicament containing Emc3 protein, the amino acid sequence of said Emc3 protein being shown in SEQ ID No:

1.

2. The medicament for treating or improving protein deposition-related diseases or symptoms as described in claim 1, characterized in that, The amino acid sequence of the Emc3 protein also includes 80%-99% homologous sequences.

3. The medicament for treating or improving protein deposition-related diseases or symptoms as described in claim 1, characterized in that, The Emc3 protein also includes its fusion protein, conjugate, nucleic acid encoding its fusion protein or conjugate, and vector expressing the aforementioned nucleic acid molecules.

4. The medicament for treating or improving protein deposition-related diseases or symptoms as described in claim 1, characterized in that, The drug may also contain a pharmaceutically acceptable carrier.

5. The medicament for treating or improving protein deposition-related diseases or symptoms as described in claim 1, characterized in that, The drug can be administered via injection or oral administration.

6. The medicament for treating or improving protein deposition-related diseases or symptoms as described in claim 1, characterized in that, The drug dosage forms include gels, suspensions, emulsions, polymers, nanoparticles, microspheres, rectal capsules, enemas, oral solutions, and implants, which can optionally be controlled-released and / or sustained-released via dosage forms or devices.

7. The medicament for treating or improving protein deposition-related diseases or symptoms as described in claim 1, characterized in that, The protein deposition-related diseases or symptoms are preferably protein deposition-related diseases of the heart, including: rheumatic heart disease, endocarditis, myocarditis, restrictive heart disease, hypertrophic heart disease, dilated cardiomyopathy, and other heart diseases with abnormal protein deposition rates.

8. The use of an Emc3 protein in the preparation of a medicament for treating or improving protein deposition-related diseases or symptoms, said Emc3 protein as described in claim 1.

9. The use of an Emc3 protein or a nucleotide sequence of the Emc3 protein in screening drugs for treating protein deposition-related diseases, wherein the Emc3 protein is as described in claim 1; the nucleotide sequence of the Emc3 gene is shown in SEQ ID No:2; wherein, When Emc3 protein or Emc3 gene nucleotides are highly expressed, the occurrence and progression of protein deposition-related diseases are suppressed.

10. A kit for screening drugs to treat protein deposition-related diseases, said kit containing reagents for detecting Emc3 protein or Emc3 protein nucleotide expression, wherein, When the Emc3 protein or the nucleotides of the Emc3 gene are highly expressed, the occurrence and progression of protein deposition-related diseases are inhibited, meaning that the drug to be screened can be used to treat protein deposition-related diseases; the Emc3 protein is as described in claim 1; the nucleotide sequence of the Emc3 gene is shown in SEQ ID No:2.

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