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WO2025116539A1 - Utilisation de peptides de type grp78 - Google Patents

Utilisation de peptides de type grp78 Download PDF

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Publication number
WO2025116539A1
WO2025116539A1 PCT/KR2024/019031 KR2024019031W WO2025116539A1 WO 2025116539 A1 WO2025116539 A1 WO 2025116539A1 KR 2024019031 W KR2024019031 W KR 2024019031W WO 2025116539 A1 WO2025116539 A1 WO 2025116539A1
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Prior art keywords
peptide
grp78
muscle
disease
amino acid
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Korean (ko)
Inventor
정종평
박윤정
이주연
이동우
조범수
박윤신
최다현
이경은
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SNU R&DB Foundation
Nibec Co Ltd
Chungbuk National Univiversity CBNU
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Seoul National University R&DB Foundation
Nibec Co Ltd
Chungbuk National Univiversity CBNU
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Publication of WO2025116539A1 publication Critical patent/WO2025116539A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the use of GRP78 and analogous peptides thereof or nucleic acids encoding same, and more particularly, to a composition comprising GRP78 and analogous peptides thereof or nucleic acids encoding same for inhibiting or reversing aging of cells, tissues or organs, or restoring or regenerating the function of aged or damaged cells, tissues or organs, a pharmaceutical composition for preventing or treating muscle diseases, and a cosmetic composition for preventing or reversing aging.
  • Aging refers to the gradual deterioration of the body's structure and function as it ages. First, differentiation and proliferation at the cellular level decrease, causing changes in the structure of specific molecules and changes in response pathways. Then, unavoidable changes occur in anatomy and physiology beyond the cellular and molecular levels. Homeostasis of organs and organ systems deteriorates, and this leads to a rapid increase in susceptibility to external stress, disease, and death (Cynthia J Kenyon, Nature. 2010 Mar 25;464(7288):504-12).
  • Telomeres are DNA terminal regions located at the end of chromosomes, which are composed of DNA and proteins, and play a role in protecting the ends of chromosomes.
  • telomeres When a parent cell divides to produce daughter cells, there is a limit to how much DNA molecule ends can be completely replicated, and as a result, each time a cell divides, the telomeres gradually lose their ends. This is an aging-specific phenomenon, and unless there is another mechanism to prevent telomere wear, telomeres will continue to shorten each time a cell divides.
  • telomeres When telomeres become shorter than a certain length, cells reach senescence and no longer divide, resulting in cell death, which accelerates human aging (Saliha Rizvi, et al., Curr Aging Sci. 2014;7(3):161-7).
  • deficiency of telomerase which is involved in the formation of telomeres, can cause various diseases, such as pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia, and these diseases are caused by the lack of the ability of specific tissues to regenerate (Joao Pedro de Magalhaes, Joao F Passos, Mech Ageing Dev. 2018 Mar:170:2-9).
  • telomeres are protected by a protein complex called ‘shelterin’ that binds to the ends of DNA. If there is a problem with this shelterin protein, even if the length of the telomeres does not shorten, the aging and survival of cells can be affected due to an abnormality in the protective structure. In some cases, a problem with the shelterin protein can damage the protective structure of telomeres and cause chromosomes to fuse incorrectly. In such cases, the tissue’s ability to recover can be reduced, aging can be accelerated, and cell survival can be affected.
  • mice manipulated to have shorter or longer telomere lengths had shorter or longer life spans, respectively (Bruno Bernardes de Jesus, et al., EMBO Mol Med. 2012 Aug;4(8):691-704).
  • Telomere length related factor-1 encodes a telomere-specific protein that regulates telomere length and acts as a telomerase inhibitor, and its expression is reduced in senescent cells (Keiji Okamoto, et al., J Biol Chem. 2008 Aug 29;283(35):23981-8).
  • Telomere length related factor-2 exists in telomeres, forms the shelterin nucleoprotein complex, and plays a role in regulating telomere length, and its expression is reduced in senescent cells (Giacomo Buscemi, et al., Curr Biol. 2009 May 26;19(10):874-9).
  • POT1 Telomere Protection Of Telomeres 1 (POT1) encodes a nuclear protein involved in telomere maintenance, binds to TTAGGG repeats to regulate telomere length, and its expression is reduced in senescent cells (Tomas Aramburu, et al., Comput Struct Biotechnol J. 2020 Jul 3:18:1939-1946).
  • Human telomerase reverse transcriptase (hTERT) is a human telomerase reverse transcriptase and a component of the telomerase complex, and its expression is reduced in senescent cells (Anastasia I Palamarchuk, et al., Biomedicines. 2023 Apr 4;11(4):1091).
  • Glucose-regulated protein 78 plays a role in maintaining protein folding and status when newly synthesized proteins are translocated to the endoplasmic reticulum. In young cells, it is abundant on the cell surface, in aged cells, it is abundantly distributed in the nucleus, and in aged cells, its expression decreases (Da Hyeon Choi, et al., Biomaterials. 2021 Nov:278:121156).
  • the inventors of the present invention have conducted extensive research efforts to discover a novel substance capable of reversing aging cells into young cells, and as a result, have discovered a peptide that has the effect of increasing biomarkers whose expression is reduced in aging cells.
  • GRP78-like peptides when GRP78-like peptides are expressed in aging cells, it was confirmed that the expression pattern of aging-related biomarkers changed to that in young cells.
  • GRP78-like peptides were administered to aged mice, it was confirmed that the expression pattern of aging-related biomarkers in aged tissue changed to that in young tissue, and at the same time, the function of the tissue was restored, thereby completing the present invention.
  • the purpose of the present invention is to provide a peptide and a use thereof that can inhibit or reverse aging and restore or regenerate the function of cells, tissues or organs associated with aging by increasing biomarkers whose expression is reduced in aging cells.
  • the present invention provides a composition for inhibiting or reversing aging of a cell, tissue or organ, or restoring or regenerating the function of an aged or damaged cell, tissue or organ, comprising GRP78, a similar peptide thereof or a nucleic acid encoding the same as an effective ingredient.
  • Figure 1 shows the plasmid map of the GRP78 expression vector and the control vector.
  • Figure 2 shows the expression level of GRP78 mRNA after injecting the GRP78 expression vector into different cells.
  • NT indicates the untreated experimental group
  • Mock indicates the experimental group into which only the control vector was introduced
  • GRP78 indicates the experimental group into which the GRP78 expression vector was introduced.
  • Figure 3 shows the results of confocal microscopy after transfection of cells (young and aged cells) with a vector carrying GRP78-EGFP.
  • NT is the untreated experimental group
  • Mock is the experimental group introduced only with the control vector
  • GRP78 is the experimental group introduced with the GRP78-EGFP expression vector (blue: nucleus, red: F-actin, green: GRP78-EGFP).
  • Figure 4 shows the results of measuring the cluster ratio of cells expressing GRP78-EGFP using FACS after injecting the GRP78-EGFP expression vector into young and aged cells.
  • Figure 5 is a graph showing the change in the mRNA expression level of TERT, a factor involved in telomere length, when GRP78 was expressed in DPSC and PDLSC, which are dental stem cells.
  • NT indicates the untreated experimental group
  • pcDNA indicates the experimental group into which only the control vector was introduced
  • GRP78 indicates the experimental group into which the GRP expression vector was introduced (*p ⁇ 0.05, **p ⁇ 0.01).
  • Figure 6 illustrates the overall process of measuring the histological changes in muscles when the GRP78 expression vector was introduced into the muscles of mice of each age group.
  • Figure 7 is a confocal micrograph showing the expression of GRP78, confirmed by fluorescence, when the GRP78-EGFP expression vector was injected into the muscle of a mouse.
  • Figure 8 is a graph showing the results of confirming the mRNA expression level of GRP78 when the GRP78 expression vector was introduced into mice of each age group (****p ⁇ 0.0001).
  • Figure 9 shows the results of confirming the level of GRP78 protein expression in tissues using Western blotting in mice of each age group.
  • Figure 10 shows the results comparing the changes in muscle bundle thickness when GRP78 was expressed in mice of each age group.
  • Figure 11 shows the results of confirming the changes in cell characteristics according to aging of periodontal ligament tissue-derived cells of young rats (11 weeks old, control group) and aged rats (96 weeks old, aged group).
  • Figure 11A shows the results of measuring the cell length and width of the control group and the aged group (**p ⁇ 0.01).
  • Figure 11B shows the results of measuring the absorbance using the SA- ⁇ gal staining method for the expression of beta galactosidase, which is highly expressed in aged cells (***p ⁇ 0.001).
  • FIG 11C shows the results of measuring the gene expression levels of telomere length related factor-1 (TRF-1), telomere length related factor-2 (TRF-2), protection of telomeres 1 (POT1), and glucose-regulated protein 78 (GRP78), which are aging-related biomarkers, by RT-PCR (**p ⁇ 0.01, ***p ⁇ 0.001).
  • Figure 11D shows the results of measuring cell proliferation (***p ⁇ 0.001).
  • Figure 12 shows the results of observing the cell permeability of peptides of sequence numbers 1 to 4 using a confocal microscope.
  • Figure 12A shows the cell permeability result of young rPDL
  • Figure 12B shows the cell permeability result of old rPDL.
  • Sequence 1 is a group treated with the peptide of sequence number 1
  • Sequence 2 is a group treated with the peptide of sequence number 2
  • Sequence 3 is a group treated with the peptide of sequence number 3
  • Sequence 4 is a group treated with the peptide of sequence number 4.
  • Figure 13 shows the results of measuring changes in the expression of aging-related biomarkers by peptides of sequence numbers 1 to 4.
  • Figure 13A shows the results of measuring changes in the expression of aging-related biomarkers TRF-1, TRF-2, and human telomerase reverse transcriptase (hTERT) by peptides of sequence numbers 1 to 4 in TMSC human tonsil tissue-derived stem cells (passage 20) aged by continuous culture, by RT-PCR (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figure 13B shows the results of measuring the expression changes of aging-related biomarker genes TRF-1, TRF-2, POT1, and GRP78 by peptides of sequence numbers 1 to 4 in periodontal ligament tissue-derived cells extracted from young rats (12 weeks old) and aged rats (96 weeks old), by RT-PCR ($p ⁇ 0.001, compared to Young rPDL_control and Old rPDL_control, p ⁇ 0.001, *** p ⁇ 0.001, ** p ⁇ 0.01, *p ⁇ 0.05, compared to the control when peptide treated).
  • Seq 1 is a group treated with the peptide of sequence number 1
  • Seq 2 is a group treated with the peptide of sequence number 2
  • Seq 3 is a group treated with the peptide of sequence number 3
  • Seq 4 is a group treated with the peptide of sequence number 4.
  • Figure 14 shows the results of measuring the time that 8-week-old, 48-week-old, and 72-week-old mice maintain without falling on a rotating rod.
  • NT is an untreated experimental group
  • sequence 3 is a group treated with the peptide of sequence number 3
  • sequence 4 is a group treated with the peptide of sequence number 4.
  • Figure 15 is a peptide administration schedule for mice at each age. Seq 19 represents the muscle regeneration peptide of sequence number 19. The above description applies equally to subsequent drawings.
  • Figure 16 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissue administered peptides of sequence number 3 or sequence number 4 for 4 weeks.
  • Figure 17 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissues that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered with the peptide of sequence number 3 or sequence number 4 alone or in combination with the muscle regeneration peptide (SEQ ID NO: 19) for 4 weeks (*p ⁇ 0.05, **p ⁇ 0.01).
  • Figure 18 shows the results of measuring fluorescence intensity after confocal microscopy observation of the expression of MyoD1 in muscle tissue of mice administered peptides of sequence number 3 and sequence number 4 for 4 weeks (**p ⁇ 0.01, ****p ⁇ 0.0001).
  • Figure 19 shows the results of measuring fluorescence intensity after confocal microscopy observation of the expression of MYH in muscle tissue of mice administered peptides of sequence number 3 and sequence number 4 for 4 weeks (***p ⁇ 0.001, ****p ⁇ 0.0001).
  • Figure 20 shows the results of measuring fluorescence intensity after confocal microscopy observation of the expression of MyoD1 in the muscles of mice that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered the peptide of sequence number 3 or sequence number 4 alone or in combination with the muscle regeneration peptide (SEQ ID NO: 19) for 4 weeks (*p ⁇ 0.05, ***p ⁇ 0.001, ****p ⁇ 0.0001).
  • Figure 21 shows the results of measuring fluorescence intensity after confocal microscopy observation of the expression of MYH in the muscles of mice that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered the peptide of sequence number 3 or sequence number 4 alone or in combination with the muscle regeneration peptide (SEQ ID NO: 19) for 4 weeks (****p ⁇ 0.0001).
  • Figure 22a is a graph showing changes in human telomerase gene expression when young and aged TMSC cells were treated with a GRP78-like peptide.
  • Figure 22b shows the activity of telomerase compared in young and aged cells using the TRAP assay, which can visually compare the length of telomerase, an enzyme that elongates telomeres (***p ⁇ 0.001).
  • Figure 22c shows the length of telomeres, a representative indicator of aging, measured by the TRF assay, which measures the length of TRF (telomere restriction fragment) in young and aged cells (*p ⁇ 0.05).
  • Figure 22d shows the results of examining the length of visible telomeres using Southern blotting in young and aged cells.
  • Figure 23a shows the results of changes in mitochondrial protein expression by treating young and aged TMSC cells with a GRP78-like peptide labeled with a fluorescent material (Rhodaime B).
  • Figure 23b is a graph showing the results of quantitative analysis of the protein expression results shown in Figure 12A using the Image J program.
  • Figure 23c is a graph showing the results of measuring changes in the copy number of mtDNA using qRT-PCR to confirm quantitative changes in mitochondrial DNA (mtDNA).
  • Figure 23d shows the results of changes in protein expression of factors related to mitochondrial activity and biogenesis in young and aged cells (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figure 23e shows the results of changes in gene and protein expression caused by GRP78-like peptides of PPAR ⁇ , PGC1 ⁇ , and p53, which are factors associated with mitochondria and telomeres (**p ⁇ 0.01, ****p ⁇ 0.0001).
  • aging refers to a decrease in the differentiation or proliferation ability of cells due to an increase in the age of an individual, and changes in the functions and conditions of the body that progress with the passage of time. Since the onset of aging varies from individual to individual, aging typically refers to a decline in the normal functions of cells, tissues, or organs over time. Aging progresses independently of disease. Aging increases the incidence of disease, while it can further accelerate the decline in the function of damaged cells, tissues, or organs in individuals with disease.
  • aging can mean a decrease in a cell’s ability to differentiate or proliferate due to an increase in the number of passages.
  • the term “passage” refers to a method of continuously culturing cells in a healthy state for a long period of time by periodically transferring a portion of cells to a new culture vessel and then changing the culture medium to continue culturing the cells for a long period of time, wherein the culture vessel is replaced or a cell group is divided and cultured.
  • One culture vessel replacement or cell group division and culture is referred to as one passage.
  • the expression of aging-related biomarkers TRF-1, TRF-2, POT1, hTERT, and GRP78 genes was decreased in aged periodontal ligament (PDL) cells and tonsil-derived mesenchymal stem cells (TMSC), and it was confirmed that when the aged cells were treated with the GRP78-like peptide according to the present invention, the expression of the biomarker genes whose expression had been decreased was increased again.
  • PDL periodontal ligament
  • TMSC tonsil-derived mesenchymal stem cells
  • the present invention relates to a composition for inhibiting or reversing aging of a cell, tissue or organ, or restoring or regenerating the function of an aged or damaged cell, tissue or organ, comprising GRP78, a similar peptide thereof or a nucleic acid encoding the same as an active ingredient.
  • GRP78 which stands for 78 kDa glucose-regulated protein, is an ER chaperone protein, also known as BiP or HSPA5. GRP78 is known to play a role not only in protein folding but also as an important factor in regulating the unfolded protein response (UPR). Under ER homeostasis conditions, GRP78 normally binds to three UPR transmembrane sensors, ATF6, PERK, and IRE1, and keeps them in an inactive state. When ER stress conditions occur, in which unfolded proteins accumulate in the lumen of the ER, GRP78 releases from the UPR sensors and contributes to their activation. Activated UPR alleviates ER stress by decreasing protein translation and increasing the folding capacity of the ER, and GRP78 is upregulated in this process. If ER homeostasis is not restored, UPR can induce apoptosis.
  • UPR unfolded protein response
  • GRP78 acts on a wide range of protein folding processes through two domains: the nucleotide binding domain (NBD), which binds and hydrolyzes ATP, and the substrate binding domain (SBD), which binds substrate proteins.
  • NBD nucleotide binding domain
  • SBD substrate binding domain
  • the NBD consists of two subdomains (I and II), which are each divided into two smaller subdomains (A and B). These subdomains are separated by a cleft that binds a nucleotide, a Mg2+ ion, and two K+ ions.
  • the SBD is also divided into two subdomains (alpha and beta).
  • the SBD alpha subdomain provides a pocket for substrate protein binding
  • the SBD beta subdomain acts as a lid that covers the binding pocket.
  • the activity of GRP78 is regulated by an allosteric ATPase cycle.
  • ATP binds to the NBD
  • the lid SBD beta subdomain
  • ADP now binds to the NBD
  • the lid closes on the bound substrate, which increases the binding affinity for the substrate protein and consequently prevents the bound substrate protein from folding insufficiently.
  • ADP is replaced by ATP
  • the lid opens again, releasing the substrate protein.
  • This ATPase cycle is tightly regulated by nucleotide binding and substrate protein binding. Binding of the substrate protein promotes ATP hydrolysis, whereas binding of nucleotides affects either binding or release of the substrate protein.
  • the amino acid sequence of GRP78 and the base sequence encoding it can be obtained from a known database such as NCBI's GenBank (e.g., GenBank Accession AAA52614.1).
  • the GRP78-like peptide according to the present invention may be a peptide including a part of the GRP78 amino acid sequence represented by SEQ ID NO: 21, and including amino acid residues at which a phosphorylation or oxidation reaction of GRP78 may occur.
  • the amino acid residue may preferably be selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine (C), methionine (M), tryptophan (W), glutamic acid (D), and proline (P), and the GRP78-like peptide according to the present invention may be characterized by including one or more, two or more, preferably three or more, of the above amino acids. It is preferable that the peptide have a length of 40-mer or less, 35-mer or less, 30-mer or less, or 25-mer or less.
  • the GRP78-like peptide according to the present invention may be a peptide comprising an amino acid sequence identical or similar to the NBD represented by SEQ ID NO: 22 or a portion thereof.
  • the portion of the NBD may be, for example, the IA, IB, IIA or IIB subdomain or a portion thereof, but is not necessarily limited thereto.
  • the similar amino acid sequence refers to an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with a reference amino acid sequence.
  • the GRP78-like peptide according to the present invention may be a peptide comprising an amino acid sequence identical or similar to the SBD represented by SEQ ID NO: 23 or a portion thereof.
  • the portion of the SBD may be, for example, an alpha or beta subdomain or a portion thereof, but is not necessarily limited thereto.
  • the similar amino acid sequence refers to an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with a reference amino acid sequence.
  • the GRP78-like peptide according to the present invention may be characterized by including an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 18 or a part thereof, but is not limited thereto.
  • Peptides of sequence numbers 1, 8, and 9 are peptides having amino acid sequences predicted to be susceptible to phosphorylation or oxidation among the entire sequence of the GRP78 protein based on protein structure and sequence analysis results.
  • the peptides of SEQ ID NOS: 2 to 4 and 10 to 14 are peptides having amino acid sequences corresponding to a part of the NBD of GRP78.
  • the NBD is divided into sequences of IA, IB, IIA, and IIB, and the peptides of SEQ ID NOS: 2 and 3 include the amino acid sequence of IA, the peptides of SEQ ID NOS: 10 and 11 include the amino acid sequence of IB, the peptides of SEQ ID NOS: 12 and 13 include the amino acid sequence of IIA, and the peptides of SEQ ID NOS: 4 and 14 include the amino acid sequence of IIB.
  • the above peptides recognize and bind to ATP, a substrate of GRP78.
  • the peptides of SEQ ID NOS: 15 to 18 are peptides having an amino acid sequence corresponding to a part of the SBD of GRP78.
  • the SBD is divided into the SBD alpha domain and the SBD beta domain, and the peptides of SEQ ID NOS: 15 and 16 include the amino acid sequence of the SBD alpha domain, and the peptides of SEQ ID NOS: 17 and 18 include the amino acid sequence of the SBD beta domain.
  • the above peptides bind to peptides that bind to other substrates, which are decomposition products generated when ATP regulated by GRP78 is decomposed.
  • the GRP78-like peptide according to the present invention may be characterized by comprising an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 18, and more preferably, the GRP78-like peptide according to the present invention may be characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 18.
  • the GRP78-like peptide according to the present invention may be characterized by comprising an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and more preferably, the GRP78-like peptide according to the present invention may be characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
  • the GRP78-like peptide according to the present invention is interpreted to mean a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18, but also including variants or fragments thereof in which amino acid residues are conservatively substituted at specific amino acid residue positions.
  • conservative substitution means a modification of a peptide including substituting one or more amino acids with amino acids having similar biochemical properties that do not cause loss of the biological or biochemical function of the peptide.
  • a “conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain.
  • Classes of amino acid residues having similar side chains are well known and defined in the art. These classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine,
  • GRP78 or a similar peptide of the present invention may still retain activity even if it has conservative amino acid substitutions.
  • the GRP78-like peptide of the present invention may act as a substance that mimics the effect of GRP78 protein by regulating unique biochemical interactions of GRP78 protein (e.g., interactions for regulating homeostasis of ATP and calcium, binding to substrate proteins for regulating protein unfolding, etc.) by including amino acid residues at which phosphorylation or oxidation reaction of GRP78 is likely to occur, including amino acid residues included in the NBD of GRP78, or including amino acid residues included in the SBD of GRP78.
  • unique biochemical interactions of GRP78 protein e.g., interactions for regulating homeostasis of ATP and calcium, binding to substrate proteins for regulating protein unfolding, etc.
  • the effective ingredient according to the present invention may preferably be characterized by increasing the expression of GRP78 when delivered to a cell.
  • increasing the expression of GRP78 includes directly or indirectly increasing the expression of GRP78.
  • Such a substance may be a peptide comprising a NBD represented by SEQ ID NO: 22 or a part thereof, an SBD represented by SEQ ID NO: 23 or a part thereof, or a part of the GRP78 amino acid sequence represented by SEQ ID NO: 21, as described above, and comprising three or more amino acids selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine (C), methionine (M), tryptophan (W), glutamic acid (D), and proline (P).
  • the above peptide has a length of 40-mer or less, 35-mer or less, 30-mer or less, or 25-mer or less.
  • the nucleic acid may be DNA, mRNA, plasmid DNA, etc., and may be delivered into cells by a cell-penetrating functional nanocarrier.
  • the nucleic acid may be used together with various carriers such as lipid nanoparticles (LNPs) and liposomes, which are known to effectively deliver oligonucleotides into cells, but are not limited thereto.
  • LNPs lipid nanoparticles
  • liposomes which are known to effectively deliver oligonucleotides into cells, but are not limited thereto.
  • GRP78 according to the present invention, a peptide analog thereof or a nucleic acid encoding the same may be useful for inhibiting or reversing aging of a cell, tissue or organ, or for restoring or regenerating the function of an aged or damaged cell, tissue or organ.
  • the cell, tissue or organ may be a cell, tissue or organ derived from a patient suffering from a disease or condition selected from the group consisting of muscle disease, bone disease, joint disease, skin disease, metabolic disease, urinary system disease, neurological disorder, cardiovascular disease, pulmonary dysfunction, cancer, immune disease, dental disease, periodontal disease and oral tissue dysfunction.
  • the patient may be an adult patient, and may be characterized as being, for example, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older or 70 years of age or older.
  • Muscle diseases for which the GRP78 or a similar peptide thereof according to the present invention may be useful include, but are not limited to, sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, atony, amyotrophic lateral sclerosis, cachexia, masticatory muscle disorder, temporomandibular disorder, or inflammatory myopathy.
  • Bone loss (osteoporosis) generally occurs in men in their 50s and in women after menopause.
  • the elasticity of the ligaments connecting the joints decreases, and arthritis occurs due to long-term wear and inflammatory reactions of cartilage, which is also a joint disease due to aging.
  • Bone or joint diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful include osteoporosis, Paget's disease, rickets, osteomalacia, osteolysis, renal osteodystrophy in renal failure, fractures, degenerative bone diseases, osteogenesis imperfecta, osteopenia, bone defects and hip dystrophy, osteoarthritis, degenerative arthritis, osteochondritis dissecans, osteoarthritis or arthritis after meniscus injury, malalignment of joints, avascular necrosis, arthroses, isolated chondral defects, chondromalacia patellae, synovitis, bursitis, traumatic effusion, ligamentous deficiency arthroses, osteochondritis dissecans (OCD), patellar instability, This may include, but is not limited to, rheumatoid arthritis, juvenile idiopathic arthritis, juvenile arthritis, post-traumatic arthritis, inflammatory arthritis, septic arthritis, lupus, scleroderma
  • Skin diseases for which the GRP78 or a similar peptide thereof according to the present invention may be useful include, but are not limited to, pruritus erythematosus, seborrheic dermatitis, contact dermatitis, xerosis, urticaria, acne, psoriasis, eczema, exfoliative dermatitis, hidradenitis, atopic dermatitis, drug allergy, diabetic dermatitis, bacterial dermatitis, fungal dermatitis, hidradenitis, allergic dermatitis, or neurodermatitis.
  • Metabolic diseases for which GRP78 or a similar peptide thereof according to the present invention may be useful may include, but are not limited to, obesity, diabetes, hyperlipidemia, hypertriglyceridemia, liver disease, arteriosclerosis, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, insulin resistance disease or hyperinsulinemia.
  • the weight of the kidney and the number of glomeruli decrease and harden.
  • it can increase renal vascular resistance, which can cause a decrease in blood flow to the kidney and a decrease in glomerular filtration rate, resulting in renal failure.
  • the occurrence of electrolyte abnormalities, glomerulonephritis, and chronic renal failure also increases.
  • Urinary incontinence can occur due to a decrease in the storage capacity of the bladder or weakened bladder muscles, and in men, prostate enlargement is often accompanied.
  • Urinary system diseases for which the GRP78 or a similar peptide thereof according to the present invention can be useful include, but are not limited to, urinary incontinence, cystitis, nephritis, benign prostatic hyperplasia, prostatitis, overactive cystitis, or urinary stones.
  • brain diseases such as depression, delirium, emotional and behavioral disorders, dementia, cerebrovascular diseases, and Parkinson's disease are observed.
  • the number and weight of brain neurons decrease by about 10%, and the ventricular area increases.
  • Fatty brown pigment accumulation in neurons, amyloid accumulation in cerebral blood vessels, senile plaques, and neurofibrillary tangles increase, and the production of active oxygen increases, which are related to nervous system damage.
  • enzymes and receptors involved in the production of neurotransmitters decrease, and cerebral blood flow and glucose metabolism decrease.
  • Neurological disorders for which the GRP78 or analogue peptides thereof according to the present invention may be useful may include, but are not limited to, spinal muscular atrophy, Friedreich's ataxia, CLN3 Batten, CLN6 Batten, CLN7 Batten, epileptic encephalopathy, Leigh syndrome, Charico-Marie-Tooth disease, giant axonal neuropathy, Lafora disease, SLC13A5 epileptic encephalopathy, congenital glycosylation disorder, Type Iq, Carridge syndrome, Angelman syndrome, Rett syndrome, spastic paraplegia, childhood alternating hemiplegia or Zellweger spectrum disorder.
  • cardiovascular diseases such as hypertension, aortic disease, heart failure, ischemic heart disease, arrhythmia, peripheral vascular disease, and valvular disease increases.
  • the heart shows left ventricular hypertrophy and left atrial dilatation with increasing age. This can be caused by myocardial cell necrosis and size increase, other growth hormone decrease, and local collagen deposition.
  • the elasticity of blood vessels decreases, which is due to a decrease in the production of vascular endothelial cells and a decrease in NO, a vasodilator, during the aging process.
  • Cardiovascular diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful may include, but are not limited to, myocardial infarction, angina pectoris, hypertension, heart failure, cardiomyopathy, primary cardiac arrest, congestive heart disease, ischemic heart failure, arteriosclerosis, arrhythmia, coronary artery disease, stroke, or peripheral vascular disease.
  • Age-related lung disease is associated with a decline in lung function, such as weakening of the respiratory muscles and diaphragm, decreased elastic recoil of the lungs, and decreased diffusing capacity (the rate of gas exchange between the lungs and the blood) of the lungs.
  • the increased risk of airway obstruction in the elderly increases the mortality rate of chronic obstructive pulmonary disease, cardiovascular disease, and increases the incidence of lung cancer.
  • Lung dysfunction diseases for which the GRP78 or a similar peptide according to the present invention may be useful include, but are not limited to, pulmonary hypertension, chronic obstructive pulmonary disease, pulmonary fibrosis, acute respiratory distress syndrome, bronchial asthma, inflammatory lung disease, pneumonia, or emphysema.
  • Cancer is also called a disease of aging. For example, it is known that the probability of developing cancer increases by more than 10 times when you are over 65 years old. As you age, the phenomenon of cell death decreases, which may increase the occurrence of cancer.
  • Cancer or immune-related diseases for which the GRP78 or a similar peptide thereof according to the present invention may be useful may include, but are not limited to, cancer, rheumatoid arthritis, insulin-dependent diabetes, juvenile diabetes, systemic lupus erythematosus, atopic dermatitis, Crohn's disease, psoriasis, multiple sclerosis, hyperthyroidism, anemia, Behcet's disease, and autoimmune diseases including autoimmune encephalomyelitis, and transplant rejection diseases including graft-versus-host disease.
  • Aging occurs everywhere in the body, but especially in oral tissues that play an important role in maintaining independent living, aging threatens the quality of life and life of the elderly population by causing functional decline, tooth extraction, recession, and degenerative loss of function due to physiological decline in teeth, surrounding tissues, and secretory glands (salivary glands, etc.).
  • Diseases that damage periodontal tissues due to periodontitis, decrease in oral muscles (masticatory muscles), and loss of teeth, gum bones, and masticatory function are known to be common diseases related to the elderly.
  • skeletal muscles decrease overall, causing various complications including decrease in oral muscles (masticatory muscles), falls, fractures, and metabolic syndrome.
  • Dental or periodontal diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful may include, but are not limited to, gingivitis, periodontitis, gingival pain, gingival bleeding, severe bad breath, gingival discoloration, tooth loosening, tooth movement, gingival recession, gingival edema, periodontal pocket formation, pericoronitis or periodontal abscess.
  • the dental or periodontal disease for which the GRP78 or analogue peptide according to the present invention may be useful may be characterized by impaired function of oral tissues, and may have one or more characteristics selected from the group consisting of, but not necessarily limited to, tooth wear, tooth loss, gingival recession, periodontal ligament attachment loss, alveolar bone loss, and decreased saliva secretion.
  • Such dental or periodontal diseases may be, but are not necessarily limited to, periodontitis, gingivitis, pulpitis, mucositis, exfoliative oral disorder, oral lichen planus, pemphigus vulgaris, stomatitis, root caries, alveolar bone defect, inflammatory fibrous hyperplasia, temporomandibular joint disorder, peri-implantitis, xerostomia, or oral cancer.
  • GRP78 or analogue peptides thereof according to the present invention may be useful may be characterized by aged muscle cells or muscle tissue, and may be accompanied by one or more features selected from the group consisting of, but not limited to, muscle dysfunction, muscle wasting, muscle wasting, and muscle degeneration.
  • Such diseases or conditions may be selected from the group consisting of, but not necessarily limited to, dystonia, muscular atrophy, muscular degeneration, muscular rigidity, amyotrophic lateral sclerosis, muscular dystrophy, cachexia, masticatory muscle disorders, temporomandibular disorder, and sarcopenia.
  • the GRP78-like peptide according to the present invention is thought to potentially reduce endoplasmic reticulum stress in a manner similar to the GRP78 protein that regulates endoplasmic reticulum stress, and thus exerts utility in inhibiting or reversing aging of cells, tissues or organs, or in restoring
  • the GRP78-like peptide according to the present invention particularly, a peptide comprising an NBD represented by SEQ ID NO: 22 or a part of an amino acid thereof, or a peptide comprising an SBD represented by SEQ ID NO: 23 or a part of an amino acid thereof, or a peptide comprising a part of the GRP78 amino acid sequence represented by SEQ ID NO: 21, wherein the peptide comprises three or more amino acids selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine (C), methionine (M), tryptophan (W), glutamic acid (D), and proline (P), or a peptide capable of increasing the expression of GRP78, may be useful for the prevention or treatment of various diseases associated with endoplasmic reticulum stress through such a mechanism.
  • endoplasmic reticulum in the present invention refers to a very important cellular organelle where protein folding occurs during protein biosynthesis, and plays a very important role in maintaining cellular homeostasis, and the endoplasmic reticulum stress response occurring in the endoplasmic reticulum is closely related to cell survival or death, similar to the autophagy phenomenon.
  • the endoplasmic reticulum is a membrane-component structure that extends from the nuclear envelope and looks like it has branches, and there are two types: the rough endoplasmic reticulum with attached ribosomes and the smooth endoplasmic reticulum without ribosomes. About 1/3 of the proteins in the cell are transformed into active protein structures through posttranslational modifications, such as folding, assembly, glycosylation, and disulfide bonds, from mRNA in the rough endoplasmic reticulum.
  • the smooth endoplasmic reticulum is a site for the synthesis of lipids and steroid hormones, and plays an important role in regulating intracellular calcium concentrations as a calcium storage.
  • ER stress endoplasmic reticulum stress
  • ER stress response means that immature proteins exceeding the ability of the ER to process are introduced into the cell body due to a physiological or pathological environment, or that calcium in the ER is depleted, causing a disruption in the function of the ER. That is, in a state of ER stress, abnormal proteins in an unfolded state are accumulated in the ER, which can cause various pathological conditions.
  • Causative factors of ER stress include environmental changes within the cell due to diseases or addition of chemical reagents, decreased glucose concentration, addition of inhibitors of sugar chain repair enzymes, and excessive accumulation of viral proteins due to viral infection. When ER stress occurs, cells have a defense mechanism to survive, which is called the ER stress response.
  • the ER stress response is caused by a signal transmission through the ER transmembrane receptors present in the ER membrane, pancreatic ER kinase (PKR)-like ER kinase (PERK), activation transcription factor 6, and inositol requiring enzyme 1 (IRE1).
  • PLR pancreatic ER kinase
  • PERK pancreatic ER kinase
  • IRE1 inositol requiring enzyme 1
  • these three ER stress receptors are inactivated by binding to the ER chaperone GRP78/BiP.
  • GRP78/BiP When unfolded proteins accumulate in the ER, GRP78/BiP dissociates from the receptor, activating the receptor and resulting in the ER stress response.
  • the ER stress response induced in this way restores the function of the ER and reduces the accumulation of unfolded proteins.
  • the apoptosis pathway is activated, eventually resulting in cell death.
  • the ER stress response is linked to mitochondrial function.
  • increased calcium release from the ER due to ER stress leads to decreased mitochondrial function, including increased mitochondrial ROS production.
  • the degree of ER stress can be determined by measuring changes in mitochondrial copy number and changes in the degree of mitochondrial synthesis.
  • endoplasmic reticulum stress-related disease refers to a disease caused by the accumulation of unfolded proteins in the endoplasmic reticulum due to endoplasmic reticulum stress, and includes neurodegenerative diseases, chronic metabolic diseases, cancer, viral infections, etc.
  • neurodegenerative diseases occur as protein tangles occur with the progression of aging, and endoplasmic reticulum stress is frequently found here.
  • neurodegenerative diseases include Alzheimer's, Parkinson's, Huntington's, hypoxia of the brain and heart, cardiac hypertrophy, and arteriosclerosis.
  • PERK PLR Like ER Kianse
  • eIF2a eukaryotic translation initiation factor 2 alpha
  • Parkin an E3 ligase protein
  • endoplasmic reticulum stress itself increases the expression of Parkin.
  • glutamine polymers form protein tangles that are difficult for the cell's decomposition capacity to handle, and if this situation persists for a long time, endoplasmic reticulum stress occurs and nerve cell death is induced.
  • Brain and heart hypoxia Hypoxia (ischemia) symptoms that occur in the brain and heart induce endoplasmic reticulum stress by increasing unfolded proteins while disrupting the cell's redox control function.
  • hypoxia ischemia symptoms that occur in the brain and heart cause an increase in unfolded proteins while disrupting the redox regulatory function of cells, thereby inducing endoplasmic reticulum stress, and the reperfusion situation after hypoxia increases the amount of nitric oxide, which disrupts the balance of active oxygen and s-nitrosylation in cells, thereby increasing oxidative stress within the cells again.
  • both type 1 diabetes and type 2 diabetes are known to be related to endoplasmic reticulum stress.
  • stress enzymes stress kinases
  • endoplasmic reticulum stress disrupt cell signaling initiated by insulin, thereby making cells insensitive to insulin and preventing normal glucose uptake.
  • immunoglobulin light chain amyloidosis amyotrophic lateral sclerosis (ALS), haemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Creutzfeldt-Jakob disease, familial hypercholesterolaemia, alpha1 antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses, Wolcott-Rallison syndrome, Wolfram syndrome, inflammatory It is known that endoplasmic reticulum stress is involved in the development of various diseases, including intestinal diseases, coronary disease, ulcerative colitis, breast cancer, and prostate cancer.
  • composition in the present specification may be a pharmaceutical composition or a cosmetic composition.
  • the present invention relates to a pharmaceutical composition for preventing or treating muscle diseases comprising GRP78, a peptide analog thereof or a nucleic acid encoding the same.
  • the present invention relates to a method for preventing or treating muscle disease, comprising a step of administering the peptide or nucleic acid.
  • the present invention relates to the use of the peptide or nucleic acid for preventing or treating muscle diseases.
  • the present invention relates to the use of said peptide or nucleic acid for the manufacture of a medicament for preventing or treating muscle diseases.
  • the muscle disease may be characterized by being selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, atony, amyotrophic lateral sclerosis, cachexia, masticatory muscle disorder, temporomandibular disorder, and inflammatory myopathy, but is not limited thereto.
  • the pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives.
  • the additives include, but are not limited to, stabilizers, surfactants, lubricants, solubilizers, buffers, sweeteners, bases, adsorbents, coagulants, binders, suspending agents, curing agents, antioxidants, brighteners, flavoring agents, flavoring agents, pigments, coating agents, humectants, moisture regulators, fillers, antifoaming agents, refreshing agents, chewing agents, antistatic agents, colorants, sugar coating agents, isotonic agents, softeners, emulsifiers, adhesives, thickeners, foaming agents, pH regulators, excipients, dispersants, disintegrants, waterproofing agents, preservatives, preservatives, solubilizing agents, solvents, fluidizing agents, and the like.
  • the pharmaceutical composition of the present invention may include pharmaceutically acceptable carriers, excipients, and diluents.
  • the carriers, excipients, and diluents are commonly used and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • composition of the present invention can be formulated as an injectable formulation such as an aqueous solution, suspension, or emulsion, a pill, a capsule, a granule, or a tablet, and thus, the composition of the present invention can be an injection, a patch, a liquid, a capsule, a granule, a tablet, a powder, a spray, an ointment, a gel, a mucosal administration formulation, a suppository, etc.
  • These formulations can be prepared by a conventional method used in formulation in the art or by a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA, and can be formulated into various formulations depending on each disease or on the ingredients.
  • composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
  • the present invention relates to a cosmetic composition for preventing or reversing aging comprising GRP78, a peptide analog thereof or a nucleic acid encoding the same.
  • the composition may be characterized by having at least one function selected from the group consisting of improving skin wrinkles, changing the color of skin or hair, and alleviating or preventing hair loss and reversing such changes, but is not limited thereto.
  • the cosmetic composition of the present invention may be any cosmetic composition commonly used in the art, but preferably, it can be provided for purposes such as a skin ointment, a basic cosmetic, a makeup cosmetic, a body cosmetic, or a shaving cosmetic.
  • the cosmetic composition according to the present invention may be a cosmetic composition mixed with other cosmetic compositions, specifically, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisture lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisture cream, a hand cream, an essence, a nutrition essence, a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, an emulsion, a pressed powder, a loose powder, an eye shadow, etc., and may be used after being diluted, if necessary.
  • the cosmetic composition of the present invention may further include, in addition to the peptide according to the present invention as an effective ingredient, a functional material having an effect of improving wrinkles or whitening, or in one embodiment, a functional material having a UV blocking or moisturizing effect, or an excipient or diluent for improving physical properties.
  • the cosmetic composition may further include a fragrance, a pigment, a sterilizer, an antioxidant, a preservative, a moisturizer, a thickener, an inorganic salt, or a synthetic polymer material for improving physical properties.
  • the compounding ingredients that may be added may include a fat component, an emollient, a surfactant, an organic pigment, an inorganic pigment, an organic powder, an ultraviolet absorber, a pH adjuster, alcohol, a blood circulation promoter, a cooling agent, an antiperspirant, or purified water.
  • Example 1 Evaluation of the ability of aged cells to revert to young cells by inducing increased expression of GRP78 in aged cells
  • GRP78 overexpression vector containing the nucleic acid sequence of sequence number 20.
  • the GRP78 gene was overexpressed in aged model stem cells, and 72 hours after transfection of the model stem cells, the intracellular GRP78 overexpression and the changes in the expression of genes related to aging and stem cell capacity according to GRP78 overexpression were investigated using qRT-PCR.
  • the constructed vector is shown in Fig. 1.
  • Example 2 Evaluation of molecular genetic changes through introduction of intracellular anti-aging factors
  • a GRP78-EGFP expression vector with a fluorescent material attached was provided by Chungbuk National University, and an experiment was performed. After establishing young stem cells (10 passages or less) and aged stem cells (15 passages or more) through subculture of DPSCs, the vector was introduced into each cell using a transfection reagent, and 72 hours after transfection, the cells were fixed and fluorescently stained, and it was confirmed using a confocal microscope whether the introduced GRP78-EGFP in the cells was expressed.
  • the GRP78 expression vector was transfected into DPSC and PDLSC, which are dental stem cells, and 72 hours later, qPCR was performed using Sybr green to confirm the amount of TERT mRNA, a factor involved in telomere length, expressed.
  • Example 3 Evaluation of tissue changes through introduction of anti-aging factors in aging animal models
  • the GRP78 expression vector was transfected into the gastrocnemius muscle of mice using an in vivo transfection reagent.
  • the experimental groups were set to three groups of naturally aging mice: 8-week-old young mice, 20-week-old middle-aged mice, and 48-week-old old mice (Fig. 6). After mixing the transfection reagent and the vector, 50 ⁇ l was injected per gastrocnemius muscle.
  • the transfection reagent-vector was injected into the gastrocnemius muscle using the GRP78-EGFP expression vector, and 72 hours after the injection, the mice were sacrificed, the gastrocnemius muscle was obtained, and cryosectioning was performed using an OCT compound. After that, the tissue nuclei were stained with DAPI and confirmed using a confocal microscope. As a result, fluorescence was confirmed in the gastrocnemius muscle of animals injected with the GRP78-EGFP vector, and it was confirmed that the GRP78 expression vector could be normally injected using the transfection reagent (Fig. 7).
  • the gastrocnemius muscle was lysed using RIPA buffer, and the protein expression level was confirmed using the Western blot technique. As a result, it was confirmed that the protein expression level increased by up to 50% or more in the GRP78 expression vector injection experimental group compared to the other experimental groups (Fig. 9).
  • tissue sections were made using the gastrocnemius muscle to determine whether there were changes in the thickness of the muscle bundle histologically.
  • tissue sections the tissue was dehydrated and paraffin-embedded, and the tissue was embedded using paraffin. After embedding the tissue, 5 ⁇ m sections were made and hematoxylin and eosin (H&E) staining was performed.
  • H&E hematoxylin and eosin
  • Peptides of sequence numbers 1 to 18 of the above Table 1 were synthesized sequentially from the N-terminus by F-moc solid phase peptide synthesis.
  • the synthesized peptide sequences were cleaved from the resin, washed, lyophilized, and then separated and purified by liquid chromatography. The molecular weight of the purified peptides was confirmed using MALDI-TOF analysis.
  • Example 5 Comparison of expression of aging-related biomarkers
  • Rat periodontal ligament (rPDL) tissue cells were seeded at a total of 4.5 ⁇ 10 4 cells per 60 mm culture dish using culture medium MEM alpha modification 1X and then cultured. After obtaining the amount of cells required for the experiment, the culture medium was removed, washed twice with DPBS, and compared under an optical microscope. SA- ⁇ gal staining was performed using Senescence ⁇ -Galactosidase Staining Kit#9860 from Cell Signaling Technology. Quantitative RT-PCR was performed using a Magnetic Induction Cycler (Mic qPCR) from Bio Molecular Systems. Real-time cell proliferation curves were performed using a Live-Cell Imaging and Analysis Instrument: Incucyte® S3 from Satorius.
  • Fig. 11A shows the results of measuring cell length and width in the control and aged groups. As aging occurred, cell length significantly increased (**p ⁇ 0.01).
  • Figure 11B shows the results of observing the expression of beta galactosidase, which is highly expressed in aged cells, using the SA- ⁇ gal staining (increased blue staining) method.
  • Beta galactosidase expressed in cells was stained using the Senescence ⁇ -Galactosidase Staining Kit, dissolved in 70% glycerol for 30 minutes, and the absorbance was measured at a wavelength of 405 nm. As the number of senescent cells increased, the degree of beta galactosidase staining increased, and the absorbance value significantly increased (***p ⁇ 0.001).
  • FIG 11C shows the results of measuring the gene expression levels of aging-related biomarkers, telomere length related factor-1 (TRF-1), telomere length related factor-2 (TRF-2), Protection Of Telomeres 1 (POT1), and glucose-regulated protein 78 (GRP78), by RT-PCR.
  • TRF-1 telomere length related factor-1
  • TRF-2 telomere length related factor-2
  • POT1 Protection Of Telomeres 1
  • GRP78 glucose-regulated protein 78
  • Figure 11D shows the results of measuring cell proliferation.
  • the control group showed 100% cell density (cell confluency) 5 days after cell division at the number of 5.3 ⁇ 10 4 , but the aged group was investigated to have a proliferation rate of about 72% in the same period of 5 days due to aging, showing that the proliferation rate of periodontal ligament cells extracted from aged rats (old rPDL) was significantly lower than that of periodontal ligament cells extracted from young rats (young rPDL) (***p ⁇ 0.001).
  • old rPDL the cell density was investigated to be 82% even at 8 days, the end point of the experiment.
  • rPDL cells show aging-specific phenomena according to age, and can be used as model cells for aging reversal.
  • Peptides of sequence numbers 1 to 4 were labeled with Rhodamine B fluorescence. After seeding a total of 5.0 ⁇ 10 4 cells, the culture medium was removed and washed twice with DPBS. The prepared fluorescent peptides were mixed at a concentration of 200 ⁇ g/ml in serum-free culture medium and incubated for 1 hour. After 1 hour of treatment, the cells were washed five times with DPBS and mounted with a mounting solution containing DAPI, and the degree of intracellular penetration and distribution of the peptides was examined using a confocal microscope (inverted).
  • Figure 12 shows the results of observing the cell permeability of peptides of sequence numbers 1 to 4 using a confocal microscope.
  • Figure 12A shows the cell permeability result of periodontal ligament cells (young rPDL) extracted from young rats
  • Figure 12B shows the cell permeability result of periodontal ligament cells (old rPDL) extracted from aged rats.
  • the cell permeability of peptides of sequence numbers 1, 3, and 4 was high in both young rPDL and old rPDL, and the permeability of the peptides was observed to be lower in old rPDL than in young rPDL. Therefore, it was confirmed that the cell permeability of aged cells was reduced compared to young cells.
  • Figure 13 shows the results of measuring changes in the expression of aging-related biomarkers by peptides of sequence numbers 1 to 4.
  • Figure 13A shows the results of measuring the expression changes of senescence-related biomarkers TRF-1, TRF-2, and hTERT (human telomerase reverse transcriptase) by peptides of SEQ ID NOS: 1 to 4 in serially cultured aged human tonsil-derived mesenchymal stem cells (TMSC) (passage 20) by RT-PCR. All genes of senescence-related biomarkers were significantly reduced in expression in senescent cells. The expression of the GRP78 gene was also significantly reduced in senescent cells.
  • the expression increase rate of TRF-2 was 2.86 ⁇ 0.10 when treated with the peptide of sequence number 1, which was significantly increased compared to senescent cells that were not treated with the peptide (***p ⁇ 0.001).
  • the expression increase rate was 1.23 ⁇ 0.04, which was significantly increased compared to senescent cells that were not treated with the peptide (*p ⁇ 0.05).
  • Figure 13B shows the results of RT-PCR measurements of changes in the expression of aging-related biomarkers TRF-1, TRF-2, POT1, and GRP78 when peptides of SEQ ID NOS: 1 to 4 were treated to periodontal ligament tissue-derived cells extracted from young (12 weeks old) and aged rats (96 weeks old). Overall, the expression of all genes was significantly reduced in aged rat cells than in young rat cells ($p ⁇ 0.001, compare Young rPDL_control and Old rPDL_control).
  • TRF-1 significantly increased in the young rPDL group by 1.34 ⁇ 0.12 and 1.21 ⁇ 0.11 times, respectively, when treated with the peptides of SEQ ID NO: 2 and 4 (***p ⁇ 0.001).
  • the expression significantly increased by 0.83 ⁇ 0.28 and 0.78 ⁇ 0.18 times, respectively, compared to old rPDL (0.58 ⁇ 0.01) that was not treated with the peptides (***p ⁇ 0.001).
  • POT1 significantly increased in the old rPDL group treated with peptides of sequence numbers 2 and 4 by a factor of 0.34 ⁇ 0.20 (p ⁇ 0.05) and 0.66 ⁇ 0.31 (**p ⁇ 0.01), respectively, compared to the old rPDL control group (0.25 ⁇ 0.01) that was not treated with peptides.
  • GRP78 was significantly increased in the old rPDL group by 0.52 ⁇ 0.18 (p ⁇ 0.05), 0.65 ⁇ 0.04 (p ⁇ 0.001), and 0.69 ⁇ 0.30 (p ⁇ 0.01) when treated with peptides of sequence numbers 2, 3, and 4, respectively, compared to old rPDL (0.56 ⁇ 0.01) that was not treated with peptides.
  • Example 8 Evaluation of muscle tissue functional recovery by peptides
  • muscle function was examined using a natural aging mouse model.
  • C57BL/6J male mice were used, and the ages were 8 weeks (0.16 year, young), 48 weeks (1 year, middle-aged), and 72 weeks (1.5 year, elderly).
  • the peptides of SEQ ID NOS: 3 and 4 were injected intramuscularly into the gastrocnemius muscle of the mice at a dose of 10 mg/kg three times a week for 4 weeks.
  • the grip strength test and rotarod test were performed before and for 8 weeks after drug injection.
  • the grip strength test was performed using a grip strength meter (JD-A-22, Jeung Do Bio and Plant Co., Ltd.) to measure the change in grip strength of the mouse forelimb.
  • the rotarod test was conducted using a rotarod (JD-A-07TS, Jeung Do Bio and Plant Co., Ltd.) at a speed of 25 rpm, and the time for which the mice remained on the rotating rod without falling was measured. After the start of administration, the time for which the mice remained on the rod was measured for up to 8 weeks.
  • the 48-week-old and 72-week-old mice administered the peptides of SEQ ID NO: 3 and SEQ ID NO: 4 for 8 weeks showed no significant difference in retention time on the rod compared to the 8-week-old (8 w NT) mice.
  • the 48-week-old mice administered the peptides of SEQ ID NO: 3 and SEQ ID NO: 4 showed an increased retention time on the rod.
  • the 72-week-old mice Compared to the 72-week-old mice (72 w NT) that were not administered the peptides, the 72-week-old mice administered the peptides of SEQ ID NO: 3 and SEQ ID NO: 4 showed an increased retention time on the rod, and in particular, the increase was significant by the peptide of SEQ ID NO: 3 (*p ⁇ 0.05, Fig. 14).
  • the statistical significance between each group was analyzed by one-way ANOVA. As a result, it means that the muscle function of aged mice was restored by the peptides of SEQ ID NO: 3 and 4.
  • muscle function was examined using a natural aging mouse model.
  • C57BL/6J male mice were used, and the ages were 8 weeks (0.16 year, young), 48 weeks (1 year, middle-aged), and 72 weeks (1.5 year, elderly).
  • the peptides of SEQ ID NO: 3 or SEQ ID NO: 4 were intramuscularly injected into the gastrocnemius muscle of the mouse hind limb at a dose of 4 mg/kg, three times a week for 4 weeks.
  • mice of each age were sacrificed, and tissue sections were prepared to evaluate the recovery of muscle tissue and stained with hematoxylin and eosin (H&E).
  • H&E hematoxylin and eosin
  • Figure 16 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissue administered peptides of sequence number 3 or sequence number 4 for 4 weeks.
  • Figure 17 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissues that were injected with peptides of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered peptides of sequence number 3 or sequence number 4 alone or in combination with muscle regeneration peptides of sequence number 3 or sequence number 4 for 4 weeks.
  • tissue sections prepared in Example 9 were subjected to immunofluorescence (IF) staining using MyoD1 antibody (Ab16148, Abcam) and myosin heavy chain (MYH) antibody (sc-376157, Santa Cruz Biotechnology). After embedding in paraffin, the tissue sections were placed in xylene to remove paraffin, and then sequentially immersed in 100%, 95%, and 70% ethanol to hydrate the tissue and washed with distilled water. The slides were immersed in a citrate buffer-based antigen retrieval buffer at pH 6.0 and then heat-treated at 95°C for 15 minutes to perform antigen retrieval. After antigen retrieval, the slides were cooled to room temperature and washed three times for 5 minutes using a PBS solution.
  • IF immunofluorescence
  • MYH myosin heavy chain
  • the slides were blocked in PBS solution containing 5% BSA (bovine serum albumin) for 1 hour at room temperature, and MYH and MyoD1 primary antibodies were diluted 1:200 each, treated on the slides, and reacted for 16 hours at 4°C.
  • the secondary antibody containing a fluorescent label (A-31570, Thermo Fisher Scientific) was diluted 1:500, treated on the slides, and reacted for 1 hour at room temperature, protected from light.
  • the slides were washed three times with PBS for 5 minutes, stained with DAPI (D1306, Thermo Fisher Scientific) for 5 minutes, and then washed with PBS. After that, the slides were mounted using fluorescent mounting medium (F4680, Sigma-Aldrich) and sealed with a cover glass.
  • MyoD1 is a marker expressed in the early stage of muscle regeneration
  • MYH is a marker expressed in the late stage of muscle regeneration
  • Figure 18 shows the results of measuring fluorescence intensity after confocal microscopy of the expression of MyoD1 in muscle tissue of mice administered with peptides of sequence number 3 and sequence number 4 for 4 weeks.
  • Figure 19 shows the results of measuring fluorescence intensity after confocal microscopy of the expression of MYH in muscle tissue of mice administered with peptides of sequence number 3 and sequence number 4 for 4 weeks.
  • the expression levels of MyoD1 and MYH in muscle tissue were the highest.
  • the expression levels of MyoD1 and MYH proteins increased when administered SEQ ID NO: 3 or SEQ ID NO: 4 compared to 48-week-old mice not administered peptides.
  • the expression levels of MyoD1 and MYH proteins increased when administered SEQ ID NO: 3 or SEQ ID NO: 4 compared to 72-week-old mice not administered peptides (Figs. 18 and 19).
  • Figure 20 shows the results of measuring fluorescence intensity after confocal microscopy observation of the expression of MyoD1 in the muscles of mice that were injected with a peptide of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered a peptide of sequence number 3 or sequence number 4 for 4 weeks, or co-administered a peptide of sequence number 3 and a muscle regeneration peptide (SEQ ID NO: 19), or co-administered a peptide of sequence number 4 and a muscle regeneration peptide (SEQ ID NO: 19).
  • Figure 21 shows the results of measuring fluorescence intensity after confocal microscopy observation of the expression of MYH in the muscles of mice that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered the peptide of sequence number 3 or sequence number 4 for 4 weeks, or co-administered the peptide of sequence number 3 and a muscle regeneration peptide (SEQ ID NO: 19), or co-administered the peptide of sequence number 4 and a muscle regeneration peptide (SEQ ID NO: 19).
  • the muscle tissue expression levels of MyoD1 and MYH were the highest in 8-week-old mice that were not administered peptides.
  • the protein expression levels of MyoD1 and MYH increased compared to 48-week-old mice that were not administered peptides.
  • the expression levels of MyoD1 and MYH significantly increased compared to when only the peptides of SEQ ID NO: 3 or SEQ ID NO: 4 were administered (**p ⁇ 0.01).
  • mice co-administered with SEQ ID NO: 3 and the muscle regeneration peptide, or SEQ ID NO: 4 and the muscle regeneration peptide the expression levels of MyoD1 and MYH were higher than those of the youngest 8-week-old mice that were not administered peptides (Figs. 20 and 21).
  • MyoD1 and MYH protein expression levels increased when administered SEQ ID NO: 3, SEQ ID NO: 3 plus muscle regeneration peptide, SEQ ID NO: 4, SEQ ID NO: 4 plus muscle regeneration peptide compared to 72-week-old mice that were not administered peptides.
  • the expression level of MyoD1 was similar when only the peptide of SEQ ID NO: 3 was administered and when SEQ ID NO: 3 plus muscle regeneration peptide was administered, but the expression level of MYH increased more when SEQ ID NO: 3 plus muscle regeneration peptide was administered.
  • the expression level of MyoD1 increased more when SEQ ID NO: 4 plus muscle regeneration peptide was administered than when only the peptide of SEQ ID NO: 4 was administered.
  • the expression level of MYH was similar.
  • Example 11 Evaluation of the effect of reversing aging through restoration of telomere length regulators
  • TMSCs 3.5 ⁇ 10 5 TMSCs were dispensed into a 60 mm 2 dish, the culture medium was removed, and the cells were washed twice with DPBS. Afterwards, the prepared GRP78 peptide (SEQ ID NO: 4) solution was treated for 24 hours and samples were obtained. Specific primers were synthesized using the nucleotide sequence of hTERT, and qRT-PCR was performed using them. As a result, the expression of hTERT tended to be lower in the control group of aged TMSCs compared to young TMSCs, and the expression of hTERT was restored when treated with the peptide of SEQ ID NO: 4 (Fig. 22a).
  • TMSCs 3.5 ⁇ 10 5 TMSCs were dispensed into a 60 mm 2 dish, and cell culture and peptide treatment were performed in the same manner as in the experiment of Fig. 22a. Afterwards, the experiment was performed using TRAPezeTM RT Telomerase Detection Kit according to the method suggested by Sigma-Aldrich. As a result, the expression of telomerase was significantly decreased in the control group of aged TMSCs compared to young TMSCs (***p ⁇ 0.001), and the expression of telomerase was found to increase when treated with the peptide of sequence number 4 (Fig. 22b).
  • TMSCs 3.5 ⁇ 10 5 TMSCs were seeded on a 60 mm 2 dish and cultured for 2 days until the cell density reached 70–80%.
  • Serum-free culture medium and GRP78 peptide SEQ ID NO: 4
  • TeloTAGGGTM Telomere Length Assay was performed in the same manner as the method suggested by Sigma-Aldrich.
  • telomere length in the control group of aged TMSCs was reduced by 0.87-fold compared to young TMSCs, and when the peptide of SEQ ID NO: 4 was treated to aged TMSCs, the telomere length significantly increased by 4.94-fold compared to the aged control group (*p ⁇ 0.05) (Fig. 22c).
  • TMSCs that had undergone replicative aging had telomeres of slightly shorter length compared to young TMSCs (Fig. 22d).
  • the GRP78-like peptide according to the present invention restores the expression of direct telomere length-related factors decreased due to replicative senescence of TMSCs caused by passage culture, which means that it has the ability to improve or reverse aging.
  • Example 12 Evaluation of the effect of GRP78-like peptide on the recovery of mitochondrial-related factors
  • TMSCs 3.5 ⁇ 10 5 TMSCs were seeded in a 60 mm 2 dish, the culture medium was removed, and the cells were washed twice with DPBS.
  • the prepared GRP78 peptide (SEQ ID NO: 4) solution was treated and cultured for 24 h. The medium was removed and fixed using 4% paraformaldehyde. After treatment with a mixture of phenol-free DMEM and 1 mM stock mitotracker for 30 min, mitochondria distributed in TMSCs were observed using a confocal microscope (inverted).
  • mtDNA mitochondrial DNA
  • the GRP78, GRP78-like peptide or nucleic acid encoding the same according to the present invention can increase the expression of a biomarker whose expression is decreased in aging cells, and thus is useful for restoring the function of muscle tissue, etc., whose function is decreased due to aging.

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Abstract

La présente invention concerne une utilisation de GRP78 et d'un peptide similaire de celle-ci ou d'un acide nucléique codant pour celle-ci. Le peptide ou l'acide nucléique selon la présente invention peuvent augmenter l'expression d'un biomarqueur ayant une expression réduite dans des cellules sénescentes et est ainsi utile pour restaurer des fonctions, et similaires, de tissu musculaire dont les fonctions sont réduites par le vieillissement.
PCT/KR2024/019031 2023-11-28 2024-11-27 Utilisation de peptides de type grp78 Pending WO2025116539A1 (fr)

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* Cited by examiner, † Cited by third party
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KR102129380B1 (ko) * 2017-10-13 2020-07-02 주식회사 나이벡 단백질표지자 grp78을 이용한 고효율 줄기세포의 선별 방법
KR102210543B1 (ko) * 2017-10-13 2021-02-01 주식회사 나이벡 고효율 줄기세포 선별을 위한 grp78 유래 펩타이드

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102129380B1 (ko) * 2017-10-13 2020-07-02 주식회사 나이벡 단백질표지자 grp78을 이용한 고효율 줄기세포의 선별 방법
KR102210543B1 (ko) * 2017-10-13 2021-02-01 주식회사 나이벡 고효율 줄기세포 선별을 위한 grp78 유래 펩타이드

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AKINYEMI AMOS OLALEKAN, SIMPSON KENDALL ELIZABETH, OYELERE SUNDAY FAITH, NUR MARIA, NGULE CHRISPUS MUTUKU, OWOYEMI BOLAJI CHARLES : "Unveiling the dark side of glucose-regulated protein 78 (GRP78) in cancers and other human pathology: a systematic review", HISTONE DEACETYLASE INHIBITORS IN THE TREATMENT OF MUSCULAR DYSTROPHIES: EPIGENETIC DRUGS FOR GENETIC DISEASES., vol. 29, no. 1, XP093319170, ISSN: 1528-3658, DOI: 10.1186/s10020-023-00706-6 *
DATABASE PROTEIN 21 February 2022 (2022-02-21), XP093319169, Database accession no. NP_001253525.1 *
SALGANIK MAXIM; SERGEYEV VALERIY G.; SHINDE VISHAL; MEYERS CRAIG A.; GORBATYUK MARINA S.; LIN JONATHAN H.; ZOLOTUKHIN SERGEY; GORB: "The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons", NEUROBIOLOGY OF AGING, TARRYTOWN, NY, US, vol. 36, no. 6, 1 January 1900 (1900-01-01), US , pages 2213 - 2223, XP029233709, ISSN: 0197-4580, DOI: 10.1016/j.neurobiolaging.2015.02.018 *

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