WO2020045349A1 - Agent permettant d'activer ou d'inhiber la communication cellule-cellule à l'aide d'une protéine fonctionnelle, anticorps ou aptamère, méthode de criblage dudit agent, protéine de fusion, acide nucléique codant pour ladite protéine, vecteur recombinant, cellule hôte transformée, modulateur de la fonction de l'exosome, et exosome - Google Patents
Agent permettant d'activer ou d'inhiber la communication cellule-cellule à l'aide d'une protéine fonctionnelle, anticorps ou aptamère, méthode de criblage dudit agent, protéine de fusion, acide nucléique codant pour ladite protéine, vecteur recombinant, cellule hôte transformée, modulateur de la fonction de l'exosome, et exosome Download PDFInfo
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Definitions
- the present invention relates to an agent capable of improving intercellular communication via a functional protein, capable of improving intercellular communication, an antibody or an aptamer usable for the agent, and a method for screening the agent.
- the present invention also relates to a fusion protein capable of modifying an exosome, a nucleic acid encoding the fusion protein, a recombinant vector, a transformed host cell, an agent for controlling the function of an exosome containing the fusion protein or the nucleic acid, and It relates to exosomes containing fusion proteins.
- sEV Small extracellular vesicles
- Exosomes are a type of sEV derived from multivesicular bodies (hereinafter, simply referred to as “MVB”) (see, for example, Non-Patent Documents 2 and 3), and transport cells, mRNA, and miRNA to communicate between cells.
- MVB multivesicular bodies
- Delivery of functional proteins between cells by sEVs containing exosomes is associated with tumor progression and neurodegenerative diseases (see, for example, Non-Patent Documents 5 and 6).
- the molecular mechanism by which functional proteins are sorted into sEVs has not been completely elucidated.
- proteins are generally known to undergo various post-translational modifications by post-translational modifiers such as ubiquitin, small ubiquitin-like modifier (SUMO), and Nedd87-10. It is known that post-modifications can affect various cellular processes.
- post-translational modifiers such as ubiquitin, small ubiquitin-like modifier (SUMO), and Nedd87-10. It is known that post-modifications can affect various cellular processes.
- Ubiquitin-like protein 3 also known as membrane-bound Ub-fold protein (MUB)
- UBL3 membrane-bound Ub-fold protein
- UBL ubiquitin-like domain
- UBL3 was not known as a post-translational modifier.
- the present invention has been made in view of the above-mentioned problems of the prior art, and can improve cell-to-cell communication, activate or suppress cell-to-cell communication via a functional protein, an antibody or an aptamer that can be used for the agent, and It is intended to provide a method for screening an agent.
- the present invention also provides a fusion protein, a nucleic acid encoding the fusion protein, a recombinant vector containing the nucleic acid, and a host cell transformed with the recombinant vector, which can modify exosomes in view of the above-described problems of the related art. , An agent for regulating the function of an exosome containing the fusion protein or the nucleic acid, and an exosome containing the fusion protein.
- the present inventors have found that post-translational modification by UBL3 can sort a functional protein into a sEV in a state in which a functional protein can be expressed, and can sort into a recipient cell in a state in which a functional protein can be expressed. It was completed. That is, the present invention is as follows.
- the agent according to (1) wherein the functional protein is a disease-causing substance or a disease-suppressing substance.
- the causative substances are KRAS, HRAS, RRAS, TGFBR1, TGFBR2, RB1, ITGA6, ITGB4, mTOR, TSC2, APLP2, IRF3, IKBKG, RPTOR, NOTCH1, NOTCH2, NOTCH3, BMPR1A, BMPRXN, ENMPR2PS.
- the agent according to (2) which is at least one member selected from the group consisting of HIP1R and NPC1.
- a fusion protein of a UBL3 protein and a functional protein is any one of the following (a) to (c).
- An exosome function regulator comprising the fusion protein according to (7) or (8) or the nucleic acid according to (3).
- an agent capable of improving cell-cell communication, activating or suppressing cell-cell communication via a functional protein, an antibody or an aptamer usable for the agent, and a method for screening the agent can be provided.
- the present invention is suitable as an in vitro research tool for developing a therapeutic tool for sEV-related diseases such as tumor formation, tumor progression, tumor metastasis, and neurodegenerative disease.
- FIG. 3 shows the results of phylogenetic tree analysis of UBL domain-containing proteins from various species. It is a figure which shows the analysis result of new post-translational modification factor UBL3. It is a figure which shows the outline
- FIG. 2 shows the intracellular localization of UBL3.
- FIG. 4 shows that UBL3 localization to MVB depends on UBL3 modification.
- FIG. 4 shows that total protein levels in sEV are reduced in UBL3 knockout mice.
- FIG. 4 shows the analysis of UBL3 in sEV.
- FIG. 4 shows that UBL3 modification is involved in protein sorting into sEV.
- FIG. 4 shows post-translational modification of tubulin by UBL3 and sorting to sEV.
- FIG. 4 shows post-translational modification of Ras protein by UBL3 and sorting into sEV.
- FIG. 3 shows intracellular localization of UBL and sorting to sEV.
- FIG. 2 shows that EGFP- and biotinylated proteins tagged by UBL3 are sorted into sEV.
- the UBL3 protein is any one of the following (a) to (c).
- a protein having an activity of sorting proteins and / or an activity of localizing as a membrane protein The protein of (A) a protein comprising the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing, and
- the 110th to 117th positions of SEQ ID NO: 1 or 2 (preferably the 111th to 117th positions, more preferably the 111th to 116th positions, still more preferably the 112th to 115th positions, Particularly preferably, the activity of binding to any protein via at least one amino acid residue at position 113 or 114).
- SEQ ID NO: 1 represents the amino acid sequence of human UBL3 protein.
- SEQ ID NO: 2 represents the amino acid sequence of mouse UBL3 protein.
- amino acid sequence having 90% or more homology means that amino acid homology is 90% or more, and the homology is preferably 93% or more, more preferably 95% or more. , More preferably 97% or more, particularly preferably 98% or more.
- All proteins having the activity of sorting any protein and / or the activity of localizing as a membrane protein are within the scope of the present invention.
- the side chains of the amino acids that are the constituents of proteins differ in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein.
- glycine (Gly) and proline (Pro) For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), and the like.
- Glycine (Gly) and proline (Pro) Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and
- the mutant protein is a mutation caused by substitution, insertion, deletion, or the like on the amino acid sequence of UBL3 described in SEQ ID NO: 1 or 2, the mutation is a highly conserved mutation in the three-dimensional structure of UBL3.
- the method for obtaining the UBL3 protein is not particularly limited, and may be a protein synthesized by chemical synthesis, a naturally-occurring protein isolated from a biological sample or cultured cells, or a recombinant protein produced by a genetic recombination technique. .
- UBL3 gene All genes encoding UBL3 protein (for example, a protein having the amino acid sequence represented by SEQ ID NO: 1 or 2) belong to the UBL3 gene.
- SEQ ID NO: 3 shows the nucleotide sequence of the coding region (CDS) encoding the human UBL3 gene.
- SEQ ID NO: 4 shows the CDS nucleotide sequence encoding the mouse (Mus musculus (house mouse)) UBL3 gene.
- the UBL3 gene includes the genes described in any of the following (d) and (e). From the viewpoint that a human or mouse-derived gene can be used as it is and unnecessary transformation is not required, It is preferably the gene of the following (d).
- E an activity of modifying any protein, consisting of a nucleotide sequence in which one or several bases are deleted, substituted and / or added in the nucleotide sequence of SEQ ID NO: 3 or 4 in the sequence listing, and the above-mentioned modification; Encoding a protein having an activity of sorting an arbitrary protein into MVB or sEV and / or an activity of localizing as a membrane protein
- the range of "one or several" in the "base sequence in which one or several bases are deleted, substituted and / or added in the base sequence" as used herein is not particularly limited, but is preferably 1 to 20. , More preferably about 1 to 10, even more preferably about 1 to 5.
- Examples of the degree of the DNA mutation include those having 80% or more homology with the base sequence of the UBL3 gene described in SEQ ID NO: 3 or 4 in the sequence listing, preferably 85% or more, more preferably DNAs having a homology of 90% or more, more preferably 95% or more, particularly preferably 98% or more are mentioned.
- the method for obtaining the UBL3 gene is not particularly limited. Proper probes and primers are prepared based on the amino acid sequence and base sequence information described in SEQ ID NOs: 1 to 4 in the sequence listing of the present specification, and are used to prepare a human cDNA library (for expressing the UBL3 gene).
- UBL3 gene can be isolated by selecting a desired clone from those prepared from suitable cells according to a conventional method. The UBL3 gene can also be obtained by the PCR method.
- a gene encoding a protein having an activity of sorting any protein into MVB or sEV by the above modification and / or an activity of localizing as a membrane protein can be obtained by chemical synthesis, genetic engineering techniques or It can also be made by any method known to those skilled in the art, such as mutagenesis.
- the “agent that activates or suppresses intercellular communication via a functional protein” of the present invention includes a substance that increases or decreases the expression of the UBL3 gene or UBL3 protein, or a substance that enhances or inhibits the activity of the UBL3 protein.
- “intercellular communication” refers to intercellular information transmission via sEV or the like of exosomes or the like (preferably, the above-mentioned sEV or the like containing the functional protein), specifically, wave, delivery of protein and delivery of the protein. Function expression in the recipient cells.
- the functional protein examples include a membrane protein (eg, a transmembrane protein), a cytoskeleton-forming protein (eg, tubulin), a labeled protein (eg, GFP (green fluorescen protein), an improved GFP (EGFP), and glutathione-protein).
- a membrane protein eg, a transmembrane protein
- a cytoskeleton-forming protein eg, tubulin
- a labeled protein eg, GFP (green fluorescen protein), an improved GFP (EGFP)
- glutathione-protein examples include any protein such as S-transferase (GST), but it is preferably a disease-causing substance or a disease-suppressing substance.
- KRAS, HRAS, RRAS, TGFBR1, TGFBR2, RB1, ITGA6, ITGB4, mTOR, TSC2, APLP2, IRF3, IKBKG, RPTOR, NOTCH1, NOTCH2, NOTCH3, BMPR1A, BMPRPS At least one selected from the group consisting of HIP1R and NPC1 is preferred, and at least one selected from the group consisting of KRAS, HRAS and RRAS is more preferred.
- neurodegenerative disease-related proteins such as amyloid ⁇ , tau, ⁇ -synuclein, prion and the like are packaged in sEV and distributed in the brain (Rajendran, L. et al. Alzheimer '). s disease beta-amyloid peptides are released in association with exosomes. Proc Natl Acad Sci USA 103,11172-11177 (2006)., Fevrier, B. et al. Cells release prions in association with exosomes. Proc Natl Acad Sci U S A 101,9683- 9688 (2004), Lee, H. J., Bae, E. J. & Lee, S. J. (2014)., Kanmert, D. et al. C-terminally trunked forms of Tau, but not full-length Tau or its C-terminal department representative representation. 35,10851-10865 (2015).), It may be used as the functional protein.
- RNA substance that increases or decreases the expression of UBL3 gene or UBL3 protein
- siRNA double-stranded RNA
- siRNA small interfering RNA
- RNA preferably a double-stranded RNA containing at least 21 consecutive nucleotides of CDS in the nucleotide sequence of RNA transcribed from the nucleotide sequence of the UBL3 gene, or a DNA encoding the double-stranded RNA. It is preferably a double-stranded RNA containing 30 consecutive nucleotides or less of CDS in the nucleotide sequence of the RNA transcribed from the nucleotide sequence of the UBL3 gene, or a DNA encoding the double-stranded RNA.
- RNA More preferably, it is a double-stranded RNA containing 25 consecutive nucleotides or less of the CDS in the nucleotide sequence of the RNA to be transcribed, or a DNA encoding the double-stranded RNA.
- RNAi refers to a phenomenon in which expression of a target gene is suppressed when double-stranded RNA (dsRNA), which is a part of an mRNA encoding a part of a target gene, is introduced into a cell.
- dsRNA double-stranded RNA
- Examples of the DNA encoding the double-stranded RNA include a DNA having an inverted repeat sequence of the UBL3 gene or a partial sequence thereof.
- the inverted repeat sequence of the target gene can be expressed in mammalian cells by incorporating the inverted repeat sequence of the target gene downstream of the promoter sequence operable in the mammal.
- the promoter sequence used in the present invention is not particularly limited as long as it can operate in mammals.
- the substance that enhances or inhibits the activity of UBL3 protein includes any substance such as an antibody, a high molecular compound (such as nucleic acid), or a low molecular compound, as long as it directly or indirectly enhances or inhibits the activity of UBL3 protein.
- antibodies or aptamers that selectively bind to the UBL3 protein are preferred.
- the above antibody or aptamer has at least the 110th to 117th positions of SEQ ID NO: 1 or 2 (preferably the 111th to 117th positions, more preferably the 111th to 116th positions, still more preferably the 112th to 115th positions, particularly preferably the 113th or 114th positions).
- Antibodies or aptamers that selectively bind to the UBL3 protein using a region containing one amino acid residue as an epitope are preferred.
- a polyclonal antibody can be prepared by separating and purifying serum obtained from an animal immunized with an antigen (UBL3 protein).
- Monoclonal antibodies are prepared by fusing antibody-producing cells obtained from animals immunized with the antigen (UBL3 protein) with myeloma cells to prepare hybridomas, and culturing the hybridomas or administering the hybridomas to animals to ascites cancer. And the above-mentioned culture solution or ascites is separated and purified.
- the aptamer refers to a nucleic acid drug composed of single-stranded RNA or DNA and capable of acting as an agonist (enhancing activity) or an antagonist (inhibiting activity) by binding to a target protein by its three-dimensional structure (Drug Delivery System 31). -1,016, pp10-14). Aptamers have high binding and specificity to target proteins, low immunogenicity, can be produced by chemical synthesis, and have high storage stability.
- the base length of the aptamer that selectively binds to the UBL3 protein is not particularly limited as long as it specifically binds to the UBL3 protein, but is preferably 15 to 60 bases, more preferably 20 to 50 bases. , 25 to 47 bases, and particularly preferably 26 to 45 bases.
- the “method of screening for a modulator of intercellular communication via a functional protein” of the present invention uses a change in the expression of a UBL3 gene or UBL3 protein or a change in the activity of a UBL3 protein by a test substance as an index.
- screening means at least narrowing down a population of test substances.
- the regulator of intercellular communication through a functional protein include an agent that activates intercellular communication through a functional protein, an agent that suppresses intercellular communication through a functional protein, and the like.
- the above-mentioned screening method preferably includes a step of using a UBL3-expressing cell, a UBL3 gene or a UBL3 protein.
- the UBL3-expressing cells may be any animal (eg, human, mouse) cells (eg, cerebral cortex-derived cells, cerebellar-derived cells, hippocampus-derived cells), or express UBL3 by genetic recombination techniques.
- Human breast cancer cells MDA-MB-231-luc-D3H2LN cells
- human fetal kidney cells eg, HEK293 cells, HEK293T cells
- human Cervical cancer cells eg, HeLa cells, HeLa-S3 cells
- hamster ovary cells eg, CHO cells, CHO-K1 cells and the like may be used.
- the activity of the UBL3 protein includes an activity of modifying any protein, an activity of sorting any protein into MVB or sEV by the above modification, and / or an activity of localizing as a membrane protein.
- the screening method comprises culturing UBL3-expressing cells in the presence and absence of a test substance, and increasing the number of UBL3-expressing cells in the presence of the test substance relative to the case where the test substance is absent.
- at least one selected from the group consisting of an increase in the expression of a UBL3 protein or a gene and an increase in the activity of a UBL3 protein is detected, it is possible to screen for an agent that activates intercellular communication via a functional protein. it can.
- the degree of the increase or enhancement is not particularly limited as long as it is a statistically significant increase or enhancement.
- the expression or activity of the UBL3 gene or protein in the control is preferably 1.5 times or more, and more preferably 2 times or more the expression or activity of the UBL3 gene or protein. More preferably, there is.
- the screening method comprises culturing UBL3-expressing cells in the presence and absence of a test substance, and reducing the number of UBL3-expressing cells in the presence of the test substance relative to the absence of the test substance.
- test substance for example, the system before the administration of the test substance (for example, wild type), Or less than ⁇ , preferably less than 4, the expression or activity of the UBL3 gene or protein in the control) system
- ⁇ preferably less than 4
- the expression or activity of the UBL3 gene or protein in the control system to which a substance that does not affect the expression or function of the protein is administered. It is more preferably 1/10 or less, and particularly preferably the expression or activity is eliminated.
- any screening method such as in vivo, in vitro, or in silico may be used as long as the above is used as an index.
- UBL3 expressing cells are cultured in the presence and absence of a test substance, and the number of UBL3 expressing cells is changed according to the presence or absence of the test substance, and the change in UBL3 protein or gene expression is changed. And at least one selected from the group consisting of changes in the activity of UBL3 protein.
- a change in the number of UBL3-expressing cells under administration of the test substance relative to the number of UBL3-expressing cells in any tissue without administration of the test substance is measured.
- the expression level of the UBL3 protein at the mRNA level can be measured by a conventional method such as Northern blot, Southern blot or RT-PCR. Specifically, it can be carried out by a conventional method known to those skilled in the art described in Molecular Cloning Second Edition or Current Protocols in Molecular Biology.
- the expression level of the UBL3 protein can be measured by ordinary immunoassay such as Western blot using an antibody or ELISA. Specifically, it can be carried out by a conventional method known to those skilled in the art described in Molecular Cloning Second Edition or Current Protocols in Molecular Biology.
- the expression of the UBL3 gene in various human tissues can be detected even in silico.
- the expression of the UBL3 gene in various human tissues can be detected, for example, by using a probe or a primer having a partial or entire nucleotide sequence of the gene.
- UBL3 gene expression can be detected by a conventional method such as RT-PCR, Northern blot, or Southern blot.
- the screening method comprises culturing UBL3-expressing cells in the presence and absence of a test substance, and changing the number of UBL3-expressing cells according to the presence or absence of the test substance, changing the expression of UBL3 protein or gene, and Preferably, the method comprises a step of detecting at least one selected from the group consisting of changes in the activity of the protein.
- a method for detecting at least one selected from the group consisting of a change in the number of UBL3-expressing cells depending on the presence or absence of a test substance, a change in the expression of a UBL3 protein or a gene, and a change in the activity of a UBL3 protein for example, A method for detecting the activation of signal transduction induced by the binding between a test substance and UBL3 may be mentioned.
- test substance can be used as the test substance to be subjected to the screening method.
- the type of the test substance is not particularly limited, and may be an antibody, a nucleic acid molecule, an individual low-molecular synthetic compound, a compound present in a natural product extract, or a synthetic peptide.
- the test compound may also be a compound library, phage display library or combinatorial library. Construction of compound libraries is known to those skilled in the art, and commercially available compound libraries can also be used.
- the test substance is preferably a small molecule compound (eg, a compound library), an antibody, or a nucleic acid molecule.
- the UBL protein is as described above.
- the fusion protein of the present invention is preferably a protein in which a UBL protein gene and a functional protein gene are transcribed and expressed as a single body to form one protein.
- the functional protein examples include a causative substance of a disease or a suppressor of a disease, a membrane protein (for example, a transmembrane protein), a cytoskeleton-forming protein (for example, tubulin), and a labeled protein (for example, GFP (green fluorescein protein)).
- a membrane protein for example, a transmembrane protein
- a cytoskeleton-forming protein for example, tubulin
- a labeled protein for example, GFP (green fluorescein protein)
- GFP green fluorescein protein
- Modified GFP EGFP
- GST glutathione-S-transferase
- a disease-causing substance or a disease-suppressing substance is preferable.
- a fusion protein of a UBL protein and a label protein is used in the examples section described later.
- KRAS, HRAS, RRAS, TGFBR1, TGFBR2, RB1, ITGA6, ITGB4, mTOR, TSC2, APLP2, IRF3, IKBKG, RPTOR, NOTCH1, NOTCH2, NOTCH3, BMPR1A, BMPRPS At least one selected from the group consisting of HIP1R and NPC1 is preferred, and at least one selected from the group consisting of KRAS, HRAS and RRAS is more preferred.
- proteins in sEV are known to play an important role in tumor progression and organic metastasis, and can be used as the functional proteins.
- neurodegenerative disease-related proteins such as amyloid ⁇ , tau, ⁇ -synuclein, and prion are also packaged in sEV and are known to be distributed in the brain. Can be used as
- the nucleic acid of the present invention encodes the above fusion protein, and the UBL3 gene and the functional protein gene can be transcribed and expressed together to form one fusion protein.
- the UBL3 gene may be on the N-terminal side or the C-terminal side, but is preferably on the N-terminal side.
- the UBL3 gene portion in the nucleic acid the gene described in any of the above (d) and (e) is preferable, and the gene (d) is more preferable.
- a nucleic acid encoding a fusion protein of a UBL protein and a labeled protein or the like is used in the Examples section described later.
- the nucleic acids of the present invention may or may not further include expression control regions such as promoters, start codons, stop codons, polyadenylation signals and enhancers.
- the recombinant vector of the present invention contains the above nucleic acid.
- a “recombinant vector” is an expression vector capable of expressing the above fusion protein in a suitable host cell, wherein the gene comprises a regulatory region operably linked to express a gene insert. Refers to a product.
- "operably linked” means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding the fusion protein are operably linked to perform a general function.
- the operative ligation with the recombinant vector can be produced using a gene recombination technique known in the technical field to which the present invention pertains. Can be easily carried out using an enzyme or the like generally known.
- An appropriate expression vector used in the present invention can include a signal sequence for membrane targeting or secretion in addition to expression control regions such as a promoter, start codon, stop codon, polyadenylation signal and enhancer.
- Initiation codons and stop codon are generally considered to be part of a nucleotide sequence that encodes the immunogenic target protein, and must have an effect on the individual when the gene product is administered, and must be compatible with the coding sequence. Should be in frame.
- Common promoters are constitutive or inducible.
- Prokaryotic cells include, but are not limited to, lac, tac, T3 and T7 promoters.
- Eukaryotic cells include simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), eg, the long terminal repeat (LTR) promoter of HIV, molony virus, cytomegalovirus (CMV) ), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoters, as well as ⁇ -actin promoter, human hemoglobin, human muscle creatine, human metallothionein-derived promoters, but are not limited thereto.
- the expression vector can include a selectable marker for selecting a host cell containing the vector.
- the selection marker is used to select cells transformed with the vector, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein is used. Can be. In an environment where the selection agent has been treated, only cells expressing the selection marker survive, so that transformed cells can be selected.
- the vector can include a replication origin, which is a specific nucleic acid sequence at which replication is initiated.
- various types of vectors such as plasmids, viruses, cosmids, and the like can be used.
- the type of the recombinant vector is not particularly limited as long as it expresses the target gene in various host cells such as prokaryotic cells and eukaryotic cells and performs the function of producing the target protein.
- a vector capable of producing a large amount of a foreign protein in the same form as in the natural state while having the expression ability is preferable.
- Expression vectors suitable for eukaryotic hosts include SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and expression control sequences derived from retrovirus, and the like. However, the present invention is not limited to this.
- Examples of expression vectors usable for bacterial hosts include bacterial plasmids derived from Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vector, col @ E1, pCR1, pBR322, pMB9 and derivatives thereof; Plasmids with a wider host range such as RP4, phage DNA exemplified by very diverse phage ⁇ (phage @ lambda) derivatives such as ⁇ gt10 and ⁇ gt11, NM989, and others such as M13 and filamentous single-stranded DNA phage DNA phages are included.
- Useful expression vectors for yeast cells are 2 ° C plasmids and derivatives thereof.
- a useful vector for insect cells is pVL941.
- the host cell of the present invention is a host cell transformed with the above recombinant vector.
- the above-mentioned recombinant vector can be inserted into a host cell to form a transformant.
- suitable host cells include Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus phylla. (Staphylococcus sp.), Fungi such as Aspergillus sp., Pichia pastoris, Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the like.
- yeast such as asa
- other lower eukaryotic cells such as asa
- higher eukaryotic cells such as cells from insects.
- the host cell can be preferably derived from a plant or mammal, but can be derived from human breast cancer cells (MDA-MB-231-luc-D3H2LN cells), human fetal kidney cells (eg, HEK293 cells, HEK293T). Cells), human cervical cancer cells (eg, HeLa cells, HeLa-S3 cells), hamster ovary cells (eg, CHO cells, CHO-K1 cells), and the like, but are not limited thereto.
- human breast cancer cells MDA-MB-231-luc-D3H2LN cells
- human fetal kidney cells eg, HEK293 cells, HEK293T
- Cells human cervical cancer cells (eg, HeLa cells, HeLa-S3 cells), hamster ovary cells (eg, CHO cells, CHO-K1 cells), and the like, but are not limited thereto.
- ⁇ transformation '' into a host cell includes any method for incorporating a nucleic acid into a cell or a tissue. It can be carried out. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation using silicon carbide fibers, and Agrobacteria. Transformation methods include, but are not limited to, transformation methods via PEG, dextran sulfate, lipofectamine and drying / suppression.
- the exosome function regulator of the present invention includes the fusion protein or the nucleic acid.
- the function of the exosome may be not only the function originally possessed by the natural exosome, but also a function artificially added to the exosome, such as visibility (for example, fluorescent labeling), discrimination (for example, tagging). ), A function directed to the target recipient cell, a function taken up by the target recipient cell, and the like.
- the exosome function regulator of the present invention can regulate (eg, enhance or suppress) the exosome function by including the fusion protein or the nucleic acid.
- Exomes to which the agent for controlling exosome function of the present invention is applied include natural products (for example, natural compounds, proteins, peptides, nucleic acids (miRNA, etc.)) and artificial products (for example, synthetic compounds, recombinant or synthetic proteins, recombinant or synthetic). It may or may not contain a peptide, a recombinant or synthetic nucleic acid (such as a recombinant or synthetic miRNA).
- an exome derived from a cell that highly expresses the natural product or the artificial product can be used (for example, Nikkei Biotech, March 14, 2016, “Special Feature”). "Exome drug discovery has begun.)
- the exosome of the present invention contains the fusion protein in a state where the functional protein can express its function.
- the exosome of the present invention may or may not further contain the natural product and the artificial product.
- As the exome further containing the natural product or the artificial product for example, an exome derived from a cell that highly expresses the natural product or the artificial product can be used.
- the function of the functional protein is, for example, membrane localization when the functional protein is a membrane protein (for example, a transmembrane protein), and when the functional protein is a cytoskeleton-forming protein.
- the expression of the cause of the disease or the suppression of the disease may be mentioned.
- an arbitrary function can be expressed in the recipient cell according to the function of the functional protein.
- a protein in which oncogenic RasG12V and UBL3 protein are fused or bound can activate Ras signaling in recipient cells.
- sEVs such as exosomes contain the fusion protein in a state where a functional protein (oncogenic RasG12V) can express its function, and can express its function (activate Ras signaling) in recipient cells. ing.
- Cell culture MDA-MB-231 in Roswell Park Memorial Institute (RPMI) 1640 medium or Dulbecco's modified Eagle medium (DMEM) with PS (100 units / mL penicillin G, 10 ⁇ g / mL streptomycin sulfate) and 10% fetal bovine serum (FBS).
- RPMI Roswell Park Memorial Institute
- DMEM Dulbecco's modified Eagle medium
- PS 100 units / mL penicillin G, 10 ⁇ g / mL streptomycin sulfate
- FBS fetal bovine serum
- Various expression vectors can be prepared by conventional molecular biology techniques and PCR (Yao, I. et al. SCRAPPER-dependent ubiquitination of active zone protein RIM1 regulations synaptic vesicle release, Cell U.S.A., U.S.A. 7, U.S.A. 7, U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A., U.S.A
- Anti-GAPDH antibody manufactured by Cell Signaling, 14C10
- Flag-M2 antibody manufactured by Sigma, F3165
- Flotillin-1 antibody manufactured by BD, clone 18
- CD63 antibody manufactured by Invitrogen, Ts63; manufactured by BD, H5C6
- EEA1 antibody BD, 610456
- Rab11 antibody BD, 610656
- COXIV antibody Cell Signaling, 4850
- calnexin antibody Enzo, ADI-SPA-860
- GM130 antibody BD, 610822
- PMP70 antibody Thermo, PA1-650
- Lamin B1 antibody Abcam, ab16048
- GP96 antibody Enzo, 9G10
- alpha actinin-4 antibody GeneTe
- C2C3 calreticulin antibody
- CD9 antibody CD9 antibody
- HRP Horseradish peroxidase
- CD63 and CD9 antibodies for sEV markers were used.
- Flotillin-1 and Alix antibodies for multiple EV markers Flotillin-1 and Alix antibodies for multiple EV markers
- Antibodies and GAPDH antibodies were used.
- MDA-MB-231 cells, HEK293T cells, and HeLa cells were transfected with DNA using Lipofectamine 2000 (Invitrogen). After 24 or 72 hours (FIG. 8f), cells are washed, scraped and collected in ice-cold PBS, pelleted by centrifugation at 400 ⁇ g for 1 minute at 4 ° C., and pelleted with 1% Triton buffer (50 mM Tris). -HCl (pH 7.4), 100 mM NaCl, and 1% (v / v) Triton X-100) at 20 ° C. for 20 minutes.
- Triton buffer 50 mM Tris
- -HCl pH 7.4
- 100 mM NaCl 100 mM NaCl
- 1% (v / v) Triton X-100) at 20 ° C. for 20 minutes.
- Crude nuclei and unbroken cells were removed by centrifugation at 800 ⁇ g for 5 minutes at 4 ° C.
- cell lysates were incubated with 20 ⁇ L of protein G-Sepharose beads (GE @ Healthcare) for 1 hour at 4 ° C. with rotation. Incubate the resulting supernatant with 2 ⁇ g of anti-Flag antibody in 15 ⁇ L of protein G-Sepharose beads for 4 hours at 4 ° C. while rotating or with 20 ⁇ L GFP-Trap (manufactured by Chromotek) without precleaning at 4 ° C. Incubated for hours.
- cell lysates were incubated with 40 ⁇ L of protein G-Sepharose beads for 1 hour at 4 ° C. with rotation.
- the resulting supernatant was incubated with the anti-UBL3 antibody (1: 1000 dilution) in 20 ⁇ L of Protein G-Sepharose beads at 4 ° C. for 10 hours while rotating.
- sEV was purified from the culture medium of biotin-tagged UBL3 transfected MDA-MB-231 cells, and PBS or SDS buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, After washing with 1% sodium deoxycholate, 1% NP-40, 2% SDS), the cells were treated with streptavidin beads (manufactured by Thermo, 29202). Pull-down (PD) and flow-through (FT) fractions were analyzed by Western blotting using streptavidin-HRP or anti-CD63 antibody.
- PBS or SDS buffer 50 mM Tris-HCl [pH 8.0], 150 mM NaCl
- Tissue homogenization and Western blot analysis Tissue homogenization and Western blot analysis were performed using conventional methods (Yao, I. et al. SCRAPPER-dependent ubiquitination of active zone protein RIM1 regulatory synthesizing vesicle release 130, 2003, Cellular release, 70, 1991). , Uezumi, A., Fukada, S., Yamamoto, N., Takeda, S. & Tsuchida, K.
- Isolation of membrane protein and cytoplasmic protein fraction Isolation of the membrane protein and the cytoplasmic protein fractions was performed according to the manufacturer's protocol (Promento JET, manufactured by Fermentas; Mem-PER Plus kit, manufactured by Thermo). RNA purification, reverse transcription reaction, and real-time qPCR analysis were performed by conventional methods (Yao, I. et al. SCRAPPER-dependent ubiquitination of active zone protein RIM1 regulatory synthetic release, 2003 Cell. 7, Cell. Uezumi, A., Fukada, S., Yamamoto, N., Takeda, S. & Tsuchida, K. Mesenchal progenitors dissect from telecommunications cells contracts. 143-152 (2010) .Hitachi, K., Nakatani, M. & Tsuchida, K. Myostatin signaling regulations Akt activity via the regulation of the physician's review. ).
- UBL domain-containing proteins were classified into subfamilies as follows. First, the distance between each UBL domain including the branch length of the inner node was investigated. If the distance was less than 0.46, the same group included two domains and defined the group as a subfamily of UBL domains. Next, a subfamily of UBL domains that were highly conserved between either worms, flies, or yeast and humans and mice was defined. The boxes in the figure are the sub-families of the highly conserved UBL domains, namely ubiquitin, Nedd8, ubiquitin-like 5 (UBL5), ubiquitin, ubiquitin-like 3 (UBL3), SUMO-1, SUMO-2 / 3, ubiquitin. Specific protease 14 (USP14), MGC10067, and Elongin-B are shown.
- Glycine residues at the carboxyl terminus of ubiquitin, SUMO and Nedd8 are generally known to be covalently linked to lysine residues of target proteins (Welchman, RL, Gordon, C. & Mayer, RJ). .. Ubiquitin and ubiquitin-like proteins as multifunctional signals Nat Rev Mol Cell Biol 6,599-609 (2005), Flotho, A & Melchior, F.Sumoylation:... a regulatory protein modification in health and disease Annu Rev Biochem 82, 357-385 (201 )., Watson, I.R., Irwin, M.S. & Ohh, M.NEDD8 pathways in cancer, Sine Quibus Non. Cancer Cell 19,168-176 (2011).).
- UBL3 in FIG. 1 modifies the target protein by a disulfide bond via a cysteine residue at the carboxyl terminus, and although UBL3 has a ubiquitin-like domain, Has been found to be a completely different mechanism from conventional ubiquitin and ubiquitin-like modification as described below.
- FIG. 2a shows that UBL3-dependent post-translational modifications were detected by immunoprecipitation (IP) with anti-Flag antibody from MDA-MB-231 cells transfected with Flag-UBL3, and Western blotting with UBL3 antiserum. (Right panel) (20 ⁇ g / lane). The estimated molecular weight of Flag-UBL3 is 16 kDa.
- the UBL3 signal was observed as a smear band up to high molecular weight only under non-reducing conditions.
- the smear signal disappeared after adding 2-mercaptoethanol ( ⁇ -ME +) to the sample before loading on an SDS-polyacrylamide gel (FIG. 2a, right panel).
- FIG. 3 is a diagram showing the production of UBL3 KO mouse.
- FIG. 3a is a diagram showing a schematic structure of a targeting vector for removing exon 2 of the UBL3 gene including an initiation codon.
- the arrow Neo indicates the neomycin resistance (Neo) gene.
- Arrow DT-A indicates the diphtheria toxin A (DT-A) gene.
- Other arrows indicate the positions of PCR primers for genotyping.
- BS indicates pBluescript. Target clones were confirmed by Southern blot analysis of genomic DNA digested with KpnI-HindIII.
- the target construct was probed with a 5 'probe using a 750 bp fragment downstream of the KpnI site outside the 5' position.
- Genomic DNA digested with NcoI was probed with a 3 'probe using an 800 bp fragment between the NdeI site and the NcoI site at the 3' end.
- the target construct was linearized by SpeI digestion.
- CCE 129 / Sv / Ev
- ES embryonic stem
- the chimeric mice were backcrossed to C57BL / 6J mice for 10 generations. Genotype was determined by Southern blotting or PCR using the following three primers.
- UBL3-KO-3 ' 5'-ACCCAGGTCCTCATGCATCGGGTAGA-3' (SEQ ID NO: 5)
- UBL3-KO-5 ' 5'-CCACCCACTGCCCTTTCCCAGAAAC-3'
- UBL3-KO-Neo 5'-GCTGCAGGGTCGCTCGGGTGTT-3 '(SEQ ID NO: 7) All procedures related to animal care and treatment were in accordance with institutional and National Institutes of Health guidelines and were approved by the Animal Care and Use Committee of Fujita Health University.
- FIG. 3b is a diagram showing the genotype analysis results of UBL3 KO mice.
- FIG. 3d is a diagram showing the results of examining the tissue distribution of endogenous UBL3 protein by Western blotting analysis using UBL3 antiserum (50 ⁇ g / lane). GAPDH was used as a loading control. After Western blot analysis, the gel was SYPRO Ruby stained.
- Ht heart, Lg: lung, Lv: liver, Sp: spleen, Pc: pancreas, Si: small intestine, Cl: colon, Ki: kidney, Ms: skeletal muscle, Te: testis, Cx: cerebral cortex, Cb: cerebellum, Hp: hippocampus.
- FIG. 3c is a diagram showing the results of Western blot using UBL3 anti-UBL3 antibody in Ubl3 + / + (wild-type; WT) mouse and Ubl3-/ ⁇ (knockout; KO) mouse-derived brains (20 ⁇ g / lane). Based on the above results, UBL3-dependent post-translational modifications in cerebral cortex lysates were analyzed below.
- FIG. 2b is a diagram showing immunoprecipitation (IP) of cerebral cortex lysates of wild-type (WT) and UBL3 KO mice with an anti-UBL3 antibody (20 ⁇ g / lane). As is evident from the results shown in FIG. 2b, the degree of post-translational modification is reduced in UBL3 KO mice.
- FIG. 2c is a diagram showing the schematic structure of a Flag-tagged wild type or UBL3 mutant in lanes 1 to 5 in FIGS. 2d and e (20 ⁇ g / lane).
- FIG. 2d shows detection of UBL3 modification in cells containing Flag-tagged wild type or UBL3 mutant in FIG. 2c (20 ⁇ g / lane).
- the IP product was boiled without 2-mercaptoethanol ( ⁇ ME-) before loading, and a part of the sample was treated with 2-mercaptoethanol ( ⁇ ME +).
- FIG. 4a shows the results of analyzing UBL3 modification in HEK293T cells and HeLa cells. IP products were boiled without loading 2-mercaptoethanol ( ⁇ ME-) (10 ⁇ g / lane) before loading. As is evident from the results shown in lane 5 in FIG. 2d and in FIG.
- the UBL3 modification in the UBL3C113 / 114A mutant, was not only in MDA-MB-231 cells, but also in each of the cell lines tested (HEK293T cells and HeLa cells). It can be seen that in FIG. On the other hand, as is clear from the results shown in lanes 3 and 4 in FIG. 2d and FIG. 4a described later, in some cells, the UBL3 modification is reduced in the UBL3C113A mutant and the UBL3C114A mutant, It turns out that it still exists.
- FIG. 2e shows the intracellular localization of UBL3 in MDA-MB-231 cells transfected with the Flag-UBL3 wild type or mutant shown in FIG. 2c (20 ⁇ g / lane).
- FIG. 4b shows the subcellular localization of UBL3 in HEK293T cells and HeLa cells transfected with Flag-UBL3 wild type and mutants (10 ⁇ g / lane).
- FIG. 2f shows the schematic structure of the Flag-tagged wild type or UBL3 mutant in lanes 1, 2 and 6-8 in FIGS. 2g and h (20 ⁇ g / lane).
- FIG. 2g shows detection of UBL3 modification in cells containing Flag-tagged wild type or UBL3 mutant in FIG. 2f (20 ⁇ g / lane).
- the IP product was boiled without 2-mercaptoethanol ( ⁇ ME-) and a portion of the sample was treated with 2-mercaptoethanol ( ⁇ ME +).
- FIG. 2h shows the intracellular localization of UBL3 in MDA-MB-231 cells transfected with Flag-UBL3 wild type or mutant (20 ⁇ g / lane).
- UBL3 ⁇ 1 lacking only one carboxyl terminal amino acid has lost the ability to modify UBL3.
- UBL3 ⁇ 1 is retained in the membrane fraction, whereas UBL3 ⁇ 2 and UBL3 ⁇ 3 are not retained in the membrane fraction.
- FIG. 5a shows that post-translational modification products by UBL3 are reduced in the cytoplasm of cells. As is clear from the results shown in FIG. 5a, it can be seen that UBL3 modification is reduced in the cytoplasm fraction.
- MDA-MB-231 cells were co-transfected with GFP-UBL3 (wild type or mutant) and iRFP670 (morphological marker) using Lipofectamine 2000. After 24 hours, cells were fixed with 4% PFA / PBS for 15 minutes. Quenched with 0.1 M glycine / PBS; permeabilized / blocked with 0.2% BSA, 2% goat serum, and 0.05% saponin / PBS for 1 hour; incubated with primary antibody for 18 hours at 4 ° C. The secondary antibody was incubated for 1 hour. It was mounted with Aqua-Poly / Mount (manufactured by Polysciences).
- the lysosome was labeled with 50 ⁇ M LysoTracker Red (manufactured by Thermo) at 37 ° C. for 1 hour.
- a confocal image was acquired on a confocal laser microscope (LSM-780, manufactured by Carl Zeiss) with a 63 ⁇ objective lens (NA1.4). Representative images projected from several 0.5 ⁇ m spaced cross sections are shown, each having a single confocal image in the inset. Fluorescence intensity and cell morphology were measured in Fiji (Image J).
- FIG. 6 shows that the localization of UBL3 to MVB depends on UBL3 modification
- FIG. 6a shows that GFP transfected with improved GFP (hereinafter also simply referred to as “EGFP”)-UBL3 and MVB
- FIG. 4 is a diagram showing a representative projection image of MDA-MB-231 cells co-stained with a marker (CD63), an early endosomal marker (EEA1), a lysosomal marker (LysoTracker), or a recycled endosomal marker (Rab11).
- CD63 marker
- EAA1 early endosomal marker
- LysoTracker a lysosomal marker
- Rab11 recycled endosomal marker
- FIG. 6B is a diagram showing the results of quantitative analysis of the fluorescence intensity of EGFP-UBL3 in FIG. 6A.
- n number 10 ⁇ 4
- * is p ⁇ 0.05 by Kruskal-Wallis / Dunn multiple comparison test
- *** is p ⁇ 0.001.
- FIG. 5b shows transfected EGFP-UBL3 and shared with mitochondrial marker (COXIV), endoplasmic reticulum marker (Calnexin), Golgi marker (GM130), peroxisome marker (PMP70), or nuclear envelope marker (lamin B1).
- UBL3 is an endosomal marker (Rab11), a mitochondrial marker (COXIV), an endoplasmic reticulum marker (Calnexin), a Golgi marker (GM130), a peroxisome marker (PMP70), and It turns out that it does not show co-localization with the nuclear membrane recycling marker (LaminB1). On the other hand, it was found that co-localization with the early endosomal marker (EEA1) and the lysosomal marker was detected. However, as is clear from the results shown in FIGS. You can see that it is weak.
- MDA-MB-231 cells transfected with 3 ⁇ Flag-UBL3 (wild-type or UBL3 ⁇ 1) or mock-transfected MDA-MB-231 cells were fixed with 4% PFA / PBS at 4 ° C. for 30 minutes, followed by 4% PFA And fixed in 0.1% glutaraldehyde (GA) / PBS at 4 ° C. for 30 minutes.
- GAC glutaraldehyde
- FIG. 5c is a diagram showing immunoelectron microscopic images of Flag-UBL3 in mitochondria and nuclear envelope of MDA-MB-231 cells.
- FIG. 6d is a diagram showing an immunoelectron microscope image of wild-type UBL3 and UBL3 ⁇ 1 in MDA-MB-231 cells.
- UBL3 colocalizes with MVB to a greater extent than lysosomes.
- Cells release extracellular vesicles (EVs) of various sizes. Some of these vesicles are derived from the plasma membrane, and these vesicles contain large and medium-sized EVs purified at 2,000 ⁇ g (2K) and 10,000 ⁇ g (10K), respectively.
- sEV can be purified at 100,000 ⁇ g (100K) and partially derived from the plasma membrane (non-exosome sEV) or from MVB (exosome sEV) (Kowal, J. et al. Proteomic comparision). define novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci USA 113, E968-977.
- sEV was isolated from the conditioned media according to conventional protocols (Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of the system. Cell Biol Chapter 3, Unit 322 (2006).). Briefly, exosome-depleted FBS (centrifugation at 100,000 xg for 16 hours) was used to prepare conditioned media. After 24 hours of incubation, the culture medium (about 90 mL from 3 ⁇ 10 7 cells) was collected and centrifuged at 4 ⁇ C at 300 ⁇ g for 10 minutes. The supernatant was centrifuged at 2,000 ⁇ g for 20 minutes at 4 ° C.
- the supernatant was centrifuged again at 10,000 xg for 30 minutes at 4 ° C.
- the supernatant was filtered through a 0.22 ⁇ m filter (Millipore, Millex-GV) to remove cell debris.
- sEV pellets were collected by ultracentrifugation at 100,000 ⁇ g (24,000 rpm, SW32Ti rotor, manufactured by Beckman) at 4 ° C. for 70 minutes.
- the sEV pellet was washed with 1 mL of PBS and collected by ultracentrifugation at 100,000 ⁇ g (42,900 rpm, TLA-110 rotor, manufactured by Beckman) at 4 ° C. for 60 minutes.
- the sEV pellet was washed again with 1 mL of PBS, collected by ultracentrifugation at 100,000 ⁇ g for 60 minutes at 4 ° C., and then resuspended in PBS.
- a differential ultracentrifugation protocol was used according to the previous literature 13 .
- filtration with a 0.22 ⁇ m filter (Millipore, Millex-GV) was further performed.
- whole blood was allowed to stand at room temperature for 30 minutes to coagulate. Whole blood was centrifuged twice at 8,000 ⁇ g for 10 minutes to remove clots.
- heparin solution (Mochida Pharmaceutical, 5000 units of heparin / 5 mL), spun slowly, centrifuged at 9,100 ⁇ g for 5 minutes, Was used as plasma.
- the volume of serum from UBL3 KO mice was consistent with the serum volume of its wild-type littermates (about 350-500 ⁇ L).
- the collected serum was immediately diluted with an equal volume of PBS and centrifuged at 2,000 ⁇ g for 30 minutes at 4 ° C.
- the supernatant was centrifuged at 12,000 ⁇ g for 45 minutes at 4 ° C.
- the supernatant was diluted 5-fold with PBS and filtered through a 0.22 ⁇ m filter.
- the EV pellet was recovered by ultracentrifugation at 110,000 ⁇ g (34,000 rpm, SW55Ti rotor, manufactured by Beckman) at 4 ° C. for 70 minutes.
- RNA RNA @ 6000 @ Pico @ kit, manufactured by Agilent.
- RNA 6000 @ Pico @ kit, manufactured by Agilent.
- cDNA complementary DNA
- the serum, plasma and cell lysate protein concentrations were measured by BCA (Thermo).
- BCA Thermo
- each pellet was diluted with 2% SDS and measured with Micro-BCA (manufactured by Thermo).
- FIG. 7 shows that total protein levels in sEV are reduced in UBL3 knockout mice
- FIG. 7 a shows cells from conditioned medium of MDA-MB-231 cells transfected with 3 ⁇ Flag-UBL3 vector.
- FIG. 4 shows the results of blotting lysate (CL) and centrifuged pellets of 2K, 10K, and 100K with various antibodies.
- the Flag antibody, the Flotillin-1 antibody, the GP96 antibody, the Actinin-4 antibody, the Calreticulin antibody, the GAPDH antibody, and the Alix antibody are under ⁇ ME + conditions
- the CD63 and CD9 antibodies are under ⁇ ME ⁇ conditions.
- EV was purified from 2K, 10K and 100K centrifuged pellets from MDA-MB-231 cells, and UBL3 was more concentrated in the 100K fraction than in the 2K and 10K fractions. You can see that there is.
- FIG. 8a shows the results of electron microscopic analysis of a 100K pellet (sEV) isolated from a conditioned medium of MDA-MB-231 cells by a differential ultracentrifugation protocol.
- the right panel is an area enlarged from the white box on the left panel.
- the sEV purified from the 100K pellet contains nanometer-sized vesicles ranging in diameter from 50 to 100 nm.
- FIG. 8b is a diagram showing the results of immunoseparation.
- PD pull-down
- FT flow-through
- * is a non-specific signal from the heavy chain (50 kDa) of the immunoglobulin used for immunoprecipitation. As is evident from the results shown in FIG. 8b, most of UBL3 was found to be present in sEV containing CD63.
- FIG. 8c is a diagram showing detection of UBL3 in sEV. Equal amounts of PD and FT fractions were loaded on a gel for Western blot analysis. As shown in FIG. 8c, under PBS treatment, biotinylated UBL3 was identified in the FT fraction but not in the PD fraction. On the other hand, most transfected biotinylated UBL3 was identified in the PD fraction with SDS treatment, indicating that UBL3 was packaged in sEV. As is evident from the results shown in FIG. 8c, UBL3 was found to be packaged in sEV.
- FIG. 8d is a diagram showing the results of Western blot analysis of sEV from the cell culture medium of the primary stromal vascular (SVF) fraction and the primary myotube (Myotube) fraction isolated from WT and UBL3 KO mice. .
- SVF primary stromal vascular
- Myotube primary myotube
- sEV was isolated from the culture medium of MDA-MB-231 cells transfected with Flag-tagged UBL3. The same amount of sEV (1 ⁇ g protein) was blotted with UBL3 antiserum. IP products from cell lysates were loaded as a positive control for UBL3 modification. The gel was stained with SYPRO Ruby.
- FIG. 8e shows UBL3 modification in sEV. As is clear from the results shown in FIG. 8e, it can be seen that UBL3 modification was observed not only in cell lysates but also in sEV.
- FIG. 7b shows the presence of UBL3 and its variants in sEV.
- the Flag, Flotillin-1 and GAPDH antibodies are in ⁇ ME + condition
- the CD9 antibody is in ⁇ ME ⁇ condition.
- the purified 100K pellet contains both exosomes and non-exosomes sEV.
- the latter are known to contain vesicles that are directly secreted from the plasma membrane (Kowal, J. et al. Proteomic comparisson definitions Novell markers to characterize heterogeneous petroleum serocellulose petroleum serocellulose petroleum serocellulose petroleum serocellulose petroleum serocellulose petroleum estrocelle estrocelle estrocelle estrocelle estrocelle estrospectres. -977 (2016), Bobrie, A., Colombo, M., Krumich, S., Rapso, G. & Terry, C.
- FIG. 7b illustrates why UBL3 ⁇ 1 localized to the plasma membrane but not to the MVB was detected in small amounts in the 100K pellet (see FIGS. 2h, 6d, e, and 7b). Also, the above findings indicate that UBL3 modification is important for UBL3 sorting into sEV.
- FIG. 8f is a diagram showing the inhibitory effect of sEV release by Rab27a shRNA on UBL3 modification at the gene level. As can be seen from FIG. 8f, when exosome release was inhibited by Rab27a shRNA15, the level of UBL3 modified product increased.
- FIG. 7c is a diagram showing an electron microscopic analysis result of serum-derived purified sEV from WT mice and UBL3 KO mice by negative staining.
- the genotype WT or UBL3 KO
- the genotype does not affect the particle concentration of sEV particles or the average vesicle diameter.
- FIG. 7d shows the protein staining of serum-derived EV in WT mice and UBL3 KO mice.
- Nanoparticle tracking analysis was performed on isolated serum sEV particles diluted 25-fold in PBS using NanoSight LM10HS with blue laser system (NanoSight, Amesbury) (Yoshioka, Y. et al.). J. Extracellular Vehicles 2, 20424 (2013) .Comparative marker analysis of extracellular vesicles in different human cancer types.
- the system focuses the laser beam on a suspension of particles of interest. They were visualized by light scattering using a conventional light microscope arranged perpendicular to the beam axis collecting light scattered from each particle in the field of view.
- the 60 second video records all events for further analysis by the NTA software.
- the Brownian motion of each particle was tracked between frames and its size was calculated using the Stokes-Einstein equation. Each sample was measured three times and the average value was used for statistical analysis.
- FIG. 7d shows the results of blotting purified serum sEV with anti-tubulin antibody and CD9 antibody together with ⁇ ME.
- FIG. 7e shows total RNA levels in serum sEV.
- n 5 ⁇ 2, and n. s. Indicates p> 0.05 by Mann-Whitney test.
- FIG. 9d is a diagram showing electropherogram data on total RNA in serum sEV.
- FIG. 9e is a diagram showing the results of measuring the total RNA amount in plasma sEV using Bioanalyzer 2100.
- n. s. Indicates p> 0.05 by Mann-Whitney test.
- FIG. 7e and FIGS. 9d and e there is no significant difference between the genotypes in the RNA profile and the RNA level.
- FIG. 9f shows the results of real-time qPCR analysis of miRNA in serum sEV purified from WT and UBL3 KO mice.
- n 5, and n. s. Indicates p> 0.05 by Mann-Whitney test.
- the level of the specific sEV-enriched miRNA is not statistically significant.
- the above results show that UBL3 KO mice produce sEV with each protein reduced in serum and normal levels of miRNA. In other words, the sorting of proteins in serum sEVs involves UBL3.
- MDA-MB-231 cells were transfected with the plasmid using Lipofectamine 2000. Twenty-four hours later, cells were washed, scraped in ice-cold PBS, pelleted by centrifugation at 400 ⁇ g for 1 minute at 4 ° C., and lysed in 1% Triton buffer at 4 ° C. for 20 minutes. Crude nuclei and unbroken cells were removed by centrifugation at 20,000 xg for 5 minutes at 4 ° C. For pre-cleaning, the supernatant was incubated twice with 40 ⁇ L protein G-Sepharose beads (GE Healthcare, 50% slurry in lysis buffer) for 1 hour at 4 ° C.
- 40 ⁇ L protein G-Sepharose beads GE Healthcare, 50% slurry in lysis buffer
- the supernatant was then incubated with 50 ⁇ l of anti-Flag M2 affinity gel (Sigma, 50% slurry in lysis buffer) at 4 ° C. for 16 hours.
- the beads were washed with 1% Triton buffer, washed three times with wash buffer (50 mM Tris-HCl [pH 7.5]) to remove detergent, and 200 ⁇ L digestion buffer (2 M urea, 50 mM Tris-HCl). [PH 7.4], resuspended in 1 mM DTT, 5 mM iodoacetamide, 1 ⁇ g of trypsin [Promega, V5280]), and incubated by shaking at 37 ° C. for 16 hours at 1,200 rpm.
- HCD high energy collisional dissociation
- the raw MS files were generated using the MaxQuant environment (version 1.5.9) using the MaxLFQ algorithm for label-free quantification and using the integrated Andromeda search engine with FDR ⁇ 0.01 at the protein and peptide levels. .3) (Cox, J. & @Mann, ⁇ M. ⁇ MaxQuant ⁇ enables ⁇ high ⁇ peptide ⁇ identification ⁇ rates, ⁇ individualized@p.p.b .- ⁇ r ⁇ . 2008)., Cox, J. et al. Accurate p. oteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13,2513-2526 (2014).).
- the search included oxidized methionine (M), acetylation (protein N-terminus), and variable modifications to fixed modifications of carbamidomethyl (C). Up to 2 missed cleaves were allowed for protease digestion. Peptides having at least 7 amino acids are considered for identification, allowing a "match between runs" with a 0.7 minute match time window, allowing quantification of MS1 features not identified in each single measurement I made it. Peptides and proteins were identified using a human (2015) UniProt @ FASTA database containing 21,051 entries.
- FIGS. 10a and b show that all three conditions with three biological replications (3 ⁇ Flag-UBL3, or 3 ⁇ Flag empty vector, or 3 ⁇ Flag-UBL3C113 / 114A) interact and are significantly regulated.
- FIG. 10a and b show that all three conditions with three biological replications (3 ⁇ Flag-UBL3, or 3 ⁇ Flag empty vector, or 3 ⁇ Flag-UBL3C113 / 114A) interact and are significantly regulated.
- FIG. 10a shows that a heat map of z-scored label-free quantification (LFQ) intensity of the identified proteins reveals UBL3 interacting protein (Cluster_1).
- the data set contained a total of 3,882 proteins with a false positive rate (FDR) of 1%.
- the average Pearson correlation coefficient was 0.988 within the three pulldowns.
- UBL3 interacting proteins were grouped based on the subcellular localization of UBL3 interacting proteins as defined by the GO term "cellular component” (GOCC). As shown in FIG. 10b, 31% of the proteins (454 out of 1,447 proteins, p value 0.00405) were found to be classified as GOCC term "extracellular vesicle exosomes".
- FIG. 11a shows profiles of various tubulin proteins under three conditions (3 ⁇ Flag-UBL3, or 3 ⁇ Flag empty vector, or 3 ⁇ Flag-UBL3C113 / 114A) having three biological replications in proteomic analysis.
- FIG. 10c and 11a As is clear from the results shown in FIGS. 10c and 11a, among these UBL3 interacting proteins, tubulin (TUBA1A, 1B, 1C and 4A; and TUBB, B2A, B3, B4A, B4B, B6 and B8).
- tubulin ⁇ Since endogenous tubulin ⁇ can be detected in sEV using a commonly used specific antibody (DM1A), tubulin ⁇ is used as a model case for sorting endogenous proteins into sEV via UBL3 modification. It investigated using. MDA-MB-231 cells were transfected with GFP-tagged UBL3 or GFP-tagged UBL3C113 / 114A. The cell lysate was subjected to immunoprecipitation (IP) by GFP-trap, and the obtained immunoprecipitate was subjected to Western blot using anti- ⁇ -tubulin antibody or anti-GFP antibody. 2 ⁇ sample buffer (no ⁇ ME) was added to the beads and the beads were boiled ( ⁇ ME-) for 3 minutes.
- IP immunoprecipitation
- FIG. 11b shows UBL3-dependent post-translational modification by GFP trap analysis (1 ⁇ g / lane).
- tubulin was modified by UBL3 and shifted only under non-reducing conditions. Observed as a band.
- the tubulin signal was specifically enhanced by UBL3 expression in the 100K fraction, but not in the 2K or 10K fractions. Under non-reducing conditions, most of the tubulin in the 100K sEV pellet was found to have a higher molecular weight than under reducing conditions.
- FIG. 11d shows the results of blotting sEV from conditioned medium of MDA-MB-231 cells transfected with mock vector, 3 ⁇ Flag-UBL3 UBL3C113 / 114A vector or UBL3 ⁇ 1 vector with anti-tubulin antibody.
- the increase in tubulin in sEV is specific for wild-type UBL3, and it can be seen that the UBL3C113 / 114A and UBL3 ⁇ 1 mutants did not show such an effect.
- FIG. 7d it was observed that the protein level of tubulin in serum sEV was reduced in UBL3 KO mice. From the above results, it can be concluded that UBL3 modifies endogenous tubulin after translation and controls sorting to sEV.
- FIG. 12a shows a disease identified as a UBL3 interacting protein in three conditions (3 ⁇ Flag-UBL3, 3 ⁇ Flag empty vector, or 3 ⁇ Flag-UBL3C113 / 114A) with three biological replications by proteomic analysis. It is a figure which shows the profile of a related protein. As shown in FIGS. 10c and 12a, at least 22 disease-related molecules were also found to be included as UBL3 interacting proteins. Interestingly, some of these molecules are involved in tumorigenesis and tumor progression / metastasis, namely HRAS, KRAS, TGFBR1, TGFBR2, RB1, ITGA6, ITGB4, mTOR, TSC2 and APLP2.
- IRF3 and IKBKG immune response molecules (IRF3 and IKBKG), mTOR signaling molecules (mTOR, RPTOR and TSC2), Notch signaling molecules (NOTCH1, NOTCH2, NOTCH3), BMP signaling molecules (BMPR1A and BMPR2), and neurodegeneration / neurons Molecules involved in the disease (PSEN1, ATXN10, HIP1R, APLP2 and NPC1) have been identified as UBL interacting proteins.
- the Ras family members are proto-oncogenes (Weinberg, RA The Biology of Cancer. Galland Science (2007).) And are reported to be abundant in sEV (Demory Beckler, M. et al.). al. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifiers intercellular transfer of mutated KRAS.13 Cell.
- FIG. 12b shows the results of transfecting MDA-MB-231 cells with exogenous wild-type Ras or oncogenic RasG12V and transfected with either 3 ⁇ Flag-tagged UBL3 or UBL3C113 / 114A.
- arrows indicate UBL3-modified Ras or RasG12V.
- Cell lysates were subjected to immunoprecipitation (IP) with anti-Flag antibody, and the obtained immunoprecipitates were subjected to Western blot analysis using anti-Ras antibody.
- IP immunoprecipitation
- FIG. 12c shows cell lysates and sEV from conditioned media of MDA-MB-231 cells transfected with mock vector or wild-type Ras vector and transfected with either mock vector or 3 ⁇ Flag-UBL3 vector. It is a figure which shows the result of blotting with anti-Ras antibody. As is clear from the results shown in FIG. 12c, it can be seen that the exogenous wild-type Ras protein was more sorted into sEV by UBL3.
- FIG. 10d is a diagram showing the results of blotting the sEV pellet with an anti-Ras antibody. As is evident from the results shown in FIG. 10d, wild-type UBL3 but not UBL3C113 / 114A enhances the sorting of RasG12V to sEV.
- ⁇ Activation test of Ras signaling in recipient cells (cells receiving sEV) >> To determine whether sEV encapsulating UBL3 and oncogenic RasG12V causes activation of Ras signaling in recipient cells, either mock, 3 ⁇ Flag-UBL3 or 3 ⁇ Flag-UBL3C113 / 114A, PKH67-labeled sEV purified from MDA-MB-231 cells transfected with RasG12V was introduced into recipient MDA-MB-231 cells. The phosphorylation level of ERK, a downstream signaling molecule, was measured as an indicator of Ras activation in recipient cells (Weinberg, RA The Biology of Cancer. Garland Science (2007).).
- Phosphorylated ERK (pERK) activity in PKH67-labeled sEV uptake cells was tested by antibody staining.
- MDA-MB-231 cells were fixed with 4% PFA / PBS for 15 minutes, permeated / blocked with 5% goat serum and 0.3% Triton X-100 / PBS for 20 minutes, and mixed with anti-pERK antibody at 4 ° C. After 18 hours of incubation with the secondary antibody for 1 hour, the cells were mounted using Aqua-Poly / Mount (manufactured by Polysciences). The data were analyzed using Fiji (Image J), and the value of pERK signal in cells incorporating PKH67-labeled sEV was the average signal measured over a region of 2.5-5 ⁇ m 2 in the cytoplasmic region adjacent to the nucleus. Expressed as intensity, and then normalized in each image to the average pERK of two adjacent cells that did not incorporate PKH67-labeled sEV.
- FIG. 10e shows phosphorylated ERK (pERK) in PKH67-labeled sEV-incorporated MDA-MB-231 cells.
- FIG. 10f each plot shows pERK fluorescence in PKH67-labeled sEVs-incorporated cells normalized to the average of pERK in two adjacent PKH67-labeled sEV-non-uptake cells from each image in FIG. 10e. .
- the n number is 18 to 21, and ** indicates p ⁇ 0.01 by Kruskal-Wallis / Dunn multiple comparison test, and *** indicates p ⁇ 0.001.
- FIGS. 10c-f identify 1,241 UBL3-interacting proteins that depend on two C-terminal cysteine residues, including at least 22 disease-associated molecules, and that the oncogenic RasG12V protein is modified by UBL3 modification.
- FIG. 13a shows a representative image of MDA-MB-231 cells transfected with either EGFP-ubiquitin, EGFP-SUMO1, EGFP-SUMO2 or EGFP-UBL3 and co-stained with the MVB marker (CD63).
- * indicates p ⁇ 0.05 by Kruskal-Wallis / Dunn multiple comparison test, and *** indicates p ⁇ 0.0001.
- FIG. 13c shows that only EGFP-UBL3, but not EGFP, EGFP-ubiquitin, EGFP-SUMO1 or EGFP-SUMO2, was selectively enriched in sEV of the cell culture medium (before loading the sample, The sample was boiled in ⁇ ME.).
- UBL3 unlike UBL3, other UBLs such as ubiquitin, SUMO1, and SUMO2 are not abundant in either MVB or sEV.
- FIG. 14 shows the results. Samples were boiled in ⁇ ME before loading. The same amount of protein was loaded on the gel (cell lysate: 20 ⁇ g / lane; sEV: 1 ⁇ g / lane). The lower panel in the figure shows that the gel was stained with SYPRO Ruby after Western blot analysis. As is evident from the results shown in FIG.
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Abstract
La présente invention a pour objet de fournir : un agent permettant d'activer ou d'inhiber la communication cellule-cellule à l'aide d'une protéine fonctionnelle qui peut modifier la communication cellule-cellule ; un anticorps ou un aptamère qui peut être utilisé dans l'agent ; une méthode de criblage de l'agent ; une protéine de fusion capable de modifier un exosome ; un acide nucléique codant pour la protéine de fusion ; un vecteur recombinant comportant l'acide nucléique ; une cellule hôte transformée par le vecteur recombinant ; un modulateur de la fonction de l'exosome comprenant la protéine de fusion ou l'acide nucléique ; et un exosome comprenant la protéine de fusion. L'invention concerne un agent permettant d'activer ou d'inhiber la communication cellule-cellule à l'aide d'une protéine fonctionnelle, l'agent comprenant une substance capable d'augmenter ou de diminuer l'expression du gène UBL3 ou de la protéine ULB3, ou une substance capable de stimuler ou d'inhiber l'activité de la protéine ULB3. L'invention concerne une méthode permettant de cribler l'agent en utilisant en tant qu'indicateur la survenue d'une modification de l'expression du gène ULB3 ou de la protéine ULB3 sous l'effet d'une substance candidate, ou la survenue d'une modification de l'activité de la protéine ULB3 sous l'effet d'une substance candidate. L'invention concerne une protéine de fusion constituée de la protéine ULB3 et d'une protéine fonctionnelle ; un acide nucléique codant pour la protéine de fusion ; un vecteur recombinant comportant l'acide nucléique ; une cellule hôte transformée par le vecteur recombinant ; un modulateur de la fonction de l'exosome comprenant la protéine de fusion ou l'acide nucléique ; et un exosome comprenant la protéine de fusion dans un état qui permette à la protéine fonctionnelle de manifester sa fonction.
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US20110135596A1 (en) * | 2009-12-04 | 2011-06-09 | Samsung Electronics Co., Ltd. | Fusion protein comprising ubiquitin or ubiquitin-like protein, membrane translocation sequence and biologically active molecule and use thereof |
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CN113219180B (zh) * | 2021-01-29 | 2022-05-13 | 厦门大学 | 一种外泌体pd-l1的研究方法 |
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