JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST001964/files/Control_2.rawhttps://storage.jpostdb.org/JPST001964/files/Control_3.rawhttps://storage.jpostdb.org/JPST001964/files/VGLL3_2.rawhttps://storage.jpostdb.org/JPST001964/files/VGLL3_3.rawhttps://storage.jpostdb.org/JPST001964/files/VGLL3_1.rawhttps://storage.jpostdb.org/JPST001964/files/Control_1.rawhttps://storage.jpostdb.org/JPST001964/files/201222_Nakaya_VGLL3_Flag_IP_QEp_Sequest_preD.pdResultprimaryOK200ProteomicsHidetaka KosakoMus Musculus (mouse)https://repository.jpostdb.org/entry/JPST001964Tokushima UniversityjPOSTMyofibroblasts cause tissue fibrosis by producing extracellular matrix proteins, such as collagens. Humoral factors like TGF-β, and matrix stiffness are important for collagen production by myofibroblasts. However, the molecular mechanisms regulating their ability to produce collagen remain poorly characterised. Here, we show that vestigial-like family member 3 (VGLL3) is specifically expressed in myofibroblasts from mouse and human fibrotic hearts and promotes collagen production. Further, substrate stiffness triggers VGLL3 translocation into the nucleus through the integrin β1-Rho-actin pathway. In the nucleus, VGLL3 undergoes liquid-liquid phase separation via its low-complexity domain and is incorporated into non-paraspeckle NONO condensates containing EWS RNA-binding protein 1 (EWSR1). VGLL3 binds EWSR1 and suppresses miR-29b, which targets collagen mRNA. Consistently, cardiac fibrosis after myocardial infarction is significantly attenuated in Vgll3-deficient mice, with increased miR-29b expression. Overall, our results reveal an unrecognised VGLL3-mediated pathway that controls myofibroblasts' collagen production, representing a novel therapeutic target for tissue fibrosis.VGLL3 is a mechanosensitive protein that promotes cardiac fibrosis through liquid-liquid phase separation.Horii Yuma Y, Matsuda Shoichi S, Toyota Chikashi C, Morinaga Takumi T, Nakaya Takeo T, Tsuchiya Soken S, Ohmuraya Masaki M, Hironaka Takanori T, Yoshiki Ryo R, Kasai Kotaro K, Yamauchi Yuto Y, Takizawa Noburo N, Nagasaka Akiomi A, Tanaka Akira A, Kosako Hidetaka H, Nakaya Michio MVGL3, RGD1560030, 1700110N18Rik, 4832416J22, VGL-3, Vgl-3, Vito-2, C80713.DmelCG5178, dlmo, DmelCG7478, Collagens, Kinship, DLMO, Laboratory, House Mouse, DmelCG12051, dLmo, dLMO, Life Cycle, EWSR1, alpha2Int, AAF01186, Non Polyadenylated, Dmrho, A, C, D, Theory, E, Utility Theories, cyt5C, Kinship Network, myocardial infarction (disease), F9K23_11, M, Tissue, Economic Policies, DMRHO, Living Costs, beta Actin, CG3606, act88F, D17Mit170, F9K23.11, DmelCG8416, Bd, DmelCG9623, Dlmo, N-Actin, Collagen Fleece, Household Consumptions, Economic, Act, fliH, G-Actin, Pen19, act87E, Infarct, Indices, Rho-1, Index, Actin/BAP47, AV149256, Polyadenylated, Myocardial Infarcts, Infarcts, alpha Isoactin, Aptitudes, CG1004, act, Tl3, Tl2, mice C57BL/6xCBA/CaJ hybrid, act42A, Economic Condition, Utility Theory, AFFX-Dros-ACTIN_M_r_at, MIR-29B, Myocardial infarction NOS, Cirrhosis, actin, simple tissue, Macroeconomic, RHOHP1, Act57A, Polyadenylated Messenger RNA, heart attack, Non Polyadenylated mRNA, DmelCG4027, bK984G1.4, protein_coding_transcript, Caspase-14 subunit p10, D-Rho1, AI326383, Consumer Price Indices, Swiss Mouse, gamma-Actin, Caspase-14 subunit p19, RHO, Rho, Home, human, Act57b, Non-Polyadenylated mRNA, Condition, act79B, Matrix Proteins, actin5C, myocardial, rhomboid/veinlet, Collagen Felt, ARHD, Mouse, (MI), rho, Infarction, Poly(A) RNA, Act5c, 3.4.22.-, Policies, Utility, Arhd, ACT5C, Kern, mini-ICE, Easterlin, Human, CG30294, 1700110N18Rik, Attack - heart, in, Dermodress, Collastat, Mus musculus domesticus, CG6500, nucleo atomico, Man, If, alpha-Actin, dRhoA, beadex/dLMO, RNA-binding protein 1, Avitene, mus79B, messenger RNA, Microeconomic Factors, Act57, man, alpha Collagen, anon-EST:fe2D2, Abilities, template RNA, CG18290, alphaPS2C, RhoHP1, Capital, Extracellular, PS 2, T10, Heart Attack, dRho1, ATRBP1, T11, Act-5C, DmelCG6500, RGD1560030, 42A, Rho kinase, RNA, MI - Myocardial infarction, cou, Family Research, 4832416J22, Phase Separations, Conditions, l(2)52Fa, Proteins, Messenger RNA, Factor, Hearts, BcDNA:GM09207, MI, AU018891, myocardial infarct, Family Members, Lr, Consumer Price Index, Consumer Price, beta-actin, Poly(A)+ mRNA, M32055, Collagen Hemostat, INSDC_feature:mRNA, Act42, dnRho, C80713, Phase, Cardiovascular, G Actin, Act88-F, AA407051, mRNA, Separations, mmu-mir-29, House Mice, E3.10/J3.8, nucleus of neuraxis, Myocardial, neuraxis nucleus, mus87E, Ews, EWS, Extracellular Matrix, Infarctions, Families, Talent, F Actin, Bra, alpha[[PS2(ms8)]], BAP47, Polyadenylated mRNA, Relatives, Bap47, Myocardial infarction (disorder), Extracellular Matrix Protein, Networks, act 87E, alphaPS2, Strokes, infarction (MI), Vito-2, Rho A, Mus domesticus, beta-Actin, CASP-14, Atomkern, Act5, hdp-a, l(1)G0420, DmelCG1004, act 88F, P54NRB, rho-1, gamma Actin, horsetail nucleus, DRho, Zyderm, Microeconomic, 8416, Family Life Cycle, portion of tissue, rsd, microRNA 29b-1, neuronal nucleus, nucleus atomi, Cardiac infarction, me75, N Actin, Man (Taxonomy), Household Consumption, Vgl-3, Polyadenylated Messenger, PPP1R114, Microfibril Collagen Hemostat, Swiss Mice, rhoA, T1, RHO1, Rho1, Stroke, Isoactin, collagen, act 42A, NRB54, rho1, Ac5C, CG4027, Therapies, Collagen, Theories, house mouse, Family Life Cycles, DmelCG18290, TL, P19, Heart Attacks, VGL-3, Myocardial infarction, Therapy, Ewsh, Home Economics, l(1)G0330, DmRHO-A, anon-Pen19, Hypothesis, PS2, Modern, RHOb, mouse, HSC0013, Myocardial Infarct, p19, Dm Rho1, Macroeconomic Factors, Economics, Ability, rhom, aPS2, CT27194, ACTIN, Household, RhoN19, Cardiovascular Stroke, RhoA, Mini-ICE, ve, tissue portion, cell nucleus, RHOM, RhoM, NOS, CG7478, CG5178, Filiation, CG10067, Ptd, Mus musculus, Factors, mice, l(1)G0117, Ve, CG8416, miR-29, CG9623, Actin, MICE, beta-actin/Bap47, MYOCARDIAL INFARCTION, Treatments, DMRHOa, DMRHOb, domesticus, l(1)G0486, Life Cycles, l(1)G0245, Easterlin Hypothesis, l(1)G0009, P54, Infarction of heart, Heart attack, Lmo, LMO, DmelCG3606, hdp, PSalpha2, Matrix Protein, Remittances, act57b, Dmelrho, l(1)G0010, human being, noyau atomique, Act87e, Family Member, miR29B, DRORHO, alpha-Collagen, Integrin, CG12051, Network, microRNA-29b, anon-EST:CL2c12, Rho GTPase, act57B, l(1)G0025, act57A, Microfibril, Act88f, nonA, miR-29b, Homo sapiens, DRhoA, miR-29c, House, Messenger, dttg, miR-29a, actin-12, Myocardial Infarction, Fibroses, Low, microRNA-29, SARFH, Sarfh, Mice, 143060_f_at, nuclei, act5C, Fibroses., alpha[[PS2]], DmelCG10067, Research, Swiss, nucleus of CNS, nucleo, noyau, F-Actin, pen19, Economic Conditions, DRho1, integrin, DrhoA, OPN2, Act42a, nucleus, Drho1, liquid, Kinship Networks, Family, Collagenfleece, alpha Actin, Policy, l(1)G0177, Economic Policy, Ifm(3)7, myocardial infarction, act 79B, MYOCARDIAL, iks, Production, l(2)k02107b, Living Cost, BEST:RE38067, Mus muscaris, Consumption, Mus, nervous system nucleus, Protein, TFIID, Polyadenylated RNA, NMT55, Pangen, Caz, l(1)G0079, Separation, Poly(A)+ RNA, Myofibroblast, Poly(A) Tail, Myocardial Infarctions, other collagen, act 57A, act 5C, Non-Polyadenylated, Talents, cas, Avicon, Laboratory Mice, alpha-Isoactin, inf, hld, Remittance, Paraspeckle, Therapeutic, Act(88F), Cost of Living, Modern Man, VGL3, PS2alpha, Treatment, INFARCTION (MI), n(2)k07236, Cardiovascular Strokes, Laboratory Mouse, Actin5CRGD1560030, Separation, Phase, Phase Separations., 4832416J22, protein complex, Vgl-3, Vito-2, Separations, Proteins, Gene, proteins, protein, protein-containing complex, Protein Gene Products, Gene Proteins, 1700110N18Rik, protein polypeptide chains, native protein, natural protein, polypeptide chain, Protein, VGL3, Gene Products, Cirrhosis, Fibroses, liquid, protein aggregate, VGL-3, C80713PLIP, MeCN, Raw, Laboratory, Phosphatase, hypersomnia with periodic respiration, SDH, Deoxycholic Acid, House Mouse, Type 2A-interacting protein, prevention, Long Term, 5730420M11Rik, Trifluoroacetate, SDS, Polypeptides, protein polypeptide chains, Magnesium chloride, dorsal marginal zone, Method, Protease Inhibitor, CH3-C#N, B1, 3, mixed central and obstructive sleep apnea, 2-amino-3-mercaptopropanoic acid, WMS, HTATIP, nup154, Disodium Salt, SET, C, Divorced, C79325, M, sleep, Salt, hypopnea, proteins, Divorces, T6G21.3, Phosphomonoesterases, AI047805, DmelCG4299, isolation and purification, salt, set, Peptidohydrolase Inhibitor, cPLA2, column, ACN, Acn, D1, sample, 3beta-Isomer, Half Cystine, phosphatase, TIP, Magnesium (2:1) Salt, SGS, LC-MS2, preventive therapy, Stars, Zinc Cysteinate, acetylation, e(Pc), Longterm Effect, mixed central and obstructive, mixed sleep apnea, Procedure, tip, ANAC091, mice C57BL/6xCBA/CaJ hybrid, ACMICD, MgCl2, Putative MAPK-activating protein PM10, common salt, Dihydroxycholanoic Acid, Dm1, Hydrolase Inhibitor, Methionin, Hmet, TIP60, (2R)-2-amino-3-sulfanylpropanoic acid, 5beta, Acid, DmelCG7776, HLA-DR-associated protein II, Antagonists, DI-2, I-2Dm, Swiss Mouse, Shwachman-Diamond type metaphyseal dysplasia, Shwachman syndrome, LNKN-1, Methodological, sodium hydrate, beta Trypsin, apnea syndromes, I-2PP1, 12beta-Isomer Deoxycholic Acid, Sodium Salt, Phosphohydrolase, breathing, 5alpha-Isomer, TAF-IBETA, antagonists and inhibitors, liquid chromatography-tandem mass spectroscopy, label, DmelLcp3, Phosphoric, BG:DS02740.11, DMZ, TAF-Ibeta, Mouse, Endopeptidase, Peptide Peptidohydrolase Inhibitor, methyl cyanide, tlp, STARS, TIP41, Liquimeth, sleep-disordered, 5alpha Isomer, F10B6_15, GH05739, 2-amino-4-(methylsulfanyl)butanoic acid, Effects, Endopeptidase Inhibitors, Biocatalysts, mouse <Mus musculus>, L-Zystein, precursor, thomson, protein-containing complex, 17alpha)-isomer, sleep apnea, F10B6.15, CG7826, ESA1, mass-to-charge ratio, sleep hypopneas, 1700110N18Rik, GH14582, 12-dihydroxy-, isolation, LC-MSMS, CG7835, Gene Products, CG42273, Mus musculus domesticus, Peptide Hydrolase, Half-Cystine, 12alpha)-, Technique, DmelCG2043, Cesium Trifluoroacetate, 12beta Isomer, lcp3, Acinus, Longterm, nodus primitivus, L Cysteine, E-920, Monosodium Salt, beta-Trypsin, LCP-3, 3beta Isomer, 4733401P19Rik, 2pp2a, Sciences, Hcys, Long-Term, Antagonist, CG10574, Phosphoric Monoester Hydrolase, acinusL, Study, Injectable, DmelCG4579, ms(2)zk, Enzyme, 2PP2A, CP3, 2-amino-4-(methylthio)butanoic acid, Protease Antagonists, acinusS, CYSTEINE, congenital lipomatosis of pancreas, Kochsalz, dSET, dSet, Kybella, Peptide Peptidohydrolase Inhibitors, FREE CYSTEINE, peptidos, Choleic Acid, D8Wsu49e, (2R)-2-amino-3-mercaptopropanoic acid, metionina, 4-(2-hydroxyethyl)-, PTHB1, inhibitors, RGD1560030, Magnesiumchlorid, LcpIII, 1-(14)C-labeled, 4832416J22, 5alpha-Isomer Deoxycholic Acid, BcDNA:LD21772, Proteins, Mischung, backward, acetonitrile, L-Isomer, buffer, mKIAA0670, Engine, native protein, Natriumchlorid, I-2PP2A, C18, chemical analysis, Long Term Effects, Dm I-2, 3beta-Isomer Deoxycholic Acid, 5alpha, MFS1, Sodium, soda lye, NaCl, LCP3, Magnesium chloride anhydrous, Met, C80713, Achromobacter protease I, AU020952, Col4a-1, DMLCP3, underdeveloped, mixed, ensemble, l(2)28-28-12, prophylaxis, Separations, Striated muscle activator of Rho-dependent signaling, CG4579, Chloride, Magnetic, SWDS, Monopotassium Salt, Shwachman-Bodian-Diamond syndrome, House Mice, (R)-2-amino-3-mercaptopropanoic acid, ethanenitrile, Peptidohydrolase Inhibitors, CDA08, 2-Amino-3-mercaptopropionic acid, Longterm Effects, ME-IV, achromopeptidase, plan specification, DmelCG5861, Gene Proteins, Peptidase, Magnesium chloride (MgCl2), Antiprotease, control, Pancreatic insufficiency and bone marrow dysfunction, Aetznatron, liquid chromatography tandem mass spectrometry, E920, Peptide Hydrolase Inhibitors, CG2043, reversed, liquid chromatography tandem mass spectroscopy, Nup32D, IPP2A2, Bru, Endopeptidase Inhibitor, E 920, NCMe, determination, Vito-2, Mus domesticus, Peptide Hydrolase Inhibitor, HEPES Monosodium Salt, protein, 12beta-Isomer, Phosphatases, Hydrolase, Techniques, L-Isomer Methionine, halite, Methionine, peptido, BANF, congenital, dJ69E11.3, sleep apneas, Inhibitors, Min, Cystein, Hydrolase Inhibitors, protein aggregate, prevention and control, Injectables, Effect, DmelCG42273, Svc, HEPES Monosodium, 3-hydroxy-5-estrane-17-carbonitrile, LCMSMS, reference sample, peptides, TAF-I, cisteina, Vgl-3, 2610036I19Rik, 2610510L13Rik, hypoplasia, min, mAPC, achromobacter beta-lytic protease (blp), Swiss Mice, purification, free, lipomatosis of pancreas, preventive measures, cloruro sodico, IGAAD, fSAP152, Methodological Studies, 1-Piperazineethanesulfonic acid, SOLO DANCERS, DmelCG10574, ppm, trifluoro-, Shwachman-Bodian syndrome, CG7776, hypopneas, house mouse, Natriumhydroxid, hydroxyde de sodium, GPHYSD2, Racemethionine, Long-Term Effects, VGL-3, phapii, rock salt, Peptidase Inhibitor, ZC2HC5, [MgCl2], LC-MS/MS, mouse, 2-Amino-4-(methylthio)butyric acid, Dmel_CG7826, AI836084, StF-IT-1, Th, Search, HTATIP1, Peptide Peptidohydrolase, L[[3]]CP3, Cys, lysyl endoprotease, Henson's node, sleep hypopnea, fixed, Dmel_CG7835, Acetic acid, Mnb, MNB, parent ion, Mus musculus, NaOH, MS/MS, Trifluoroacetic, L-Cysteine, MASS, CG4299, AW124434, natrii chloridum, Methodological Study, L-Cystein, domesticus, nanospray, Peptidase Inhibitors, precursor ion, Cesium, E(PC), anon-48Ac, Antiproteases, Biocatalyst, Polypeptide, mixed sleep Apneas, HEPES, Hydrolases, Inhibitor, i2pp2a, Cholan-24-oic acid, apnea syndrome, Cysteine Hydrochloride, Procedures, Lagodeoxycholic Acid, L Isomer, FBN, Gene, Protease, ACINUS, Monoester Hydrolase, PHAPII, LC-MS-MS, Buffer, method, III, reduced, polypeptide chain, Ms1, House, ECTOL1, Injection, Arabidopsis NAC domain containing protein 91, method used in an experiment, Studies, Sodium Deoxycholate, zk, L-Methionine, Protease Antagonist, Del(8)44H, tiny, Separated, Mice, apneas, Pedameth, Pro-Mega, Search Engines, l(2)10432, MS1, Swiss, MS2, ipp2a2, chlorure de sodium, inhibiteur, Acetylations, soude caustique, Desoxycholic Acid, OCTD, lysate, inhibidor, 10^[-6], taf-ibeta, stem node, male sterility 1, apnea, N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, liquid, Long-Term Effect, l(2)01501, Controlled, small, Monoammonium Salt, Controlling, DYRK1, 5E4, data, 3H-labeled, DL-Methionine, protein complex, igaad, inhibitor, Deoxycholate, Magnesium, Peptide, group, Mus muscaris, Phosphomonoesterase, LC/MS/MS, natural protein, Mus, Protein, caustic soda, I2PP2A, table salt, Dyrk1, (3alpha, ACETONITRILE, tandem MS, Zystein, cyanomethane, WMS2, Data Base, antagonists, DDA, Phosphoric Monoester, Separation, nup32D, Myofibroblast, PRSS, nodal stem, MALE STERILITY 1 PROTEIN, 2-amino-3-sulfanylpropanoic acid, CC1, Tripcellim, N 2 Hydroxyethylpiperazine N' 2' ethanesulfonic Acid, anon-35Fa, L-2-Amino-3-mercaptopropionic acid, phosphoric monoester hydrolase activity, CGI-97, sample population, Laboratory Mice, Phosphohydrolases, Protein Gene Products, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, anon-35Fc, SSKS, VGL3, TCV-interacting protein, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, assay, variable, SCH, AA407739, Laboratory Mouse, Peptide., 3-OHECNfalseNakaya_VGLL3-FLAG_IP-MS_QEplusCardiac myofibroblasts retrovirally transduced with FLAG-VGLL3 or control vector were fixed with 0.1% PFA and lysed in a HEPES-RIPA buffer (20 mM HEPES-NaOH [pH 7.5]/1 mM EGTA/1 mM MgCl2/150 mM NaCl/0.25% sodium deoxycholate/0.05% SDS/1% NP-40) containing protease inhibitors (1:100), phosphatase inhibitors (1:100), and 28 U/µL of Benzonase (1:500, Novagen) at 4 °C for 20 min. The supernatant of the cell lysate was incubated overnight at 4 °C with anti-FLAG magnetic beads. The beads were washed three times with HEPES-RIPA buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested with 200 ng of Trypsin/Lysyl Endopeptidase mixture (Promega), and the digested peptides were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 ultra-high-performance liquid chromatograph connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75 µm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4–32% ACN gradient for 0–150 min followed by an increase to 80% ACN for 20 min and finally hold at 80% ACN for 10 min. The mass spectrometer was operated in a data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured at a resolution of 70,000, an automatic gain control (AGC) target of 1e6, and a mass range from 350 to 1,500 m/z. MS/MS spectra were triggered at a resolution of 17,500, an AGC target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalised collision energy of 27. Dynamic exclusion was set to 15 s. Raw data were directly analysed against the SwissProt database, restricted to Mus musculus, using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) with the Sequest_HT search engine for identification and label-free precursor ion quantification. The search parameters were as follows: (i) trypsin as an enzyme with up to two missed cleavages; (ii) precursor mass tolerance of 10 ppm; (iii) fragment mass tolerance of 0.02 Da; (iv) carbamidomethylation of cysteine as a fixed modification; and (v) acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false-discovery rate (FDR) of 1% using the percolator node and the protein FDR validator node, respectively. Normalisation was performed such that the total sum of abundance values for each sample across all peptides was the same.Sat Dec 24 00:00:00 GMT 2022PXD0390911009036754961