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1.
Biol Open ; 13(2)2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38299702

RESUMO

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer's disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins - vimentin, α-internexin, neurofilament polypeptides - were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods.


Assuntos
Actinas , Proteínas Associadas aos Microtúbulos , Camundongos , Animais , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Actinas/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Tubulina (Proteína)/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Mamíferos/metabolismo
2.
Cells ; 13(4)2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38391954

RESUMO

The cell membrane is frequently subjected to damage, either through physical or chemical means. The swift restoration of the cell membrane's integrity is crucial to prevent the leakage of intracellular materials and the uncontrolled influx of extracellular ions. Consequently, wound repair plays a vital role in cell survival, akin to the importance of DNA repair. The mechanisms involved in wound repair encompass a series of events, including ion influx, membrane patch formation, endocytosis, exocytosis, recruitment of the actin cytoskeleton, and the elimination of damaged membrane sections. Despite the absence of a universally accepted general model, diverse molecular models have been proposed for wound repair in different organisms. Traditional wound methods not only damage the cell membrane but also impact intracellular structures, including the underlying cortical actin networks, microtubules, and organelles. In contrast, the more recent improved laserporation selectively targets the cell membrane. Studies on Dictyostelium cells utilizing this method have introduced a novel perspective on the wound repair mechanism. This review commences by detailing methods for inducing wounds and subsequently reviews recent developments in the field.


Assuntos
Dictyostelium , Dictyostelium/metabolismo , Membrana Celular/metabolismo , Actinas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo
3.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 55(1): 60-66, 2024 Jan 20.
Artigo em Chinês | MEDLINE | ID: mdl-38322534

RESUMO

Objective: To explore the relationship between the expression of plectin and the migration of hepatocellular carcinoma (HCC) cells and to elucidate the molecular mechanisms by which plectin expression affects the migration of HCC cells. Methods: First of all, Western blot was performed to determine the expression of plectin in normal hepatocytes and HCC cells. Secondly, a plectin-downregulated HCC cell strain was established and the control group (shNC group) and shPLEC group were set up. Each group was divided into a vehicle control group (shNC+DMSO group or shPLEC+DMSO group) and a F-actin cytoskeleton polymerization inducer Jasplakinolide group (shNC+Jasp group or shPLEC+Jasp group). Western blot was performed to determine the expression of plectin and epithelial-mesenchymal transition (EMT)-related proteins, including N-cadherin, vimentin, and E-cadherin. HCC cell migration was evaluated by Transwell assay. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to analyze the signaling pathways related to plectin gene. The polymerization of F-actin was analyzed by immunofluorescence assay. Results: Compared with the normal hepatocytes, HCC cells showed high expression of plectin. Compared with those in the shNC group, the expression of plectin in the shPLEC group was decreased (P<0.05), the migration ability of HCC cells was weakened (P<0.05), and the EMT process was inhibited (with the expression of N-cadherin and vimentin being decreased and the expression of E-cadherin being increased) (P<0.05). KEGG analysis showed that the regulation of cytoskeletal F-actin was most closely associated with plectin and cytoskeletal F-actin depolymerized in the shPLEC group. After treatment with Jasplakinolide, an inducer of F-actin cytoskeleton polymerization, the migration ability of HCC cells in the shPLEC+Jasp group was enhanced compared with that of shPLEC+DMSO group (P<0.05) and the EMT process was restored (with the expression of N-cadherin and vimentin being increased and the expression of E-cadherin being decreased) (P<0.05). In addition, the polymerization of cytoskeletal F-actin in HCC cells was also restored. Conclusion: Plectin is highly expressed in HCC cells. Plectin promotes the migration and the EMT of HCC cells through inducing F-actin polymerization.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Actinas/metabolismo , Vimentina/metabolismo , Linhagem Celular Tumoral , Plectina/genética , Plectina/metabolismo , Dimetil Sulfóxido , Polimerização , Movimento Celular , Caderinas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica
5.
Nat Commun ; 15(1): 1007, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38307855

RESUMO

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. PhLP2A binds to open apo-TRiC through polyvalent domain-specific contacts with its chamber's equatorial and apical regions. PhLP2A N-terminal H3-domain binding to subunits CCT3/4 apical domains displace PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to positively charged inner surface residues from CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.


Assuntos
Actinas , Proteínas do Olho , Reguladores de Proteínas de Ligação ao GTP , Fosfoproteínas , Dobramento de Proteína , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Chaperoninas/metabolismo , Trifosfato de Adenosina/metabolismo , Chaperonina com TCP-1/metabolismo
6.
Sci Adv ; 10(6): eadi7840, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38324689

RESUMO

Prolonged obstruction of the ureter, which leads to injury of the kidney collecting ducts, results in permanent structural damage, while early reversal allows for repair. Cell structure is defined by the actin cytoskeleton, which is dynamically organized by small Rho guanosine triphosphatases (GTPases). In this study, we identified the Rho GTPase, Rac1, as a driver of postobstructive kidney collecting duct repair. After the relief of ureteric obstruction, Rac1 promoted actin cytoskeletal reconstitution, which was required to maintain normal mitotic morphology allowing for successful cell division. Mechanistically, Rac1 restricted excessive actomyosin activity that stabilized the negative mitotic entry kinase Wee1. This mechanism ensured mechanical G2-M checkpoint stability and prevented premature mitotic entry. The repair defects following injury could be rescued by direct myosin inhibition. Thus, Rac1-dependent control of the actin cytoskeleton integrates with the cell cycle to mediate kidney tubular repair by preventing dysmorphic cells from entering cell division.


Assuntos
Túbulos Renais Coletores , Túbulos Renais Coletores/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Citoesqueleto/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo
7.
Cell Mol Life Sci ; 81(1): 84, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38345631

RESUMO

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.


Assuntos
Actinas , Plaquetas , Retração do Coágulo , Fator 2 de Liberação do Nucleotídeo Guanina , Animais , Actinas/metabolismo , Plaquetas/metabolismo , Exocitose/fisiologia , Hemostasia , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo
8.
Zhonghua Xin Xue Guan Bing Za Zhi ; 52(2): 185-190, 2024 Feb 24.
Artigo em Chinês | MEDLINE | ID: mdl-38326071

RESUMO

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.


Assuntos
Hiperóxia , Hipertensão Pulmonar , Ratos , Animais , Artéria Pulmonar/patologia , Metaloproteinase 2 da Matriz/genética , Hiperóxia/metabolismo , Hiperóxia/patologia , Actinas/genética , Actinas/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxigênio/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Cultivadas
9.
J Ovarian Res ; 17(1): 37, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38336796

RESUMO

BACKGROUND: Premature ovarian insufficiency (POI) is a condition defined as women developing menopause before 40 years old. These patients display low ovarian reserve at young age and difficulties to conceive even with assisted reproductive technology. The pathogenesis of ovarian insufficiency is not fully understood. Genetic factors may underlie most of the cases. Actin cytoskeleton plays a pivotal role in ovarian folliculogenesis. Calponin 2 encoded by the Cnn2 gene is an actin associated protein that regulates motility and mechanical signaling related cellular functions. RESULTS: The present study compared breeding of age-matched calponin 2 knockout (Cnn2-KO) and wild type (WT) mice and found that Cnn2-KO mothers had significantly smaller litter sizes. Ovaries from 4 weeks old Cnn2-KO mice showed significantly lower numbers of total ovarian follicles than WT control with the presence of multi-oocyte follicles. Cnn2-KO mice also showed age-progressive earlier depletion of ovarian follicles. Cnn2 expression is detected in the cumulus cells of the ovarian follicles of WT mice and colocalizes with actin stress fiber, tropomyosin and myosin II in primary cultures of cumulus cells. CONCLUSIONS: The findings demonstrate that the loss of calponin 2 impairs ovarian folliculogenesis with premature depletion of ovarian follicles. The role of calponin 2 in ovarian granulosa cells suggests a molecular target for further investigations on the pathogenesis of POI and for therapeutic development.


Assuntos
Insuficiência Ovariana Primária , Adulto , Animais , Feminino , Humanos , Camundongos , Actinas/genética , Actinas/metabolismo , /metabolismo , Menopausa Precoce , Camundongos Knockout , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/metabolismo
10.
Cell Commun Signal ; 22(1): 111, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38347641

RESUMO

Macroautophagy/autophagy is a lysosome-dependent catabolic process induced by various cellular stress conditions, maintaining the homeostasis of cells, tissues and organs. Autophagy is a series of membrane-related events involving multiple autophagy-related (ATG) proteins. Most studies to date have focused on various signaling pathways affecting ATG proteins to control autophagy. However, mounting evidence reveals that the actin cytoskeleton acts on autophagy-associated membranes to regulate different events of autophagy. The actin cytoskeleton assists in vesicle formation and provides the mechanical forces for cellular activities that involve membrane deformation. Although the interaction between the actin cytoskeleton and membrane makes the role of actin in autophagy recognized, how the actin cytoskeleton is recruited and assembles on membranes during autophagy needs to be detailed. Nucleation-promoting factors (NPFs) activate the Arp2/3 complex to produce actin cytoskeleton. In this review, we summarize the important roles of the actin cytoskeleton in autophagy regulation and focus on the effect of NPFs on actin cytoskeleton assembly during autophagy, providing new insights into the occurrence and regulatory mechanisms of autophagy. Video Abstract.


Assuntos
Citoesqueleto de Actina , Actinas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Autofagia/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo
11.
Nat Commun ; 15(1): 1187, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38331992

RESUMO

Myosin VI is the only molecular motor that moves towards the minus end along actin filaments. Numerous cellular processes require myosin VI and tight regulations of the motor's activity. Defects in myosin VI activity are known to cause genetic diseases such as deafness and cardiomyopathy. However, the molecular mechanisms underlying the activity regulation of myosin VI remain elusive. Here, we determined the high-resolution cryo-electron microscopic structure of myosin VI in its autoinhibited state. Our structure reveals that autoinhibited myosin VI adopts a compact, monomeric conformation via extensive interactions between the head and tail domains, orchestrated by an elongated single-α-helix region resembling a "spine". This autoinhibited structure effectively blocks cargo binding sites and represses the motor's ATPase activity. Certain cargo adaptors such as GIPC can release multiple inhibitory interactions and promote motor activity, pointing to a cargo-mediated activation of the processive motor. Moreover, our structural findings allow rationalization of disease-associated mutations in myosin VI. Beyond the activity regulation mechanisms of myosin VI, our study also sheds lights on how activities of other myosin motors such as myosin VII and X might be regulated.


Assuntos
Cadeias Pesadas de Miosina , Miosinas , Microscopia Crioeletrônica , Cadeias Pesadas de Miosina/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo
12.
Proc Natl Acad Sci U S A ; 121(9): e2316722121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38377188

RESUMO

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.


Assuntos
Actinas , Junções Aderentes , Humanos , Junções Aderentes/metabolismo , Actinas/metabolismo , Células CACO-2 , Caderinas/genética , Caderinas/metabolismo , Junções Intercelulares/metabolismo , Junções Íntimas/metabolismo , Cateninas/metabolismo , Células Epiteliais/metabolismo
13.
Proc Natl Acad Sci U S A ; 121(9): e2315472121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38377203

RESUMO

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in ß-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human ß, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in ß myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in ß myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not ß, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.


Assuntos
Miosinas , Miosinas Ventriculares , Humanos , Miosinas Ventriculares/genética , Miosinas/metabolismo , Adenosina Trifosfatases/metabolismo , Mutação , Actinas/metabolismo , Músculo Esquelético/metabolismo
14.
Nucleus ; 15(1): 2320656, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38384139

RESUMO

The view on nuclear filaments formed by non-skeletal ß-actin has significantly changed over the decades. Initially, filamentous actin was observed in amphibian oocyte nuclei and only under specific cell stress conditions in mammalian cell nuclei. Improved labeling and imaging technologies have permitted insights into a transient but microscopically apparent filament network that is relevant for chromatin organization, biomechanics of the mammalian cell nucleus, gene expression, and DNA damage repair. Here, we will provide a historical perspective on the developing insight into nuclear actin filaments.


Assuntos
Citoesqueleto de Actina , Núcleo Celular , Animais , Núcleo Celular/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Oócitos/metabolismo , Mamíferos/metabolismo
15.
Nat Commun ; 15(1): 1311, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38346988

RESUMO

Actin mediates insulin secretion in pancreatic ß-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a "netlike" to a "blooming" architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.


Assuntos
Actinas , Células Secretoras de Insulina , Secreção de Insulina , Actinas/metabolismo , Glucose/metabolismo , Tomografia com Microscopia Eletrônica , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Citoesqueleto de Actina/metabolismo
16.
Nat Commun ; 15(1): 1426, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365893

RESUMO

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.


Assuntos
Fatores de Despolimerização de Actina , Actinas , Humanos , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Quinases Lim/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/metabolismo , Fosforilação , Cofilina 1/genética , Cofilina 1/metabolismo
17.
Int J Mol Sci ; 25(3)2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38338749

RESUMO

Mitral valve prolapse (MVP) is a common valvular disease, affecting 2-3% of the adult human population and is a degenerative condition. A total of 5-10% of the afflicted will develop severe mitral regurgitation, cardiac dysfunction, congestive heart failure, and sudden cardiac death. Naturally occurring myxomatous MVP in dogs closely resembles MVP in humans structurally, and functional consequences are similar. In both species, valvular interstitial cells (VICs) in affected valves exhibit phenotype consistent with activated myofibroblasts with increased alpha-smooth muscle actin (αSMA) expression. Using VICs collected from normal and MVP-affected valves of dogs, we analyzed the miRNA expression profile of the cells and their associated small extracellular vesicles (sEV) using RNA sequencing to understand the role of non-coding RNAs and sEV in MVP pathogenesis. miR-145 was shown to be upregulated in both the affected VICs and sEV, and overexpression of miR-145 by mimic transfection in quiescent VIC recapitulates the activated myofibroblastic phenotype. Concurrently, KLF4 expression was noted to be suppressed by miR-145, confirming the miR-145-KLF4-αSMA axis. Targeting this axis may serve as a potential therapy in controlling pathologic abnormalities found in MVP valves.


Assuntos
Estenose da Valva Aórtica , Fator 4 Semelhante a Kruppel , MicroRNAs , Prolapso da Valva Mitral , Adulto , Animais , Cães , Humanos , Valva Aórtica/patologia , Células Cultivadas , MicroRNAs/genética , Prolapso da Valva Mitral/metabolismo , Prolapso da Valva Mitral/patologia , Actinas/metabolismo , Fator 4 Semelhante a Kruppel/metabolismo
18.
Eur J Pharmacol ; 967: 176369, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38325796

RESUMO

Hypertrophic scars are a common complication of burn injuries, yet there are no medications to prevent their formation. During scar formation, resident fibroblasts are transformed to myofibroblasts which become resistant to apoptosis. Previously, we have shown that hydroxypyridone anti-fungals can inhibit transformation of fibroblasts, isolated from hypertrophic scars, to myofibroblasts. This study aimed to investigate if these drugs can also target myofibroblast persistence. Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor beta-1 (TGF-ß1) for 72 h to induce myofibroblast transformation. The cells were then incubated with three hydroxypyridone anti-fungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine; 0.03-300 µM) for a further 72 h. The In-Cell ELISA method was utilised to quantify myofibroblast transformation by measuring alpha-smooth muscle actin (α-SMA) expression and DRAQ5 staining, to measure cell viability. TUNEL staining was utilised to assess if the drugs could induce apoptosis. When given to established myofibroblasts, the three hydroxypyridones did not reverse myofibroblast transformation, but instead elicited a concentration-dependent decrease in cell viability. TUNEL staining confirmed that the hydroxypyridone anti-fungals induced apoptosis in established myofibroblasts. This is the first study to show that hydroxypyridone anti-fungals are capable of inducing apoptosis in established myofibroblasts. Together with our previous results, we suggest that hydroxypyridone anti-fungals can prevent scar formation by preventing the formation of new myofibroblasts and by reducing the number of existing myofibroblasts.


Assuntos
Cicatriz Hipertrófica , Miofibroblastos , Humanos , Miofibroblastos/patologia , Cicatriz Hipertrófica/metabolismo , Ciclopirox/metabolismo , Ciclopirox/uso terapêutico , Fibroblastos/metabolismo , Apoptose , Fator de Crescimento Transformador beta1/metabolismo , Actinas/metabolismo , Células Cultivadas , Diferenciação Celular
19.
Bull Exp Biol Med ; 176(3): 324-327, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38336971

RESUMO

Cardiac myosin binding protein-C (cMyBP-C) located in the C-zone of myocyte sarcomere is involved in the regulation of myocardial contraction. Its N-terminal domains C0, C1, C2, and the m-motif between C1 and C2 can bind to the myosin head and actin of the thin filament and affect the characteristics of their interaction. Measurements using an optical trap showed that the C0-C2 fragment of cMyBP-C increases the interaction time of cardiac myosin with the actin filament, while in an in vitro motility assay, it dose-dependently reduces the sliding velocity of actin filaments. Thus, it was found that the N-terminal part of cMyBP-C affects the kinetics of the myosin cross-bridge.


Assuntos
Actinas , Proteínas de Transporte , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Miosinas Cardíacas/metabolismo , Ligação Proteica/fisiologia , Miocárdio/metabolismo
20.
Biomolecules ; 14(2)2024 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-38397451

RESUMO

The protein 4.1R is an essential component of the erythrocyte membrane skeleton, serving as a key structural element and contributing to the regulation of the membrane's physical properties, including mechanical stability and deformability, through its interaction with spectrin-actin. Recent research has uncovered additional roles of 4.1R beyond its function as a linker between the plasma membrane and the membrane skeleton. It has been found to play a crucial role in various biological processes, such as cell fate determination, cell cycle regulation, cell proliferation, and cell motility. Additionally, 4.1R has been implicated in cancer, with numerous studies demonstrating its potential as a diagnostic and prognostic biomarker for tumors. In this review, we provide an updated overview of the gene and protein structure of 4.1R, as well as its cellular functions in both physiological and pathological contexts.


Assuntos
Proteínas do Citoesqueleto , Proteínas de Membrana , Proteínas de Membrana/metabolismo , Proteínas do Citoesqueleto/metabolismo , Espectrina/química , Espectrina/genética , Espectrina/metabolismo , Actinas/metabolismo , Membrana Eritrocítica/metabolismo
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