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European Heart Journal Jan 2023Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of...
BACKGROUND AND AIMS
Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of disturbed flow on the regulation of vascular endothelial phosphoproteins and their contribution to therapeutic application in atherogenesis were elucidated.
METHODS
Porcine models, large-scale phosphoproteomics, transgenic mice, and clinical specimens were used to discover novel site-specific phosphorylation alterations induced by disturbed flow in endothelial cells (ECs).
RESULTS
A large-scale phosphoproteomics analysis of native endothelium from disturbed (athero-susceptible) vs. pulsatile flow (athero-resistant) regions of porcine aortas led to the identification of a novel atherosclerosis-related phosphoprotein vinculin (VCL) with disturbed flow-induced phosphorylation at serine 721 (VCLS721p). The induction of VCLS721p was mediated by G-protein-coupled receptor kinase 2 (GRK2)S29p and resulted in an inactive form of VCL with a closed conformation, leading to the VE-cadherin/catenin complex disruption to enhance endothelial permeability and atherogenesis. The generation of novel apolipoprotein E-deficient (ApoE-/-) mice overexpressing S721-non-phosphorylatable VCL mutant in ECs confirmed the critical role of VCLS721p in promoting atherosclerosis. The administration of a GRK2 inhibitor to ApoE-/- mice suppressed plaque formation by inhibiting endothelial VCLS721p. Studies on clinical specimens from patients with coronary artery disease (CAD) revealed that endothelial VCLS721p is a critical clinicopathological biomarker for atherosclerosis progression and that serum VCLS721p level is a promising biomarker for CAD diagnosis.
CONCLUSIONS
The findings of this study indicate that endothelial VCLS721p is a valuable hemodynamic-based target for clinical assessment and treatment of vascular disorders resulting from atherosclerosis.
Topics: Animals; Mice; Atherosclerosis; Endothelial Cells; Endothelium, Vascular; Mice, Knockout, ApoE; Phosphorylation; Swine; Vinculin; Humans
PubMed: 36380599
DOI: 10.1093/eurheartj/ehac647 -
Biochemistry Nov 2019Life is an emergent property of transient interactions between biomolecules and other organic and inorganic molecules that somehow leads to harmony and order.... (Review)
Review
Life is an emergent property of transient interactions between biomolecules and other organic and inorganic molecules that somehow leads to harmony and order. Measurement and quantitation of these biological interactions are of value to scientists and are major goals of biochemistry, as affinities provide insight into biological processes. In an organism, these interactions occur in the context of forces and the need for a consideration of binding affinities in the context of a changing mechanical landscape necessitates a new way to consider the biochemistry of protein-protein interactions. In the past few decades, the field of mechanobiology has exploded, as both the appreciation of, and the technical advances required to facilitate the study of, how forces impact biological processes have become evident. The aim of this review is to introduce the concept of force dependence of biomolecular interactions and the requirement to be able to measure force-dependent binding constants. The focus of this discussion will be on the mechanotransduction that occurs at the integrin-mediated adhesions with the extracellular matrix and the major mechanosensors talin and vinculin. However, the approaches that the cell uses to sense and respond to forces can be applied to other systems, and this therefore provides a general discussion of the force dependence of biomolecule interactions.
Topics: Animals; Biophysical Phenomena; Cell Adhesion; Extracellular Matrix; Focal Adhesions; Humans; Integrins; Mechanotransduction, Cellular; Protein Binding; Talin; Vinculin
PubMed: 31315399
DOI: 10.1021/acs.biochem.9b00453 -
Nature Communications Jul 2023The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear....
The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.
Topics: Actins; Talin; Vinculin; Allosteric Regulation; Focal Adhesions; Protein Binding
PubMed: 37463895
DOI: 10.1038/s41467-023-39646-4 -
The landscape of genetic variation in dilated cardiomyopathy as surveyed by clinical DNA sequencing.Genetics in Medicine : Official Journal... Aug 2014Dilated cardiomyopathy is characterized by substantial locus, allelic, and clinical heterogeneity that necessitates testing of many genes across clinically overlapping...
PURPOSE
Dilated cardiomyopathy is characterized by substantial locus, allelic, and clinical heterogeneity that necessitates testing of many genes across clinically overlapping diseases. Few studies have sequenced sufficient individuals; thus, the contributions of individual genes and the pathogenic variant spectrum are still poorly defined. We analyzed 766 dilated cardiomyopathy patients tested over 5 years in our molecular diagnostics laboratory.
METHODS
Patients were tested using gene panels of increasing size from 5 to 46 genes, including 121 cases tested with a multiple-cardiomyopathy next-generation panel covering 46 genes. All variants were reassessed using our current clinical-grade scoring system to eliminate false-positive disease associations that afflict many older analyses.
RESULTS
Up to 37% of dilated cardiomyopathy cases carry a clinically relevant variant in one of 20 genes, titin (TTN) being the largest contributor (up to 14%). Desmoplakin (DSP), an arrhythmogenic right ventricular cardiomyopathy gene, contributed 2.4%, illustrating the utility of multidisease testing. The clinical sensitivity increased from 10 to 37% as gene panel sizes increased. However, the number of inconclusive cases also increased from 4.6 to 51%.
CONCLUSION
Our data illustrate the utility of broad gene panels for genetically and clinically heterogeneous diseases but also highlight challenges as molecular diagnostics moves toward genome-wide testing.
Topics: Cardiomyopathy, Dilated; Carrier Proteins; Connectin; Desmoplakins; Female; Genetic Predisposition to Disease; Genetic Variation; Humans; Male; Sequence Analysis, DNA; Vinculin
PubMed: 24503780
DOI: 10.1038/gim.2013.204 -
Journal of the College of Physicians... Jan 2017Myofibroblast-mediated contraction is viewed as a cycle of four steps. The first step is stimulation of myofibroblasts by lysophospholipids leading to the activation of... (Review)
Review
Myofibroblast-mediated contraction is viewed as a cycle of four steps. The first step is stimulation of myofibroblasts by lysophospholipids leading to the activation of G proteins and ending with contraction of the actin-myosin complex. The next step is the transmission of the intracellular contractile force at the focal adhesions of myofibroblasts; a step that involves talin, vinculin, paxillin, Hic-5, and the integrin receptors. In the third step, fibronectin will act as the extracellular link between the integrin receptors and the extracellular collagen. Finally, "sensing" tension and the maintenance of myofibroblast activity represent the fourth step. The clinical relevance of each step is then discussed in the form of modalities to prevent excessive scarring/fibrosis.
Topics: Animals; Fibroblasts; Fibronectins; Focal Adhesions; Humans; Muscle Contraction; Muscle, Skeletal; Muscle, Smooth; Myofibroblasts; Vinculin; Wound Healing
PubMed: 28292367
DOI: No ID Found -
Biochemistry May 2015Vinculin localizes to cellular adhesions where it regulates motility, migration, development, wound healing, and response to force. Importantly, vinculin loss results in...
Vinculin localizes to cellular adhesions where it regulates motility, migration, development, wound healing, and response to force. Importantly, vinculin loss results in cancer phenotypes, cardiovascular disease, and embryonic lethality. At the plasma cell membrane, the most abundant phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), binds the vinculin tail domain, Vt, and triggers homotypic and heterotypic interactions that amplify binding of vinculin to the actin network. Binding of PIP2 to Vt is necessary for maintaining optimal focal adhesions, for organizing stress fibers, for cell migration and spreading, and for the control of vinculin dynamics and turnover of focal adhesions. While the recently determined Vt/PIP2 crystal structure revealed the conformational changes occurring upon lipid binding and oligomerization, characterization of PIP2-induced vinculin oligomerization has been challenging in the adhesion biology field. Here, via a series of novel biochemical assays not performed in previous studies that relied on chemical cross-linking, we characterize the PIP2-induced vinculin oligomerization. Our results show that Vt/PIP2 forms a tight dimer with Vt or with the muscle-specific vinculin isoform, metavinculin, at sites of adhesion at the cell membrane. Insight into how PIP2 regulates clustering and into mechanisms that regulate cell adhesion allows the development for a more definite sensor for PIP2, and our developed techniques can be applied generally and thus open the door for the characterization of many other protein/PIP2 complexes under physiological conditions.
Topics: Cell Adhesion; Cell Membrane; Chromatography, Gel; Dimerization; Lipids; Surface Plasmon Resonance; Vinculin
PubMed: 25880222
DOI: 10.1021/acs.biochem.5b00015 -
Cellular and Molecular Life Sciences :... Aug 2017Vinculin was identified as a component of focal adhesions and adherens junctions nearly 40 years ago. Since that time, remarkable progress has been made in understanding... (Review)
Review
Vinculin was identified as a component of focal adhesions and adherens junctions nearly 40 years ago. Since that time, remarkable progress has been made in understanding its activation, regulation and function. Here we discuss the current understanding of the roles of vinculin in cell-cell and cell-matrix adhesions. Emphasis is placed on the how vinculin is recruited, activated and regulated. We also highlight the recent understanding of how vinculin responds to and transmits force at integrin- and cadherin-containing adhesion complexes to the cytoskeleton. Furthermore, we discuss roles of vinculin in binding to and rearranging the actin cytoskeleton.
Topics: Actin Cytoskeleton; Adherens Junctions; Animals; Cadherins; Cell Adhesion; Cell Movement; Focal Adhesions; Humans; Integrins; Mechanotransduction, Cellular; Models, Molecular; Protein Interaction Maps; Vinculin
PubMed: 28401269
DOI: 10.1007/s00018-017-2511-3 -
Advances in Experimental Medicine and... 2017Adhesion, segregation, and cellular plasticity are regulated by actin filaments anchored at the plaques of adherens junctions, sites of mechanical stabilization, and... (Review)
Review
Adhesion, segregation, and cellular plasticity are regulated by actin filaments anchored at the plaques of adherens junctions, sites of mechanical stabilization, and interfaces of multiple signaling networks. Drebrins were originally identified in neuronal cells, but the isoform drebrin E was also detected at adherens junctions of a wide range of non-neuronal cells, including polarized epithelia, endothelia, and fibroblasts. Here the protein is enriched at actin filament bundles associated with junctional plaques. Polarized epithelial cells contain two types of actin-associated complexes, one comprising drebrin but not vinculin and the other involving vinculin, but not drebrin. At gap junctions drebrin interacts with connexin 43, stabilizes this protein at membranes, and links it to the actin cytoskeleton. In vivo drebrin is widespread in diverse non-neuronal tissues of epithelial, endothelial, and smooth muscle origin, but not ubiquitous. In intestinal cells it is involved in cell compaction, linking of actin filaments to microtubules and formation and stabilization of the terminal web. Upregulation of drebrin was noted in several types of cancers, e.g., basal cell carcinomas for which it may serve as marker, liver metastases of colon carcinomas, and bladder cancer, suggesting that it is involved in regulating actin dynamics during tumor development, progression, and metastasis.
Topics: Actin Cytoskeleton; Adherens Junctions; Animals; Biomarkers, Tumor; Connexin 43; Humans; Microtubules; Neoplasms; Neuropeptides; Protein Interaction Maps; Vinculin
PubMed: 28865028
DOI: 10.1007/978-4-431-56550-5_18 -
Biomaterials Jul 2024Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal...
Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.
Topics: Talin; Vinculin; Humans; Cell Nucleus; YAP-Signaling Proteins; Mechanotransduction, Cellular; Adaptor Proteins, Signal Transducing; Transcription Factors; Mesenchymal Stem Cells; Animals; Focal Adhesions; Mice; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Protein Binding
PubMed: 38547833
DOI: 10.1016/j.biomaterials.2024.122542 -
Methods in Cell Biology 2018Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical...
Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical functions as (1) the physical linkage between chromosomes and spindle microtubules and (2) a mechanomolecular sensor that relays a spindle assembly checkpoint signal delaying anaphase onset until chromosomes are attached to spindle microtubules and bioriented. Given its central roles in such a vital process, the kinetochore is one of the most important force-transducing structures in cells; yet it has been technically challenging to measure kinetochore forces. Barriers to measuring cellular forces have begun to be broken by the development of fluorescence-based tension sensors. In this chapter, two methods will be described for measuring kinetochore forces in living cells and strategies for applying these sensors to other force-transducing processes and molecules will be discussed.
Topics: Animals; Biomechanical Phenomena; Biosensing Techniques; Cytological Techniques; Drosophila; Fluorescence Resonance Energy Transfer; Mitosis; Photobleaching; Talin; Vinculin
PubMed: 29804669
DOI: 10.1016/bs.mcb.2018.03.007