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Cell Jan 2022Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while...
Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.
Topics: Base Sequence; Case-Control Studies; Cell Adhesion; Cell Membrane; Cell Movement; Cell Proliferation; Epithelial Cells; Focal Adhesion Protein-Tyrosine Kinases; HEK293 Cells; HT29 Cells; Humans; Inflammatory Bowel Diseases; Methotrexate; Mutation; Phosphorylation; Polymorphism, Single Nucleotide; Pore Forming Cytotoxic Proteins; Pyroptosis; Reproducibility of Results; Transcriptome; Up-Regulation; Wound Healing
PubMed: 35021065
DOI: 10.1016/j.cell.2021.12.024 -
Acta Pharmaceutica Sinica. B Feb 2021Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the... (Review)
Review
Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the neurotrophic receptor tyrosine kinase 1, 2 and 3 ( and ) genes, respectively. TRKs can regulate cell proliferation, differentiation and even apoptosis through the RAS/MAPKs, PI3K/AKT and PLC pathways. Gene fusions involving act as oncogenic drivers of a broad diversity of adult and pediatric tumors, and TRKs have become promising antitumor targets. Therefore, achieving a comprehensive understanding of TRKs and relevant TRK inhibitors should be urgently pursued for the further development of novel TRK inhibitors for potential clinical applications. This review focuses on summarizing the biological functions of TRKs and fusion proteins, the development of small-molecule TRK inhibitors with different chemotypes and their activity and selectivity, and the potential therapeutic applications of these inhibitors for future cancer drug discovery efforts.
PubMed: 33643817
DOI: 10.1016/j.apsb.2020.05.004 -
Autophagy Jan 2023In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown....
In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from KO, as well as KO and mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype ( cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cKO mice. Furthermore, the antibody rescued retinal function in cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options. ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; HO: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1β: interleukin 1 beta; IS: Inner segment; ITGB1/integrin β1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.
Topics: Animals; Humans; Mice; Antibodies, Monoclonal; Autophagy; Ferroptosis; Inflammasomes; Lipocalin-2; Macular Degeneration; Mice, Inbred NOD; Mice, SCID; Nucleotidyltransferases
PubMed: 35473441
DOI: 10.1080/15548627.2022.2062887 -
Acta Pharmaceutica Sinica. B Mar 2023The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection... (Review)
Review
The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection of internal organs, production of blood cells and regulation of calcium and phosphate metabolism. The prevalence of skeletal diseases and disorders, such as osteoporosis and bone fracture, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, increases with age, causing pain and loss of mobility and creating a huge social and economic burden globally. Focal adhesions (FAs) are macromolecular assemblies that are composed of the extracellular matrix (ECM), integrins, intracellular cytoskeleton and other proteins, including kindlin, talin, vinculin, paxillin, pinch, Src, focal adhesion kinase (FAK) and integrin-linked protein kinase (ILK) and other proteins. FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell-environment communications and modulates important processes, such as cell attachment, spreading, migration, differentiation and mechanotransduction, in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways. This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.
PubMed: 36970189
DOI: 10.1016/j.apsb.2022.09.020 -
Autophagy Dec 2021Current disease-modifying therapies for Huntington disease (HD) focus on lowering mutant HTT (huntingtin; mHTT) levels, and the immunosuppressant drug rapamycin is an...
Current disease-modifying therapies for Huntington disease (HD) focus on lowering mutant HTT (huntingtin; mHTT) levels, and the immunosuppressant drug rapamycin is an intriguing therapeutic for aging and neurological disorders. Rapamycin interacts with FKBP1A/FKBP12 and FKBP5/FKBP51, inhibiting the MTORC1 complex and increasing cellular clearance mechanisms. Whether the levels of FKBP (FK506 binding protein) family members are altered in HD models and if these proteins are potential therapeutic targets for HD have not been investigated. Here, we found levels of FKBP5 are significantly reduced in HD R6/2 and zQ175 mouse models and human HD isogenic neural stem cells and medium spiny neurons derived from induced pluripotent stem cells. Moreover, FKBP5 interacts and colocalizes with HTT in the striatum and cortex of zQ175 mice and controls. Importantly, when we decreased FKBP5 levels or activity by genetic or pharmacological approaches, we observed reduced levels of mHTT in our isogenic human HD stem cell model. Decreasing FKBP5 levels by siRNA or pharmacological inhibition increased LC3-II levels and macroautophagic/autophagic flux, suggesting autophagic cellular clearance mechanisms are responsible for mHTT lowering. Unlike rapamycin, the effect of pharmacological inhibition with SAFit2, an inhibitor of FKBP5, is MTOR independent. Further, treatment for 2 weeks with SAFit2, results in reduced HTT levels in both HD R6/2 and zQ175 mouse models. Our studies establish FKBP5 as a protein involved in the pathogenesis of HD and identify FKBP5 as a potential therapeutic target for HD. : ACTB/β-actin: actin beta; AD: Alzheimer disease; BafA1: bafilomycin A; BCA: bicinchoninic acid; BBB: blood brain barrier; BSA: bovine serum albumin; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FKBPs: FK506 binding proteins; HD: Huntington disease; HTT: huntingtin; iPSC: induced pluripotent stem cells; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MES: 2-ethanesulfonic acid; MOPS: 3-(N-morphorlino)propanesulfonic acid); MSN: medium spiny neurons; mHTT: mutant huntingtin; MTOR: mechanistic target of rapamycin kinase; NSC: neural stem cells; ON: overnight; PD: Parkinson disease; PPIase: peptidyl-prolyl -isomerases; polyQ: polyglutamine; PPP1R1B/DARPP-32: protein phosphatase 1 regulatory inhibitor subunit 1B; PTSD: post-traumatic stress disorder; RT: room temperature; SQSTM1/p62: sequestosome 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST:Tris-buffered saline, 0.1% Tween 20; TUBA: tubulin; ULK1: unc-51 like autophagy activating kinase 1; VCL: vinculin; WT: littermate controls.
Topics: Animals; Autophagy; Huntingtin Protein; Huntington Disease; Mechanistic Target of Rapamycin Complex 1; Mice; Neurons; Tacrolimus Binding Proteins
PubMed: 34024231
DOI: 10.1080/15548627.2021.1904489 -
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