scholarly journals Interactions between the N- and C- termini of mechanosensitive ion channel AtMSL10 are consistent with a three-step mechanism for activation

2019 ◽  
Author(s):  
Debarati Basu ◽  
Jennette M. Shoots ◽  
Elizabeth S. Haswell
Keyword(s):  
Cell Death ◽  
Ion Channel ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Adaptive Responses ◽  
C Terminus ◽  
Step Model ◽  
N Terminus ◽  
Mechanosensitive Ion Channel ◽  
Cellular Phenotypes

ABSTRACTAlthough a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to GFP triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag and could be also induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus are transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants.HIGHLIGHTCell death is triggered by mutations in either the cytoplasmic N- or C-terminus of AìMSLlü. Our proposed model explains how membrane tension may activate signaling through the interaction of these two domains.

scholarly journals Interactions between the N- and C-termini of the mechanosensitive ion channel AtMSL10 are consistent with a three-step mechanism for activation

2020 ◽  
Vol 71 (14) ◽  
pp. 4020-4032 ◽  
Author(s):  
Debarati Basu ◽  
Jennette M Shoots ◽  
Elizabeth S Haswell
Keyword(s):  
Cell Death ◽  
Ion Channel ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Adaptive Responses ◽  
C Terminus ◽  
N Terminus ◽  
Mechanosensitive Ion Channel ◽  
Green Fluorescent

Abstract Although a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to green fluorescent protein (GFP) triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag, and could also be induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus could be transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants.

scholarly journals Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

scholarly journals Mechanosensitive Ion Channel Piezo1 Regulates Myocyte Fusion during Skeletal Myogenesis

2020 ◽  
Author(s):  
Huascar Pedro Ortuste Quiroga ◽  
Shingo Yokoyama ◽  
Massimo Ganassi ◽  
Kodai Nakamura ◽  
Tomohiro Yamashita ◽  
...  
Keyword(s):  
Ion Channel ◽  
Molecular Mechanisms ◽  
Myoblast Fusion ◽  
Mechanical Stimuli ◽  
Muscle Satellite Cell ◽  
Myotube Formation ◽  
Mechanosensitive Ion Channel ◽  
And Function ◽  
Sirna Knockdown

AbstractMechanical stimuli such as stretch and resistance training are essential to regulate growth and function of skeletal muscle. However, the molecular mechanisms involved in sensing mechanical stress remain unclear. Here, the purpose of this study was to investigate the role of the mechanosensitive ion channel Piezo1 during myogenic progression. Muscle satellite cell-derived myoblasts and myotubes were modified with stretch, siRNA knockdown and agonist-induced activation of Piezo1. Direct manipulation of Piezo1 modulates terminal myogenic progression. Piezo1 knockdown suppressed myoblast fusion during myotube formation and maturation. This was accompanied by downregulation of the fusogenic protein Myomaker. Piezo1 knockdown also lowered Ca2+ influx in response to stretch. Conversely Piezo1 activation stimulated fusion and increased Ca2+ influx in response to stretch. These evidences indicate that Piezo1 is essential for myotube formation and maturation, which may have implications for msucular dystrophy prevention through its role as a mechanosensitive Ca2+ channel.

scholarly journals Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity

2014 ◽  
Vol 26 (7) ◽  
pp. 3115-3131 ◽  
Author(s):  
Kira M. Veley ◽  
Grigory Maksaev ◽  
Elizabeth M. Frick ◽  
Emma January ◽  
Sarah C. Kloepper ◽  
...  
Keyword(s):  
Cell Death ◽  
Ion Channel ◽  
Channel Activity ◽  
Regulated Cell Death ◽  
Mechanosensitive Ion Channel ◽  
Cell Death Signaling ◽  
Signaling Activity

scholarly journals Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Arthur Neuberger ◽  
Kirill D. Nadezhdin ◽  
Alexander I. Sobolevsky
Keyword(s):  
Ion Channel ◽  
Conformational Changes ◽  
Calcium Imaging ◽  
Molecular Mechanisms ◽  
Channel Blocker ◽  
Ruthenium Red ◽  
Allosteric Site ◽  
Structural Mechanisms ◽  
New Generation ◽  
Molecular Bases

AbstractTRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel’s periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel’s selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.

scholarly journals The Mechanosensitive Ion Channel MSL10 Modulates Susceptibility to Pseudomonas syringae in Arabidopsis thaliana

2021 ◽  
Author(s):  
Debarati Basu ◽  
Jennette M. Codjoe ◽  
Kira Veley ◽  
Elizabeth Haswell
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Death ◽  
Ion Channel ◽  
Pseudomonas Syringae ◽  
Stomatal Closure ◽  
Avirulence Genes ◽  
Loss Of Function ◽  
Susceptibility To Infection ◽  
Mechanical Signals ◽  
Mechanosensitive Ion Channel

Plants sense and respond to molecular signals associated with the presence of pathogens and their virulence factors. Mechanical signals generated during pathogenic invasion may also be important, but their contributions have rarely been studied. Here we investigate the potential role of a mechanosensitive ion channel, MscS-Like (MSL)10, in defense against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. We previously showed that overexpression of MSL10-GFP, phospho-mimetic versions of MSL10, and the gain-of-function allele msl10-3G all produce dwarfing, spontaneous cell death, and the hyperaccumulation of reactive oxygen species. These phenotypes are shared by many autoimmune mutants and are frequently suppressed by growth at high temperature in those lines. Here, we found that the same was true for all three MSL10 hypermorphs. In addition, we show that the SGT1/RAR1/HSP90 co-chaperone complex was required for dwarfing and ectopic cell death, PAD4 and SID2 were partially required, and the immune regulators EDS1 and NDR1 were dispensable. All MSL10 hypermorphs exhibited reduced susceptibility to infection by P. syringae strain Pto DC3000, Pto DC3000 expressing the avirulence genes avrRpt2 or avrRpm1, but not Pto DC3000 hrpL, and showed an accelerated induction of PR1 expression compared to wild-type plants. Null msl10-1 mutants were delayed in PR1 induction and displayed modest susceptibility to infection by COR-deficient Pst. Finally, stomatal closure was reduced in msl10-1 loss-of-function mutants in response to Pst COR−. These data show that MSL10 modulates pathogen responses and begin to address the possibility that mechanical signals are exploited by the plant for pathogen perception.

scholarly journals GSDMB promotes non-canonical pyroptosis by enhancing caspase-4 activity

2018 ◽  
Vol 11 (6) ◽  
pp. 496-508 ◽  
Author(s):  
Qin Chen ◽  
Peiliang Shi ◽  
Yufang Wang ◽  
Dayuan Zou ◽  
Xiuwen Wu ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Regulatory Mechanism ◽  
Inflammatory Diseases ◽  
Human Monocyte ◽  
Card Domain ◽  
N Terminus ◽  
Human Monocyte Cell Line ◽  
New Strategy ◽  
Monocyte Cell

Abstract Gasdermin B (GSDMB) has been reported to be associated with immune diseases in humans, but the detailed molecular mechanisms remain unsolved. The N-terminus of GSDMB by itself, unlike other gasdermin family proteins, does not induce cell death. Here, we show that GSDMB is highly expressed in the leukocytes of septic shock patients, which is associated with increased release of the gasdermin D (GSDMD) N-terminus. GSDMB expression and the accumulation of the N-terminal fragment of GSDMD are induced by the activation of the non-canonical pyroptosis pathway in a human monocyte cell line. The downregulation of GSDMB alleviates the cleavage of GSDMD and cell death. Consistently, the overexpression of GSDMB promotes GSDMD cleavage, accompanied by increased LDH release. We further found that GSDMB promotes caspase-4 activity, which is required for the cleavage of GSDMD in non-canonical pyroptosis, by directly binding to the CARD domain of caspase-4. Our study reveals a GSDMB-mediated novel regulatory mechanism for non-canonical pyroptosis and suggests a potential new strategy for the treatment of inflammatory diseases.

scholarly journals Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling

2007 ◽  
Vol 130 (4) ◽  
pp. 379-388 ◽  
Author(s):  
Nancy M. Lorenzon ◽  
Kurt G. Beam
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Conformational Changes ◽  
Ec Coupling ◽  
C Terminus ◽  
N Terminus ◽  
Electrically Evoked Contractions ◽  
Evoked Contractions

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca2+ channel and as the voltage sensor for excitation–contraction (EC coupling), triggering Ca2+ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR α1S or β1a subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the ∼60-kD streptavidin molecule had access to the β1a N and C termini and to the α1S N terminus and proximal II–III loop (residues 671–686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the α1S C terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the β1a N or C terminus, or to the α1S N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal α1S II–III loop abolished such contractions, without affecting agonist-induced Ca2+ release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the α1S II–III loop are necessary for EC coupling in skeletal muscle.

scholarly journals DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

2020 ◽  
Author(s):  
L. Robert Hollingsworth ◽  
Humayun Sharif ◽  
Andrew R. Griswold ◽  
Pietro Fontana ◽  
Julian Mintseris ◽  
...  
Keyword(s):  
Cell Death ◽  
Active Site ◽  
Protein Turnover ◽  
Ternary Complex ◽  
Inflammatory Diseases ◽  
Inflammasome Activation ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus ◽  
Dipeptidyl Peptidases

AbstractNLRP1 is a cytosolic inflammasome sensor that mediates activation of caspase-1, which in turn induces cytokine maturation and pyroptotic cell death1-6. Gain-of-function NLPR1 mutations cause skin inflammatory diseases including carcinoma, keratosis, and papillomatosis7-14. NLRP1 contains a unique function-to-find domain (FIIND) that autoproteolyzes into noncovalently associated subdomains15-18. Proteasomal degradation of the autoinhibitory N-terminal fragment (NT) activates NLRP1 by releasing the inflammatory C-terminal fragment (CT)19,20. Cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with NLRP1, and small-molecule DPP8/9 inhibitors activate NLRP1 by poorly characterized mechanisms11,19,21. Here, we report cryo-EM structures of the human NLRP1-DPP9 complex, alone and in complex with the DPP8/9 inhibitor Val-boroPro (VbP). Surprisingly, the NLRP1-DPP9 complex is a ternary complex comprised of DPP9, one intact FIIND of a non-degraded full-length NLRP1 (NLRP1-FL) and one NLRP1-CT freed by NT degradation. The N-terminus of the NLRP1-CT unfolds and inserts into the DPP9 active site but is not cleaved by DPP9, and this binding is disrupted by VbP. Structure-based mutagenesis reveals that the binding of NLRP1-CT to DPP9 requires NLRP1-FL and vice versa, and inflammasome activation by ectopic NLRP1-CT expression is rescued by co-expressing autoproteolysis-deficient NLRP1-FL. Collectively, these data indicate that DPP9 functions as a “bomb-diffuser” to prevent NLRP1-CTs from inducing inflammation during homeostatic protein turnover.

scholarly journals Insights into the molecular mechanisms underlying the inhibition of acid-sensing ion channel 3 gating by stomatin

2020 ◽  
Vol 152 (3) ◽  
Author(s):  
Robert C. Klipp ◽  
Megan M. Cullinan ◽  
John R. Bankston
Keyword(s):  
Ion Channel ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Ph Dependence ◽  
Integral Membrane Protein ◽  
Surface Expression ◽  
C Terminus ◽  
Patch Clamp Electrophysiology ◽  
A Site ◽  
Terminal Site

Stomatin (STOM) is a monotopic integral membrane protein found in all classes of life that has been shown to regulate members of the acid-sensing ion channel (ASIC) family. However, the mechanism by which STOM alters ASIC function is not known. Using chimeric channels, we combined patch-clamp electrophysiology and FRET to search for regions of ASIC3 critical for binding to and regulation by STOM. With this approach, we found that regulation requires two distinct sites on ASIC3: the distal C-terminus and the first transmembrane domain (TM1). The C-terminal site is critical for formation of the STOM–ASIC3 complex, while TM1 is required only for the regulatory effect. We then looked at the mechanism of STOM-dependent regulation of ASIC3 and found that STOM does not alter surface expression of ASIC3 or shift the pH dependence of channel activation. However, a point mutation (Q269G) that prevents channel desensitization also prevents STOM regulation, suggesting that STOM may alter ASIC3 currents by stabilizing the desensitized state of the channel. Based on these findings, we propose a model whereby STOM is anchored to the channel via a site on the distal C-terminus and stabilizes the desensitized state of the channel via an interaction with TM1.

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