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 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 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.

Flavonoids as P-gp Inhibitors: A Systematic Review of SARs

2019 ◽  
Vol 26 (25) ◽  
pp. 4799-4831 ◽  
Author(s):  
Jiahua Cui ◽  
Xiaoyang Liu ◽  
Larry M.C. Chow
Keyword(s):  
Molecular Mechanisms ◽  
Metastatic Cancer ◽  
Drug Candidates ◽  
Chemical Structures ◽  
Transporter Family ◽  
Promising Area ◽  
New Generation ◽  
Nontoxic Inhibitors

P-glycoprotein, also known as ABCB1 in the ABC transporter family, confers the simultaneous resistance of metastatic cancer cells towards various anticancer drugs with different targets and diverse chemical structures. The exploration of safe and specific inhibitors of this pump has always been the pursuit of scientists for the past four decades. Naturally occurring flavonoids as benzopyrone derivatives were recognized as a class of nontoxic inhibitors of P-gp. The recent advent of synthetic flavonoid dimer FD18, as a potent P-gp modulator in reversing multidrug resistance both in vitro and in vivo, specifically targeted the pseudodimeric structure of the drug transporter and represented a new generation of inhibitors with high transporter binding affinity and low toxicity. This review concerned the recent updates on the structure-activity relationships of flavonoids as P-gp inhibitors, the molecular mechanisms of their action and their ability to overcome P-gp-mediated MDR in preclinical studies. It had crucial implications on the discovery of new drug candidates that modulated the efflux of ABC transporters and also provided some clues for the future development in this promising area.

Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4

Glycobiology ◽  
2021 ◽  
Author(s):  
Margrethe Gaardløs ◽  
Sergey A Samsonov ◽  
Marit Sletmoen ◽  
Maya Hjørnevik ◽  
Gerd Inger Sætrom ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Interdisciplinary Approach ◽  
Product Formation ◽  
Titration Calorimetry ◽  
Binding Groove ◽  
Dynamics Simulations

Abstract Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

Quantitative analysis of the interactions between HIV-1 integrase and retroviral reverse transcriptases

2008 ◽  
Vol 412 (1) ◽  
pp. 163-170 ◽  
Author(s):  
Alon Herschhorn ◽  
Iris Oz-Gleenberg ◽  
Amnon Hizi
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Reaction Model ◽  
Protein Protein Interactions ◽  
Common Site ◽  
Leukaemia Virus ◽  
Reverse Transcriptases ◽  
Interaction Kinetics ◽  
Hiv 1

The RT (reverse transcriptase) of HIV-1 interacts with HIV-1 IN (integrase) and inhibits its enzymatic activities. However, the molecular mechanisms underling these interactions are not well understood. In order to study these mechanisms, we have analysed the interactions of HIV-1 IN with HIV-1 RT and with two other related RTs: those of HIV-2 and MLV (murine-leukaemia virus). All three RTs inhibited HIV-1 IN, albeit to a different extent, suggesting a common site of binding that could be slightly modified for each one of the studied RTs. Using surface plasmon resonance technology, which monitors direct protein–protein interactions, we performed kinetic analyses of the binding of HIV-1 IN to these three RTs and observed interesting binding patterns. The interaction of HIV-1 RT with HIV-1 IN was unique and followed a two-state reaction model. According to this model, the initial IN–RT complex formation was followed by a conformational change in the complex that led to an elevation of the total affinity between these two proteins. In contrast, HIV-2 and MLV RTs interacted with IN in a simple bi-molecular manner, without any apparent secondary conformational changes. Interestingly, HIV-1 and HIV-2 RTs were the most efficient inhibitors of HIV-1 IN activity, whereas HIV-1 and MLV RTs showed the highest affinity towards HIV-1 IN. These modes of direct protein interactions, along with the apparent rate constants calculated and the correlations of the interaction kinetics with the capacity of the RTs to inhibit IN activities, are all discussed.

scholarly journals The Primary Causes of Muscle Dysfunction Associated with the Point Mutations in Tpm3.12; Conformational Analysis of Mutant Proteins as a Tool for Classification of Myopathies

2018 ◽  
Vol 19 (12) ◽  
pp. 3975 ◽  
Author(s):  
Yurii Borovikov ◽  
Olga Karpicheva ◽  
Armen Simonyan ◽  
Stanislava Avrova ◽  
Elena Rogozovets ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Fiber Type ◽  
Point Mutations ◽  
Nemaline Myopathy ◽  
Muscle Dysfunction ◽  
Thin Filaments ◽  
Mutant Proteins ◽  
Strong Binding ◽  
Genes Encoding

Point mutations in genes encoding isoforms of skeletal muscle tropomyosin may cause nemaline myopathy, cap myopathy (Cap), congenital fiber-type disproportion (CFTD), and distal arthrogryposis. The molecular mechanisms of muscle dysfunction in these diseases remain unclear. We studied the effect of the E173A, R90P, E150A, and A155T myopathy-causing substitutions in γ-tropomyosin (Tpm3.12) on the position of tropomyosin in thin filaments, and the conformational state of actin monomers and myosin heads at different stages of the ATPase cycle using polarized fluorescence microscopy. The E173A, R90P, and E150A mutations produced abnormally large displacement of tropomyosin to the inner domains of actin and an increase in the number of myosin heads in strong-binding state at low and high Ca2+, which is characteristic of CFTD. On the contrary, the A155T mutation caused a decrease in the amount of such heads at high Ca2+ which is typical for mutations associated with Cap. An increase in the number of the myosin heads in strong-binding state at low Ca2+ was observed for all mutations associated with high Ca2+-sensitivity. Comparison between the typical conformational changes in mutant proteins associated with different myopathies observed with α-, β-, and γ-tropomyosins demonstrated the possibility of using such changes as tests for identifying the diseases.

scholarly journals Electrostatics, proton sensor, and networks governing the gating transition in GLIC, a proton-gated pentameric ion channel

2018 ◽  
Vol 115 (52) ◽  
pp. E12172-E12181 ◽  
Author(s):  
Haidai Hu ◽  
Kenichi Ataka ◽  
Anaïs Menny ◽  
Zaineb Fourati ◽  
Ludovic Sauguet ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Ion Channel ◽  
Transmembrane Domain ◽  
Hydrogen Bond Network ◽  
Transmembrane Region ◽  
Allosteric Site ◽  
Closed Channel ◽  
Open Conformation ◽  
Poisson Boltzmann ◽  
Bond Network

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure–function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson–Boltzmann/Debye–Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pKa close to the experimental pH50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pKa at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the highly conserved electrostatic triad R192-D122-D32 and then to Y197-Y119-K248, both located at the extracellular domain–transmembrane domain interface. The second triad controls a cluster of hydrophobic side chains from the M2-M3 loop that is remodeled during the gating transition. We solved 12 crystal structures of GLIC mutants, 6 of them being trapped in an agonist-bound but nonconductive conformation. Combined with previous data, this reveals two branches of a continuous network originating from E35 that reach, independently, the middle transmembrane region of two adjacent subunits. We conclude that GLIC’s gating proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site.

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 Molecular Inverse Comorbidity between Alzheimer’s disease and Lung Cancer: new insights from Matrix Factorization

2019 ◽  
Author(s):  
Alessandro Greco ◽  
Jon Sanchez Valle ◽  
Vera Pancaldi ◽  
Anaïs Baudot ◽  
Emmanuel Barillot ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Matrix Factorization ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Biological Data ◽  
Biological Data Analysis ◽  
Specific Factors ◽  
Molecular Bases

AbstractMatrix Factorization (MF) is an established paradigm for large-scale biological data analysis with tremendous potential in computational biology.We here challenge MF in depicting the molecular bases of epidemiologically described Disease-Disease (DD) relationships. As use case, we focus on the inverse comorbidity association between Alzheimer’s disease (AD) and lung cancer (LC), described as a lower than expected probability of developing LC in AD patients. To the day, the molecular mechanisms underlying DD relationships remain poorly explained and their better characterization might offer unprecedented clinical opportunities.To this goal, we extend our previously designed MF-based framework for the molecular characterization of DD relationships. Considering AD-LC inverse comorbidity as a case study, we highlight multiple molecular mechanisms, among which the previously identified immune system and mitochondrial metabolism. We then discriminate mechanisms specific to LC from those shared with other cancers through a pancancer analysis. Additionally, new candidate molecular players, such as Estrogen Receptor (ER), CDH1 and HDAC, are pinpointed as factors that might underlie the inverse relationship, opening the way to new investigations. Finally, some lung cancer subtype-specific factors are also detected, suggesting the existence of heterogeneity across patients also in the context of inverse comorbidity.

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