scholarly journals Activation Mechanisms of the VPS34 Complexes

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3124
Author(s):  
Yohei Ohashi
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Physicochemical Parameters ◽  
Future Perspectives ◽  
Downstream Effectors ◽  
Activation Mechanisms ◽  
Cellular Pathways ◽  
Upstream Regulators ◽  
Endocytic Pathways ◽  
Key Questions

Phosphatidylinositol-3-phosphate (PtdIns(3)P) is essential for cell survival, and its intracellular synthesis is spatially and temporally regulated. It has major roles in two distinctive cellular pathways, namely, the autophagy and endocytic pathways. PtdIns(3)P is synthesized from phosphatidylinositol (PtdIns) by PIK3C3C/VPS34 in mammals or Vps34 in yeast. Pathway-specific VPS34/Vps34 activity is the consequence of the enzyme being incorporated into two mutually exclusive complexes: complex I for autophagy, composed of VPS34/Vps34–Vps15/Vps15-Beclin 1/Vps30-ATG14L/Atg14 (mammals/yeast), and complex II for endocytic pathways, in which ATG14L/Atg14 is replaced with UVRAG/Vps38 (mammals/yeast). Because of its involvement in autophagy, defects in which are closely associated with human diseases such as cancer and neurodegenerative diseases, developing highly selective drugs that target specific VPS34/Vps34 complexes is an essential goal in the autophagy field. Recent studies on the activation mechanisms of VPS34/Vps34 complexes have revealed that a variety of factors, including conformational changes, lipid physicochemical parameters, upstream regulators, and downstream effectors, greatly influence the activity of these complexes. This review summarizes and highlights each of these influences as well as clarifying key questions remaining in the field and outlining future perspectives.

scholarly journals Structural insights into the activation of human calcium-sensing receptor

2021 ◽  
Author(s):  
Xiaochen Chen ◽  
Lu Wang ◽  
Zhanyu Ding ◽  
Qianqian Cui ◽  
Li Han ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Transmembrane Domains ◽  
Structural Basis ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Cryo Electron Microscopy ◽  
Activation Mechanisms ◽  
G Protein Coupled ◽  
Structural Insights

AbstractHuman calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.

scholarly journals In Silico Electrophysiology of Inner-Ear Mechanotransduction Channel TMC1 Models

2021 ◽  
Author(s):  
Sanket Walujkar ◽  
Jeffrey M Lotthammer ◽  
Collin R Nisler ◽  
Joseph C Sudar ◽  
Angela Ballesteros ◽  
...  
Keyword(s):  
Inner Ear ◽  
Conformational Changes ◽  
Hair Cells ◽  
Head Movements ◽  
Mechanical Stimuli ◽  
Ion Conduction ◽  
Sensory Hair Cells ◽  
Activation Mechanisms ◽  
Dynamics Simulations ◽  
Molecular Components

Inner-ear sensory hair cells convert mechanical stimuli from sound and head movements into electrical signals during mechanotransduction. Identification of all molecular components of the inner-ear mechanotransduction apparatus is ongoing; however, there is strong evidence that TMC1 and TMC2 are pore-forming subunits of the complex. We present molecular dynamics simulations that probe ion conduction of TMC1 models built based on two different structures of related TMEM16 proteins. Unlike most channels, the TMC1 models do not show a central pore. Instead, simulations of these models in a membrane environment at various voltages reveal a peripheral permeation pathway that is exposed to lipids and that shows cation permeation at rates comparable to those measured in hair cells. Furthermore, our analyses suggest that TMC1 gating mechanisms involve protein conformational changes and tension-induced lipid-mediated pore widening. These results provide insights into ion conduction and activation mechanisms of hair-cell mechanotransduction channels essential for hearing and balance.

scholarly journals Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR

2015 ◽  
Vol 112 (46) ◽  
pp. 14254-14259 ◽  
Author(s):  
Rajan Lamichhane ◽  
Jeffrey J. Liu ◽  
Goran Pljevaljcic ◽  
Kate L. White ◽  
Edwin van der Schans ◽  
...  
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Conformational Changes ◽  
Fluorescence Probe ◽  
Inverse Agonist ◽  
Inactive Conformation ◽  
Basal Activity ◽  
Activation Mechanisms ◽  
High Level ◽  
G Protein Coupled

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

CDC20 assists its catalytic incorporation in the mitotic checkpoint complex

Science ◽  
2020 ◽  
Vol 371 (6524) ◽  
pp. 67-71 ◽  
Author(s):  
Valentina Piano ◽  
Amal Alex ◽  
Patricia Stege ◽  
Stefano Maffini ◽  
Gerardo A. Stoppiello ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mitotic Checkpoint ◽  
Catalytic Complex ◽  
Controlled Systems ◽  
Cellular Pathways ◽  
Mitotic Checkpoint Complex ◽  
Rate Limiting

Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.

scholarly journals Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

2015 ◽  
Vol 290 (34) ◽  
pp. 20761-20773 ◽  
Author(s):  
Shaotong Zhu ◽  
Steven B. Vik
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Nadh Oxidase ◽  
Membrane Surface ◽  
Proton Translocation ◽  
Membrane Vesicles ◽  
Peripheral Region ◽  
Significant Loss ◽  
Cysteine Residues ◽  
Nadh:Ubiquinone Oxidoreductase

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441–445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu2+ ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50–90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10–20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.

scholarly journals Redox-induced activation of the proton pump in the respiratory complex I

2015 ◽  
Vol 112 (37) ◽  
pp. 11571-11576 ◽  
Author(s):  
Vivek Sharma ◽  
Galina Belevich ◽  
Ana P. Gamiz-Hernandez ◽  
Tomasz Róg ◽  
Ilpo Vattulainen ◽  
...  
Keyword(s):  
Proton Pump ◽  
Conformational Changes ◽  
Complex I ◽  
Large Scale ◽  
Reductase Activity ◽  
Proton Pumping ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Respiratory Chains ◽  
Dynamics Simulations

Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.

scholarly journals Regulators of TNFα Mediated Insulin Resistance Elucidated by Quantitative Proteomics

2020 ◽  
Author(s):  
Rodrigo Mohallem ◽  
Uma K. Aryal
Keyword(s):  
Insulin Resistance ◽  
Quantitative Proteomics ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Migration And Invasion ◽  
Downstream Effectors ◽  
Cellular Pathways ◽  
Factor Α ◽  
Necrosis Factor

AbstractObesity is a growing epidemic worldwide and is a major risk factor for several chronic diseases, including diabetes, kidney disease, heart disease, and cancer. Obesity often leads to type 2 diabetes mellitus (T2DM, via the increased production of proinflammatory cytokines such as tumor necrosis factor-α (TNFα). Our study combines different proteomic techniques to investigate the changes in the global proteome, secretome and phosphoproteome of adipocytes under chronic inflammation condition, as well as fundamental cross-talks between different cellular pathways regulated by chronic TNFα exposure. Our results show that many key regulator proteins of the canonical and non-canonical NF-κB pathways, such as Nfkb2, and its downstream effectors, including Csf-1 and Lgals3bp, directly involved in leukocyte migration and invasion, were significantly upregulated at the intra and extracellular levels, culminating in the progression of inflammation. Our data provides evidence of several key proteins that play a role in the development of insulin resistance.

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