Cryo-EM structures and functional characterization of the lipid scramblase TMEM16F

SUMMARYThe lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of TMEM16F that underlie its function as lipid scramblase and ion channel. The cryo-EM structures of TMEM16F in Ca2+-bound and Ca2+-free states display a striking similarity to the scrambling-incompetent anion channel TMEM16A, yet with distinct differences in the catalytic site and in the conformational changes upon activation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, which likely resembles an equivalent process defined in the homologue nhTMEM16, both functions appear to be mediated by alternate protein conformations, which are at equilibrium in the ligand-bound state.

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Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F

eLife ◽

10.7554/elife.44365 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 32

Author(s):

Carolina Alvadia ◽

Novandy K Lim ◽

Vanessa Clerico Mosina ◽

Gert T Oostergetel ◽

Raimund Dutzler ◽

...

Keyword(s):

Functional Data ◽

Functional Characterization ◽

Anion Channel ◽

Bound State ◽

Common Mechanism ◽

Protein Conformations ◽

Closed Conformation ◽

Ligand Free ◽

The Relationship

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

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Effect of Surface Topography Parameters on Friction and Wear of Random Rough Surface

Materials ◽

10.3390/ma12172762 ◽

2019 ◽

Vol 12 (17) ◽

pp. 2762 ◽

Cited By ~ 4

Author(s):

Ruimin Shi ◽

Bukang Wang ◽

Zhiwei Yan ◽

Zongyan Wang ◽

Lei Dong

Keyword(s):

Surface Topography ◽

Fluid Dynamic ◽

Dynamic Pressure ◽

Functional Characterization ◽

Three Dimensional ◽

Measuring System ◽

Height Distribution ◽

Worn Surface ◽

The Relationship

In order to explore the relationship between the surface topography parameters and friction properties of a rough contact interface under fluid dynamic pressure lubrication conditions, friction experiments were carried out. The three-dimensional surface topography of specimens was measured and characterized with a profile microscopy measuring system and scanning electron microscope. The friction coefficient showed a trend of decreasing first and then increasing with the increase in some surface topography parameters at lower pressure, such as the surface height arithmetic mean Sa, surface height distribution kurtosis Sku, surface volume average volume Vvv, and surface center area average void volume Vvc, which are the ISO 25178 international standard parameters. The effects of surface topographic parameters on friction were analyzed and the wear mechanism of the worn surface was presented. The wear characteristics of the samples were mainly characterized as strain fatigue, grinding, and scraping. The results provide a theoretical basis for the functional characterization of surface topography.

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Structural and Functional Characterization of a Novel Tumor-Derived Rat Galectin-1 Having Transforming Growth Factor (TGF) Activity: The Relationship between Intramolecular Disulfide Bridges and TGF Activity

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a021325 ◽

1996 ◽

Vol 119 (5) ◽

pp. 878-886 ◽

Cited By ~ 26

Author(s):

K. Yamaoka ◽

A. Ingendoh ◽

S. Tsubuki ◽

Y. Nagai ◽

Y. Sanai

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Functional Characterization ◽

Disulfide Bridges ◽

The Relationship

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NMR investigation of protein–ligand interactions for G-protein coupled receptors

Future Medicinal Chemistry ◽

10.4155/fmc-2018-0312 ◽

2019 ◽

Vol 11 (14) ◽

pp. 1811-1825 ◽

Cited By ~ 1

Author(s):

Claire Raingeval ◽

Isabelle Krimm

Keyword(s):

Conformational Changes ◽

Functional Characterization ◽

Allosteric Modulation ◽

G Protein Coupled Receptors ◽

Nmr Studies ◽

Protein Ligand Interactions ◽

Ligand Interactions ◽

Therapeutic Compounds ◽

G Protein Coupled

In this review, we report NMR studies of ligand–GPCR interactions, including both ligand-observed and protein-observed NMR experiments. Published studies exemplify how NMR can be used as a powerful tool to design novel GPCR ligands and investigate the ligand-induced conformational changes of GPCRs. The strength of NMR also lies in its capability to explore the diverse signaling pathways and probe the allosteric modulation of these highly dynamic receptors. By offering unique opportunities for the identification, structural and functional characterization of GPCR ligands, NMR will likely play a major role for the generation of novel molecules both as new tools for the understanding of the GPCR function and as therapeutic compounds for a large diversity of pathologies.

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Prokaryotic Expression and Functional Characterization of the 19 kDa Protein in Balanus albicostatus Cement

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.461.445 ◽

2013 ◽

Vol 461 ◽

pp. 445-450

Author(s):

Chao Liang ◽

Yun Qiu Li ◽

Bi Ru Hu ◽

Wen Jian Wu

Keyword(s):

Amino Acid ◽

Molecular Design ◽

Functional Characterization ◽

Blue Staining ◽

Amino Acid Residues ◽

Adhesive Ability ◽

Solid Liquid ◽

Dental Applications ◽

The Relationship

Barnacle is a unique sessile crustacean, which produces a multi-protein complex historically called barnacle cement to attach to diverse immersed materials permanently. The proteinaceous cement exhibits powerful adhesive property and special waterproof capability to cure at solid-liquid boundaries, which makes it ideal biomaterial for technical, medical and dental applications. It has been proved that a 19 kDa protein component, termed cp-19k in the cement plays a key role in surface coupling during underwater attachment. To verify whether the bacterial recombinant 19 kDa protein retains the adhesive ability, we cloned and sequenced the Bacp-19k gene in Balanus albicostatus. It encodes 173 amino acid residues, with seven biased ones, Thr, Lys, Gly, Ala, Val, Ser and Leu, comprising about 80% of the total. Two amino acid substitutions (F69L, I106L) were discovered in Bacp-19k due to the polymorphisms in barnacle cp-19ks, compared with the submitted one (GenBank: AB242295.1). Recombinant Bacp-19k was highly expressed in host strain Escherichia coli BL21 (DE3) and purified by affinity chromatography. Adsorption of recombinant Bacp-19k to glass substrata was examined by Coomassie brilliant blue staining. Future study will reveal the relationship between specific structures and functions for molecular design of novel biomimetic underwater adhesives.

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Analysis of Secreted Proteins and Potential Virulence via the ICEs-Mediated Pathway of the Foodborne Pathogen Vibrio parahaemolyticus

Frontiers in Microbiology ◽

10.3389/fmicb.2021.612166 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yu He ◽

Shuai Wang ◽

Kaiwen Wang ◽

Jinwei Zhou ◽

Zhi Han ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Biochemical Characterization ◽

Functional Characterization ◽

Foodborne Pathogen ◽

Secreted Proteins ◽

Secretion Systems ◽

Two Dimensional Gel Electrophoresis ◽

Integrative And Conjugative Elements ◽

The Relationship

Vibrio parahaemolyticus uses bacterial secretion systems and integrative and conjugative elements (ICEs) to induce various diseases and to adapt to harsh environments, respectively. Information pertaining to the identity of secreted proteins and functional characterization of ICEs has been previously reported, but the relationship between these elements remains unclear. Herein we investigated secreted proteins of V. parahaemolyticus strains JHY20 and JHY20△ICE using two-dimensional gel electrophoresis and LC-MS/MS, which led to the identification of an ICE-associated secreted protein – dihydrolipoamide dehydrogenase (DLDH). Considering the data related to its physical and biochemical characterization, we predicted that DLDH is a novel immunogenic protein and associated with virulence in JHY20. Our findings indicate a potential relationship between ICE-associated transport and secreted proteins and shed light on the function of such transport mechanisms. We believe that our data should enhance our understanding of mobile genetic elements.

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Functional characterization of a voltage-gated anion channel from teleost fish intestinal epithelium

Journal of Experimental Biology ◽

10.1242/jeb.136.1.383 ◽

1988 ◽

Vol 136 (1) ◽

pp. 383-403 ◽

Cited By ~ 3

Author(s):

C. A. Loretz ◽

C. R. Fourtner

Keyword(s):

Single Channel ◽

Basolateral Membrane ◽

Functional Characterization ◽

Anion Channel ◽

Intestinal Epithelial ◽

Channel Conductance ◽

Patch Clamp Technique ◽

Voltage Gated

An anion channel was isolated, using patch-clamp technique, from the basolateral membrane of goby intestinal epithelial cells. Single-channel conductance varied over a range from 20 to 90 pS. The channel was voltage-gated over the physiological range of cell membrane potential with depolarization increasing the proportion of time in the open state. There was no Ca2+ sensitivity. The selectivity sequence was SO4(2-) greater than Cl- greater than Mes-. The channel may function in vivo as one of several avenues of basolateral membrane Cl- exit with the voltage-gating property serving to match basolateral Cl- exit to apical entry.

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The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression

Disease Models & Mechanisms ◽

10.1242/dmm.046300 ◽

2021 ◽

Vol 14 (2) ◽

pp. dmm046300

Author(s):

Dan Wu ◽

Sailan Wang ◽

Daniel V. Oliveira ◽

Francesca Del Gaudio ◽

Michael Vanlandewijck ◽

...

Keyword(s):

Functional Characterization ◽

Extracellular Domain ◽

Infantile Myofibromatosis ◽

Independent Manner ◽

Functional Link ◽

Downstream Signaling ◽

Chloroquine Treatment ◽

Notch3 Mutation ◽

The Relationship

ABSTRACTInfantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal-dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this study, we address the molecular consequences of the NOTCH3L1519P mutation and the relationship between Notch and PDGFRB signaling in IMF. The NOTCH3L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3L1519P extracellular domain and decreases signaling. Finally, NOTCH3L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3–PDGFRB axis in IMF, in which an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.

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