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1.
Annu Rev Biochem ; 87: 809-837, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29596003

RESUMO

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.


Assuntos
Colesterol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Oxisteróis/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores de Esteroides/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação Viral/fisiologia
2.
Cell ; 155(4): 830-43, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24209621

RESUMO

Several proteins at endoplasmic reticulum (ER)-Golgi membrane contact sites contain a PH domain that interacts with the Golgi phosphoinositide PI(4)P, a FFAT motif that interacts with the ER protein VAP-A, and a lipid transfer domain. This architecture suggests the ability to both tether organelles and transport lipids between them. We show that in oxysterol binding protein (OSBP) these two activities are coupled by a four-step cycle. Membrane tethering by the PH domain and the FFAT motif enables sterol transfer by the lipid transfer domain (ORD), followed by back transfer of PI(4)P by the ORD. Finally, PI(4)P is hydrolyzed in cis by the ER protein Sac1. The energy provided by PI(4)P hydrolysis drives sterol transfer and allows negative feedback when PI(4)P becomes limiting. Other lipid transfer proteins are tethered by the same mechanism. Thus, OSBP-mediated back transfer of PI(4)P might coordinate the transfer of other lipid species at the ER-Golgi interface.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Citosol/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de Esteroides/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismo
3.
Annu Rev Biochem ; 80: 101-23, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21438688

RESUMO

Bacteria and eukaryotic cells contain geometry-sensing tools in their cytosol: protein motifs or domains that recognize the curvature, concave or convex, deep or shallow, of lipid membranes. These sensors contrast with classical lipid-binding domains by their extended structure and, sometimes, counterintuitive chemistry. Among the sensors are long amphipathic helices, such as the ALPS motif and the N-terminal region of α-synuclein, whose apparent "design defects" translate into a remarkable ability to specifically adsorb to the surface of small vesicles. Fundamental differences in the lipid composition of membranes of the early and late secretory pathways probably explain why some sensors use mostly electrostatics whereas others take advantage of the hydrophobic effect. Membrane curvature sensors help to organize very diverse reactions, such as lipid transfer between membranes, the tethering of vesicles at the Golgi apparatus, and the assembly-disassembly cycle of protein coats.


Assuntos
Membrana Celular/ultraestrutura , Lipídeos de Membrana/química , Proteínas de Membrana/química , Adsorção , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Membrana Celular/química , Proteínas Ativadoras de GTPase/metabolismo , Bicamadas Lipídicas/química , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , alfa-Sinucleína/química
4.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34878112

RESUMO

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (DHA-PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses showed that human umbilical vein endothelial cells (HUVECs) subjected to a DHA diet undergo a 6-fold enrichment in DHA-PLs at the plasma membrane (PM) at the expense of monounsaturated oleic acid-containing PLs (OA-PLs). Consequently, DHA-PL enrichment at the PM induces a reduction in cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM lead to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PL levels in membranes affect cell biomechanical properties.


Assuntos
Ácidos Docosa-Hexaenoicos , Fosfolipídeos , Animais , Membrana Celular/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Células Endoteliais/metabolismo , Humanos , Fusão de Membrana , Fosfolipídeos/metabolismo
5.
J Biol Chem ; 298(7): 102136, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35714773

RESUMO

Tumor protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54, and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical (e.g., COPI and COPII) transport vesicles. Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by four amphipathic helices (AH1-4), which could fold upon binding to lipid membranes. Limited proteolysis, CD spectroscopy, tryptophan fluorescence, and cysteine mutagenesis coupled to covalent binding of a membrane-sensitive probe showed that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. Furthermore, site-directed mutagenesis indicated that AH2 and AH3 have a predominant role in TPD54 binding to membranes both in cells and using model liposomes. We found that AH3 has the physicochemical features of an amphipathic lipid packing sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Accordingly, we observed that binding of TPD54 to liposomes is very sensitive to membrane curvature and lipid unsaturation. We conclude that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and ALPS-independent mechanisms.


Assuntos
Lipossomos , Proteínas de Neoplasias , Lipídeos , Lipossomos/química , Membranas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Vesículas Transportadoras/metabolismo
6.
Cell ; 135(7): 1163-5, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-19109885

RESUMO

During membrane fission, the GTPase dynamin forms helical assemblies at the neck of membrane buds. Although it has been proposed that fission results from the constriction of the dynamin helix, new work by Bashkirov et al. (2008) and Pucadyil and Schmid (2008) unexpectedly shows that helix disassembly is also necessary for membrane fission.


Assuntos
Dinaminas/metabolismo , Membranas Intracelulares/metabolismo , Animais , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Modelos Biológicos
7.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33666950

RESUMO

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Assuntos
COVID-19/virologia , Biblioteca de Peptídeos , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Células A549 , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem com Lapso de Tempo , Proteínas Virais/genética , Proteína Vermelha Fluorescente
8.
J Biol Chem ; 295(13): 4277-4288, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32075908

RESUMO

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.


Assuntos
Transporte Biológico/efeitos dos fármacos , Lipídeos/genética , Receptores de Esteroides/metabolismo , Estilbenos/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Fluorescência , Humanos , Lipídeos/química , Ligação Proteica/genética , Transporte Proteico/genética , Receptores de Esteroides/química , Estilbenos/química , Rede trans-Golgi/química , Rede trans-Golgi/genética
9.
EMBO J ; 36(21): 3156-3174, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-28978670

RESUMO

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIIIß triggers large periodic traveling waves of PI4P across the TGN These waves are cadenced by long-range PI4P production by PI4KIIα and PI4P consumption by OSBP Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes.


Assuntos
Colesterol/metabolismo , Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Esteroides/metabolismo , Transporte Biológico , Colestenonas/farmacologia , Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Dicarbetoxi-Di-Hidrocolidina/química , Retículo Endoplasmático/metabolismo , Células Epiteliais/citologia , Corantes Fluorescentes/química , Expressão Gênica , Células HeLa , Humanos , Gotículas Lipídicas/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/genética , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Saponinas/farmacologia , Imagem com Lapso de Tempo , Rede trans-Golgi/metabolismo
10.
EMBO J ; 35(21): 2270-2284, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27670760

RESUMO

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.


Assuntos
Membrana Celular/fisiologia , Dinaminas/fisiologia , Animais , Guanosina Trifosfato/fisiologia , Humanos
11.
Soft Matter ; 16(7): 1722-1730, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31916552

RESUMO

In the cell, membrane deformation and fission (collectively referred to as 'budding') is driven by specific protein machineries but is also influenced by lipid composition. We previously reported that phospholipids with polyunsaturated acyl chains facilitate membrane budding because they adapt their shape to membrane curvature, thereby decreasing membrane bending rigidity. The facilitating effect of polyunsaturated lipids was observed in experiments and simulations performed on membranes where the two bilayer leaflets had the same lipid composition. However, biological membranes are generally asymmetric. Here, we present coarse-grained molecular dynamics simulations on asymmetric phospholipid bilayers undergoing deformation via a pulling force along the bilayer normal. One leaflet contains monounsaturated C18:0-C18:1-phospholipids, whereas the opposite leaflet contains polyunsaturated C18:0-C22:6-phospholipids. When present in the monolayer orientated towards the pulling force and thereby in the convex face of the forming tube, C18:0-C22:6-phospholipids facilitate membrane tubulation. In contrast, C18:0-C22:6-phospholipids in the concave face of the tube have no effect. Analysis of lipid shape indicates that these contrasting effects arise from the superior ability of polyunsaturated phospholipids to swell in the convex leaflet, whereas mono and polyunsaturated phospholipids behave similarly in the concave leaflet. The leaflet-dependent effect of polyunsaturated phospholipids matches well their asymmetric distribution in biological membranes, notably in synaptic vesicles, which are produced by the fastest budding event in the body.


Assuntos
Membrana Celular/química , Endocitose , Bicamadas Lipídicas/química , Fosfolipídeos/química , Membrana Celular/ultraestrutura , Fusão de Membrana , Simulação de Dinâmica Molecular
12.
Biophys J ; 115(3): 436-444, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30055754

RESUMO

The analysis of the structural organization of lipid bilayers is generally performed across the direction normal to the bilayer/water interface, whereas the surface properties of the bilayer at the interface with water are often neglected. Here, we present PackMem, a bioinformatic tool that performs a topographic analysis of the bilayer surface from various molecular dynamics simulations. PackMem unifies and rationalizes previous analyses based on a Cartesian grid. The grid allows identification of surface regions defined as lipid-packing defects where lipids are loosely packed, leading to cavities in which aliphatic carbons are exposed to the solvent, either deep inside or close to the membrane surface. Examples are provided to show that the abundance of lipid-packing defects varies according to the temperature and to the bilayer composition. Because lipid-packing defects control the adsorption of peripheral proteins with hydrophobic insertions, PackMem is instrumental for us to understand and quantify the adhesive properties of biological membranes as well as their response to mechanical perturbations such as membrane deformation.


Assuntos
Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Propriedades de Superfície , Temperatura , Água/química
13.
J Cell Sci ; 129(12): 2368-81, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27142833

RESUMO

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.


Assuntos
Membrana Celular/metabolismo , Lipídeos/toxicidade , Saccharomyces cerevisiae/metabolismo , Membrana Celular/efeitos dos fármacos , Diglicerídeos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Metaboloma/efeitos dos fármacos , Metabolômica , Farmacogenética , Saccharomyces cerevisiae/efeitos dos fármacos , Via Secretória/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Interface Usuário-Computador
14.
Trends Biochem Sci ; 38(11): 576-84, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24054463

RESUMO

Many cellular processes require membrane deformation, which is driven by specialized protein machinery and can often be recapitulated using pure lipid bilayers. However, biological membranes contain a large amount of embedded proteins. Recent research suggests that membrane-bound proteins with asymmetric distribution of mass across the bilayer can influence membrane bending in a nonspecific manner due to molecular crowding. This mechanism is physical in nature and arises from collisions between such 'mushroom-shaped' proteins. It can either facilitate or impede the action of protein coats, for example COPII, during vesicle budding. We describe the physics of how molecular crowding can influence membrane bending and discuss the implications for other cellular processes, such as sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and production of intraluminal vesicles.


Assuntos
Membrana Celular/fisiologia , Proteínas de Membrana/fisiologia , Bicamadas Lipídicas
15.
Biochim Biophys Acta ; 1861(8 Pt B): 940-951, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26928592

RESUMO

Cholesterol levels in intracellular membranes are constantly adjusted to match with specific organelle functions. Cholesterol is kept high in the plasma membrane (PM) because it is essential for its barrier function, while low levels are found in the endoplasmic reticulum (ER) where cholesterol mediates feedback control of its own synthesis by sterol-sensor proteins. The ER→Golgi→PM concentration gradient of cholesterol in mammalian cells, and ergosterol in yeast, appears to be sustained by specific intracellular transport processes, which are mostly mediated by lipid transfer proteins (LTPs). Here we review a recently described function of two LTPs, OSBP and its yeast homolog Osh4p, which consists in creating a sterol gradient between membranes by vectorial transport. OSBP also contributes to the formation of ER/Golgi membrane contact sites, which are important hubs for the transfer of several lipid species. OSBP and Osh4p organize a counterflow transport of lipids whereby sterols are exchanged for the phosphoinositide PI4P, which is used as a fuel to drive sterol transport. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.


Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Transporte Biológico Ativo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo
16.
Proc Natl Acad Sci U S A ; 110(33): 13244-9, 2013 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-23901109

RESUMO

Intracellular trafficking between organelles is achieved by coat protein complexes, coat protomers, that bud vesicles from bilayer membranes. Lipid droplets are protected by a monolayer and thus seem unsuitable targets for coatomers. Unexpectedly, coat protein complex I (COPI) is required for lipid droplet targeting of some proteins, suggesting a possible direct interaction between COPI and lipid droplets. Here, we find that COPI coat components can bud 60-nm triacylglycerol nanodroplets from artificial lipid droplet (LD) interfaces. This budding decreases phospholipid packing of the monolayer decorating the mother LD. As a result, hydrophobic triacylglycerol molecules become more exposed to the aqueous environment, increasing LD surface tension. In vivo, this surface tension increase may prime lipid droplets for reactions with neighboring proteins or membranes. It provides a mechanism fundamentally different from transport vesicle formation by COPI, likely responsible for the diverse lipid droplet phenotypes associated with depletion of COPI subunits.


Assuntos
Complexo I de Proteína do Envoltório/química , Bicamadas Lipídicas/química , Transporte Proteico/fisiologia , Vesículas Transportadoras/química , Fator 1 de Ribosilação do ADP/genética , Animais , Escherichia coli , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica , Fosfatidilcolinas , Fosfatidiletanolaminas , Fosfolipídeos/química , Células Sf9 , Espectrometria de Fluorescência , Spodoptera , Tensão Superficial , Triglicerídeos/química , Água/química
17.
J Membr Biol ; 248(4): 611-40, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26063070

RESUMO

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.


Assuntos
Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Modelos Químicos , Animais , Humanos , Proteínas de Membrana Transportadoras/genética , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
18.
PLoS Pathog ; 9(11): e1003747, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244168

RESUMO

The intracellular bacterial pathogen Legionella pneumophila (Lp) evades destruction in macrophages by camouflaging in a specialized organelle, the Legionella-containing vacuole (LCV), where it replicates. The LCV maturates by incorporating ER vesicles, which are diverted by effectors that Lp injects to take control of host cell membrane transport processes. One of these effectors, RalF, recruits the trafficking small GTPase Arf1 to the LCV. LpRalF has a Sec7 domain related to host ArfGEFs, followed by a capping domain that intimately associates with the Sec7 domain to inhibit GEF activity. How RalF is activated to function as a LCV-specific ArfGEF is unknown. We combined the reconstitution of Arf activation on artificial membranes with cellular expression and Lp infection assays, to analyze how auto-inhibition is relieved for LpRalF to function in vivo. We find that membranes activate LpRalF by about 1000 fold, and identify the membrane-binding region as the region that inhibits the Sec7 active site. It is enriched in aromatic and positively charged residues, which establish a membrane sensor to control the GEF activity in accordance with specific lipid environments. A similar mechanism of activation is found in RalF from Rickettsia prowazekii (Rp), with a different aromatic/charged residues ratio that results in divergent membrane preferences. The membrane sensor is the primary determinant of the localization of LpRalF on the LCV, and drives the timing of Arf activation during infection. Finally, we identify a conserved motif in the capping domain, remote from the membrane sensor, which is critical for RalF activity presumably by organizing its active conformation. These data demonstrate that RalF proteins are regulated by a membrane sensor that functions as a binary switch to derepress ArfGEF activity when RalF encounters a favorable lipid environment, thus establishing a regulatory paradigm to ensure that Arf GTPases are efficiently activated at specific membrane locations.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Legionella pneumophila/química , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Legionella pneumophila/genética , Doença dos Legionários/genética , Estrutura Terciária de Proteína , Rickettsia prowazekii/genética , Rickettsia prowazekii/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/microbiologia
19.
EMBO J ; 29(2): 292-303, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-19927117

RESUMO

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1-GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R approximately 35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1-GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1-GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Lipídeos de Membrana/metabolismo , Lipossomas Unilamelares/metabolismo , Fator 1 de Ribosilação do ADP/análise , Motivos de Aminoácidos , Complexo I de Proteína do Envoltório/análise , Difusão , Proteínas Ativadoras de GTPase/análise , Complexo de Golgi/metabolismo , Lipídeos de Membrana/análise , Pinças Ópticas , Ligação Proteica , Lipossomas Unilamelares/análise
20.
Biochem Soc Trans ; 42(5): 1465-70, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25233433

RESUMO

In eukaryotic cells, a sterol gradient exists between the early and late regions of the secretory pathway. This gradient seems to rely on non-vesicular transport mechanisms mediated by specialized carriers. The oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) family has been assumed initially to exclusively include proteins acting as sterol sensors/transporters and many efforts have been made to determine their mode of action. Our recent studies have demonstrated that some ORP/Osh proteins are not mere sterol transporters, but sterol/phosphatidylinositol 4-phosphate [PI(4)P] exchangers. They exploit the PI(4)P gradient at the endoplasmic reticulum (ER)/Golgi interface, or at membrane-contact sites between these compartments, to actively create a sterol gradient. Other recent reports have suggested that all ORP/Osh proteins bind PI(4)P and recognize a second lipid that is not necessary sterol. We have thus proposed that ORP/Osh proteins use PI(4)P gradients between organelles to convey various lipid species.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Receptores de Esteroides/metabolismo , Animais , Transporte Biológico , Humanos , Ligantes , Proteínas de Membrana/química , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/química
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