Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Nature ; 604(7905): 384-390, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35388214

RESUMO

G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates1,2 and roles for lipids in signalling3,4. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal-lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin-opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments.


Assuntos
Opsinas , Rodopsina , Transducina , Isomerismo , Metabolismo dos Lipídeos , Opsinas/metabolismo , Disco Óptico , Fosfatidilcolinas , Conformação Proteica , Receptores Acoplados a Proteínas G , Rodopsina/química
2.
Nature ; 575(7781): 234-237, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31666700

RESUMO

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.


Assuntos
Microscopia Crioeletrônica , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Subunidades Proteicas/química , Animais , Galinhas , Anemia de Fanconi/enzimologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/ultraestrutura , Espectrometria de Massas , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , Ubiquitinação
3.
Nature ; 564(7736): 372-377, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30542154

RESUMO

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which-together with computational studies-elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. 'Hot spots' of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects.


Assuntos
Conexinas/química , Conexinas/ultraestrutura , Microscopia Crioeletrônica , Cristalino/citologia , Cristalino/ultraestrutura , Sequência de Aminoácidos , Catarata/congênito , Catarata/genética , Conexina 26/química , Conexinas/genética , Junções Comunicantes/química , Junções Comunicantes/genética , Junções Comunicantes/ultraestrutura , Humanos , Cristalino/química , Modelos Moleculares , Mutação
4.
Nature ; 541(7637): 407-411, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28077872

RESUMO

The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota, which has an important role in human health and nutrition. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA-SusG. Glycan degradation mainly occurs intracellularly and depends on the import of oligosaccharides by an outer membrane protein complex composed of an extracellular SusD-like lipoprotein and an integral membrane SusC-like TonB-dependent transporter. The presence of the partner SusD-like lipoprotein is the major feature that distinguishes SusC-like proteins from previously characterized TonB-dependent transporters. Many sequenced gut Bacteroides spp. encode over 100 SusCD pairs, of which the majority have unknown functions and substrate specificities. The mechanism by which extracellular substrate binding by SusD proteins is coupled to outer membrane passage through their cognate SusC transporter is unknown. Here we present X-ray crystal structures of two functionally distinct SusCD complexes purified from Bacteroides thetaiotaomicron and derive a general model for substrate translocation. The SusC transporters form homodimers, with each ß-barrel protomer tightly capped by SusD. Ligands are bound at the SusC-SusD interface in a large solvent-excluded cavity. Molecular dynamics simulations and single-channel electrophysiology reveal a 'pedal bin' mechanism, in which SusD moves away from SusC in a hinge-like fashion in the absence of ligand to expose the substrate-binding site to the extracellular milieu. These data provide mechanistic insights into outer membrane nutrient import by members of the microbiota, an area of major importance for understanding human-microbiota symbiosis.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteroides/química , Bacteroides/metabolismo , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/microbiologia , Polissacarídeos/metabolismo , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Eletrofisiologia , Humanos , Ligantes , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Nat Chem Biol ; 16(12): 1285-1292, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33199903

RESUMO

Within cell membranes numerous protein assemblies reside. Among their many functions, these assemblies regulate the movement of molecules between membranes, facilitate signaling into and out of cells, allow movement of cells by cell-matrix attachment, and regulate the electric potential of the membrane. With such critical roles, membrane protein complexes are of considerable interest for human health, yet they pose an enduring challenge for structural biologists because it is difficult to study these protein structures at atomic resolution in in situ environments. To advance structural and functional insights for these protein assemblies, membrane mimetics are typically employed to recapitulate some of the physical and chemical properties of the lipid bilayer membrane. However, extraction from native membranes can sometimes change the structure and lipid-binding properties of these complexes, leading to conflicting results and fueling a drive to study complexes directly from native membranes. Here we consider the co-development of membrane mimetics with technological breakthroughs in both cryo-electron microscopy (cryo-EM) and native mass spectrometry (nMS). Together, these developments are leading to a plethora of high-resolution protein structures, as well as new knowledge of their lipid interactions, from different membrane-like environments.


Assuntos
Membrana Celular/química , Células Eucarióticas/metabolismo , Lipídeos de Membrana/química , Proteínas de Membrana/química , Animais , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Detergentes/química , Células Eucarióticas/citologia , Humanos , Lipídeos de Membrana/classificação , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membranas Artificiais , Micelas , Modelos Moleculares , Estrutura Secundária de Proteína , Transdução de Sinais , Thermus thermophilus/metabolismo , Thermus thermophilus/ultraestrutura
6.
Proteomics ; 15(16): 2777-91, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25727951

RESUMO

Although the number of protein-encoding genes in the human genome is only about 20 000 not far from the amount found in the nematode worm genome, the number of proteins that are translated from these sequences is larger by several orders of magnitude. A number of mechanisms have evolved to enable this diversity. For example, genes can be alternatively spliced to create multiple transcripts; they may also be translated from different alternative initiation sites. After translation, hundreds of chemical modifications can be introduced in proteins, altering their chemical properties, folding, stability, and activity. The complexity is then further enhanced by the various combinations that are generated from the assembly of different subunit variants into protein complexes. This, in turn, confers structural and functional flexibility, and endows the cell with the ability to adapt to various environmental conditions. Therefore, exposing the variability of protein complexes is an important step toward understanding their biological functions. Revealing this enormous diversity, however, is not a simple task. In this review, we will focus on the array of MS-based strategies that are capable of performing this mission. We will also discuss the challenges that lie ahead, and the future directions toward which the field might be heading.


Assuntos
Espectrometria de Massas/métodos , Conformação Proteica , Subunidades Proteicas/química , Biologia Computacional/métodos , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/análise
7.
Nat Protoc ; 15(5): 1690-1706, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32238951

RESUMO

Recent applications of mass spectrometry (MS) to study membrane protein complexes are yielding valuable insights into the binding of lipids and their structural and functional roles. To date, most native MS experiments with membrane proteins are based on detergent solubilization. Many insights into the structure and function of membrane proteins have been obtained using detergents; however, these can promote local lipid rearrangement and can cause fluctuations in the oligomeric state of protein complexes. To overcome these problems, we developed a method that does not use detergents or other chemicals. Here we report a detailed protocol that enables direct ejection of protein complexes from membranes for analysis by native MS. Briefly, lipid vesicles are prepared directly from membranes of different sources and subjected to sonication pulses. The resulting destabilized vesicles are concentrated, introduced into a mass spectrometer and ionized. The mass of the observed protein complexes is determined and this information, in conjunction with 'omics'-based strategies, is used to determine subunit stoichiometry as well as cofactor and lipid binding. Within this protocol, we expand the applications of the method to include peripheral membrane proteins of the S-layer and amyloid protein export machineries overexpressed in membranes from which the most abundant components have been removed. The described experimental procedure takes approximately 3 d from preparation to MS. The time required for data analysis depends on the complexity of the protein assemblies embedded in the membrane under investigation.


Assuntos
Espectrometria de Massas/métodos , Proteínas de Membrana/análise , Vesículas Citoplasmáticas , Sonicação
8.
Science ; 366(6466)2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31699906

RESUMO

Hirst et al claim that proteins ejected directly from mitochondrial membranes in our study are degraded, are incorrectly assigned, lack lipids, and show discrepancies with "native states" mostly obtained in detergent micelles. Here, we add further evidence in full support of our assignments and show that all complexes are either ejected intact or in known intermediate states, with core subunit interactions maintained. None are degraded or rearranged.


Assuntos
Detergentes , Proteínas de Membrana , Lipídeos , Espectrometria de Massas , Membranas Mitocondriais
9.
Nat Commun ; 10(1): 1130, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850607

RESUMO

Conserved lipid transfer proteins of the Ups/PRELI family regulate lipid accumulation in mitochondria by shuttling phospholipids in a lipid-specific manner across the intermembrane space. Here, we combine structural analysis, unbiased genetic approaches in yeast and molecular dynamics simulations to unravel determinants of lipid specificity within the conserved Ups/PRELI family. We present structures of human PRELID1-TRIAP1 and PRELID3b-TRIAP1 complexes, which exert lipid transfer activity for phosphatidic acid and phosphatidylserine, respectively. Reverse yeast genetic screens identify critical amino acid exchanges that broaden and swap their lipid specificities. We find that amino acids involved in head group recognition and the hydrophobicity of flexible loops regulate lipid entry into the binding cavity. Molecular dynamics simulations reveal different membrane orientations of PRELID1 and PRELID3b during the stepwise release of lipids. Our experiments thus define the structural determinants of lipid specificity and the dynamics of lipid interactions by Ups/PRELI proteins.


Assuntos
Proteínas de Transporte/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Mitocondriais/química , Ácidos Fosfatídicos/química , Fosfatidilserinas/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Ácidos Fosfatídicos/metabolismo , Fosfatidilserinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
10.
Int J Biochem Cell Biol ; 40(10): 2040-52, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18450498

RESUMO

The strength and duration of intracellular signals must be precisely regulated, since inappropriate signaling can cause disease. Negative feedback mechanisms provide an effective means of controlling growth factor-mediated signaling, either by restricting the incoming signal or by inducing counter-regulatory mechanisms affecting signal propagation. Sef proteins represent a new class of feedback antagonists capable of regulating receptor tyrosine kinase signaling. The involvement of Sef in development, as well as in other biological processes, was demonstrated by biochemical and genetic approaches.


Assuntos
Retroalimentação Fisiológica , Proteínas de Membrana/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais , Sequência de Aminoácidos , Animais , Desenvolvimento Embrionário , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Receptores Proteína Tirosina Quinases/metabolismo
11.
Sci Rep ; 8(1): 2693, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29426917

RESUMO

Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the detailed molecular mechanisms underlying FA mechanosensitivity are still unclear. Besides talin, another key candidate for regulating FA-associated mechanosensing, is vinculin, a prominent FA component, which possesses either closed ("auto-inhibited") or open ("active") conformation. A direct experimental demonstration, however, of the conformational transition between the two states is still absent. In this study, we combined multiple structural and biological approaches to probe the transition from the auto-inhibited to the active conformation, and determine its effects on FA structure and dynamics. We further show that the transition from a closed to an open conformation requires two sequential steps that can differentially regulate FA growth and stability.


Assuntos
Adesões Focais/fisiologia , Adesões Focais/ultraestrutura , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Matriz Extracelular/metabolismo , Fibroblastos , Adesões Focais/metabolismo , Células HeLa , Humanos , Integrinas/metabolismo , Camundongos , Conformação Molecular , Ligação Proteica/fisiologia , Talina/metabolismo , Vinculina/química , Vinculina/fisiologia , Vinculina/ultraestrutura
12.
Science ; 362(6416): 829-834, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30442809

RESUMO

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the ß-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F1FO adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid-bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome.


Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Chaperonas Moleculares/metabolismo , Canais de Translocação SEC/metabolismo , Translocador 1 do Nucleotídeo Adenina/química , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Bovinos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espectrometria de Massas , Proteínas de Membrana/química , Membranas Mitocondriais/química , ATPases Mitocondriais Próton-Translocadoras/química , Chaperonas Moleculares/química , Porinas/química , Porinas/metabolismo , Conformação Proteica em Folha beta , Proteoma/química , Proteoma/metabolismo , Canais de Translocação SEC/química
13.
Nat Commun ; 8(1): 263, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28811582

RESUMO

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated ß-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.Gram-negative bacteria assemble biofilms from amyloid fibres, which translocate across the outer membrane as unfolded amyloid precursors through a secretion system. Here, the authors characterise the structural details of the amyloid transporter FapF in Pseudomonas.


Assuntos
Amiloide/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Pseudomonas/metabolismo , Amiloide/química , Amiloide/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/química , Sistemas de Secreção Bacterianos/genética , Biofilmes , Cristalografia por Raios X , Conformação Proteica , Transporte Proteico , Pseudomonas/química , Pseudomonas/genética
14.
Nat Commun ; 5: 3758, 2014 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-24781749

RESUMO

Focal adhesions (FAs) are large multi-protein complexes that act as transmembrane links between the extracellular matrix and the actin cytoskeleton. Recently, FAs were extensively characterized, yet the molecular mechanisms underlying their mechanical and signalling functions remain unresolved. To address this question, we isolated protein complexes containing different FA components, from chicken smooth muscle, and characterized their properties. Here we identified 'hybrid complexes' consisting of the actin-nucleating subunits of Arp2/3 and either vinculin or vinculin and α-actinin. We further show that suppression of p41-ARC, a central component of native Arp2/3, which is absent from the hybrid complexes, increases the levels of the Arp2/3-nucleating core in FA sites and stimulates FA growth and dynamics. In contrast, overexpression of p41-ARC adversely affects FAs. These results support the view that Arp2/3 can form modular 'hybrid complexes' containing an actin-nucleating 'functional core', and 'anchoring domains' (vinculin/p41-ARC) that regulate its subcellular localization.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Adesões Focais/fisiologia , Músculo Liso/metabolismo , Vinculina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinina/metabolismo , Animais , Galinhas , Fluoresceína-5-Isotiocianato , Células HeLa , Humanos , Imuno-Histoquímica , Imunoprecipitação , Espectrometria de Massas , Microscopia , RNA Interferente Pequeno/genética , Vinculina/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA