Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Struct Mol Biol ; 23(9): 821-9, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27478930

RESUMO

Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during endocytosis. Here, we exploited the exceptional features of these coats to test three models-Brownian ratchet, power-stroke and entropic pulling-proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models and instead support a collision-pressure mechanism whereby collisions between clathrin-coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also found that self-association augments collision pressure, thereby allowing disassembly of clathrin lattices that have been predicted to be resistant to disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.


Assuntos
Cadeias Pesadas de Clatrina/química , Proteínas de Choque Térmico HSP72/fisiologia , Multimerização Proteica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Entropia , Proteínas de Choque Térmico HSP72/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Tamanho da Partícula , Domínios Proteicos , Estabilidade Proteica , Estrutura Quaternária de Proteína , Ratos
2.
J Neurosci ; 33(18): 8009-21, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23637191

RESUMO

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. After the internalization of a clathrin-coated vesicle, the vesicle must uncoat to replenish the pool of SVs. Clathrin-coated vesicle uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo and whether they play such roles in SV endocytosis in particular is unknown. To address this question, we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:co-chaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so that it could no longer bind and inhibit Hsp110 (a NEF that we find to be highly abundant in brain cytosol), its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis.


Assuntos
Endocitose/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Vesículas Sinápticas/fisiologia , Potenciais de Ação/fisiologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Auxilinas/farmacologia , Encéfalo/citologia , Bovinos , Clatrina/metabolismo , Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP110/metabolismo , Humanos , Lampreias , Larva , Masculino , Modelos Moleculares , Mutação/genética , Ligação Proteica/genética , Medula Espinal/citologia
3.
Mol Cell ; 31(2): 232-43, 2008 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-18550409

RESUMO

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein's NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.


Assuntos
Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSP110/química , Nucleotídeos/metabolismo , Animais , Bovinos , Clatrina/metabolismo , Cristalografia por Raios X , Dimerização , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Soluções
4.
J Biol Chem ; 278(35): 33583-92, 2003 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-12807910

RESUMO

Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly. We found that although Eps15 has no intrinsic ability to assemble clathrin, it potently stimulates the ability of the clathrin adaptor protein, AP180, to assemble clathrin at physiological pH. We have also defined the binding sites for Eps15 on squid AP180. These sites contain an NPF motif, and peptides derived from these binding sites inhibit the ability of Eps15 to stimulate clathrin assembly in vitro. Furthermore, when injected into squid giant presynaptic nerve terminals, these peptides inhibit the formation of clathrin-coated pits and coated vesicles during synaptic vesicle endocytosis. This is consistent with the hypothesis that Eps15 regulates clathrin coat assembly in vivo, and indicates that interactions between Eps15 homology domains and NPF motifs are involved in clathrin-coated vesicle formation during synaptic vesicle recycling.


Assuntos
Proteínas de Ligação ao Cálcio/química , Clatrina/química , Fosfoproteínas/química , Vesículas Sinápticas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Decapodiformes , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Endocitose , Glicerol/química , Humanos , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Eletrônica , Proteínas Monoméricas de Montagem de Clatrina/química , Peptídeos/química , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...