RESUMO
Many bacterial pathogens reorganize the host actin cytoskeleton during the course of infection, including enterohemorrhagic Escherichia coli (EHEC), which utilizes the effector protein EspF(U) to assemble actin filaments within plasma membrane protrusions called pedestals. EspF(U) activates N-WASP, a host actin nucleation-promoting factor that is normally auto-inhibited and found in a complex with the actin-binding protein WIP. Under native conditions, this N-WASP/WIP complex is activated by the small GTPase Cdc42 in concert with several different SH3 (Src-homology-3) domain-containing proteins. In the current study, we tested whether SH3 domains from the F-BAR (FCH-Bin-Amphiphysin-Rvs) subfamily of membrane-deforming proteins are involved in actin pedestal formation. We found that three F-BAR proteins: CIP4, FBP17, and TOCA1 (transducer of Cdc42-dependent actin assembly), play different roles during actin pedestal biogenesis. Whereas CIP4 and FBP17 inhibited actin pedestal assembly, TOCA1 stimulated this process. TOCA1 was recruited to pedestals by its SH3 domain, which bound directly to proline-rich sequences within EspF(U). Moreover, EspF(U) and TOCA1 activated the N-WASP/WIP complex in an additive fashion in vitro, suggesting that TOCA1 can augment actin assembly within pedestals. These results reveal that EspF(U) acts as a scaffold to recruit multiple actin assembly factors whose functions are normally regulated by Cdc42.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Escherichia coli Êntero-Hemorrágica/genética , Infecções por Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Ligação a Ácido Graxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Antígenos de Histocompatibilidade Menor , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/genética , Domínios de Homologia de srcRESUMO
Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspF(U), a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspF(U) repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspF(U) are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspF(U) fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspF(U). Whereas clustering of a single EspF(U) repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspF(U) derivatives promote actin assembly more efficiently. Moreover, the EspF(U) repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3-mediated signaling pathways.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Sequências Repetitivas de Aminoácidos , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli Êntero-Hemorrágica/genética , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , SuínosRESUMO
The lack of established standards to describe and annotate biological assays and screening outcomes in the domain of drug and chemical probe discovery is a severe limitation to utilize public and proprietary drug screening data to their maximum potential. We have created the BioAssay Ontology (BAO) project (http://bioassayontology.org) to develop common reference metadata terms and definitions required for describing relevant information of low-and high-throughput drug and probe screening assays and results. The main objectives of BAO are to enable effective integration, aggregation, retrieval, and analyses of drug screening data. Since we first released BAO on the BioPortal in 2010 we have considerably expanded and enhanced BAO and we have applied the ontology in several internal and external collaborative projects, for example the BioAssay Research Database (BARD). We describe the evolution of BAO with a design that enables modeling complex assays including profile and panel assays such as those in the Library of Integrated Network-based Cellular Signatures (LINCS). One of the critical questions in evolving BAO is the following: how can we provide a way to efficiently reuse and share among various research projects specific parts of our ontologies without violating the integrity of the ontology and without creating redundancies. This paper provides a comprehensive answer to this question with a description of a methodology for ontology modularization using a layered architecture. Our modularization approach defines several distinct BAO components and separates internal from external modules and domain-level from structural components. This approach facilitates the generation/extraction of derived ontologies (or perspectives) that can suit particular use cases or software applications. We describe the evolution of BAO related to its formal structures, engineering approaches, and content to enable modeling of complex assays and integration with other ontologies and datasets.