RESUMO
Small GTPases, in association with their GEFs, GAPs and effectors, control major intracellular processes such as signal transduction, cytoskeletal dynamics and membrane trafficking. Accordingly, dysfunctions in their biochemical properties are associated with many diseases, including cancers, diabetes, infections, mental disorders and cardiac diseases, which makes them attractive targets for therapies. However, small GTPases signalling modules are not well-suited for classical inhibition strategies due to their mode of action that combines protein-protein and protein-membrane interactions. As a consequence, there is still no validated drug available on the market that target small GTPases, whether directly or through their regulators. Alternative inhibitory strategies are thus highly needed. Here we review recent studies that highlight the unique modalities of the interaction of small GTPases and their GEFs at the periphery of membranes, and discuss how they can be harnessed in drug discovery.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Membrana Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas/química , Transdução de Sinais , Animais , Sítios de Ligação , Membrana Celular/química , Movimento Celular , Citoesqueleto/metabolismo , Dimerização , Desenho de Fármacos , Descoberta de Drogas , GTP Fosfo-Hidrolases/química , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Lipídeos/química , Glicoproteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Sulfotransferases/metabolismoRESUMO
Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein-membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2-Bragsin complex and structure-activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein-membrane interactions without disruption.
Assuntos
Fator 1 de Ribosilação do ADP/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Fator 1 de Ribosilação do ADP/metabolismo , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Células HeLa , Humanos , Bicamadas Lipídicas , Glicoproteínas de Membrana/metabolismo , Nucleotídeos , Domínios de Homologia à Plecstrina/fisiologia , Ligação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Sulfotransferases/metabolismoRESUMO
Osteoporosis is caused by excessive activity of bone-degrading osteoclasts over bone-forming osteoblast. Standard antiosteolytic treatments inhibit bone resorption by inducing osteoclast loss, with the adverse effect of hindering also bone formation. Formation of the osteoclast sealing zone requires Dock5, a guanine nucleotide exchange factor for the small GTPase Rac, and C21, a chemical inhibitor of Dock5, decreases bone resorption by cultured osteoclasts. Here we show that C21 directly inhibits the exchange activity of Dock5 and disrupts osteoclast podosome organization. Remarkably, C21 administration protects mice against bone degradation in models recapitulating major osteolytic diseases: menopause, rheumatoid arthritis and bone metastasis. Furthermore, C21 administration does not affect bone formation and is not toxic. Our results validate the pharmacological inhibition of Dock5 as a novel therapeutic route for fighting osteolytic diseases while preserving bone formation.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteólise/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológico , Sulfonamidas/química , Sulfonamidas/farmacologia , BenzenossulfonamidasRESUMO
Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
Assuntos
Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Resistência a Medicamentos/genética , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Haploinsuficiência , Humanos , Farmacogenética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
The mechanisms whereby guanine nucleotide exchange factors (GEFs) coordinate their subcellular targeting to their activation of small GTPases remain poorly understood. Here we analyzed how membranes control the efficiency of human BRAG2, an ArfGEF involved in receptor endocytosis, Wnt signaling, and tumor invasion. The crystal structure of an Arf1-BRAG2 complex that mimics a membrane-bound intermediate revealed an atypical PH domain that is constitutively anchored to the catalytic Sec7 domain and interacts with Arf. Combined with the quantitative analysis of BRAG2 exchange activity reconstituted on membranes, we find that this PH domain potentiates nucleotide exchange by about 2,000-fold by cumulative conformational and membrane-targeting contributions. Furthermore, it restricts BRAG2 activity to negatively charged membranes without phosphoinositide specificity, using a positively charged surface peripheral to but excluding the canonical lipid-binding pocket. This suggests a model of BRAG2 regulation along the early endosomal pathway that expands the repertoire of GEF regulatory mechanisms. Notably, it departs from the auto-inhibitory and feedback loop paradigm emerging from studies of SOS and cytohesins. It also uncovers a novel mechanism of unspecific lipid-sensing by PH domains that may allow sustained binding to maturating membranes.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Metabolismo dos Lipídeos , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Fator 1 de Ribosilação do ADP/ultraestrutura , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/ultraestrutura , Cristalografia por Raios X , Endocitose , Endossomos , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Conformação Proteica , Estrutura Terciária de Proteína , Via de Sinalização WntRESUMO
Forkhead box O (FOXO) transcription factors favor both T cell quiescence and trafficking through their control of the expression of genes involved in cell cycle progression, adhesion, and homing. In this article, we report that the product of the fam65b gene is a new transcriptional target of FOXO1 that regulates RhoA activity. We show that family with sequence similarity 65 member b (Fam65b) binds the small GTPase RhoA via a noncanonical domain and represses its activity by decreasing its GTP loading. As a consequence, Fam65b negatively regulates chemokine-induced responses, such as adhesion, morphological polarization, and migration. These results show the existence of a new functional link between FOXO1 and RhoA pathways, through which the FOXO1 target Fam65b tonically dampens chemokine-induced migration by repressing RhoA activity.
Assuntos
Movimento Celular/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas/genética , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Moléculas de Adesão Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiocinas/farmacologia , Proteína Forkhead Box O1 , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Proteínas/metabolismo , Ativação TranscricionalRESUMO
Guanine nucleotide exchange factors (GEFs) activate the Rho GTPases by accelerating their GDP/GTP exchange rate. Some RhoGEFs have been isolated based on their oncogenic potency, and strategies to inhibit their activity are therefore actively being sought. In this study we devise a peptide inhibitor screening strategy to target the GEF activity of Tgat, an oncogenic isoform of the RhoGEF Trio, based on random mutations of the Trio inhibitor TRIP alpha, which we previously isolated using a peptide aptamer screen. This identifies one peptide, TRIP(E32G), which specifically inhibits Tgat GEF activity in vitro and significantly reduces Tgat-induced RhoA activation and foci formation. Furthermore, subcutaneous injection of cells expressing Tgat and TRIP(E32G) into nude mice reduces the formation of Tgat-induced tumors. Our approach thus demonstrates that peptide aptamers are potent inhibitors that can be used to interfere with RhoGEF functions in vivo.
Assuntos
Aptâmeros de Peptídeos/metabolismo , Aptâmeros de Peptídeos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Aptâmeros de Peptídeos/química , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Biblioteca de Peptídeos , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de ProteínaRESUMO
We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.