RESUMO
Integrin-mediated cell-extracellular matrix (ECM) interactions play crucial roles in a broad range of physiological and pathological processes. Kindlins are important positive regulators of integrin activation. The FERM-domain-containing kindlin family comprises three members, kindlin-1, kindlin-2 and kindlin-3 (also known as FERMT1, FERMT2 and FERMT3), which share high sequence similarity (identity >50%), as well as domain organization, but exhibit diverse tissue-specific expression patterns and cellular functions. Given the significance of kindlins, analysis of their atomic structures has been an attractive field for decades. Recently, the structures of kindlin and its ß-integrin-bound form have been obtained, which greatly advance our understanding of the molecular functions that involve kindlins. In particular, emerging evidence indicates that oligomerization of kindlins might affect their integrin binding and focal adhesion localization, positively or negatively. In this Review, we presented an update on the recent progress of obtaining kindlin structures, and discuss the implication for integrin activation based on kindlin oligomerization, as well as the possible regulation of this process.
Assuntos
Proteínas de Membrana , Proteínas de Neoplasias , Adesão Celular , Adesões Focais , Integrinas/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genéticaRESUMO
Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.
Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Multimerização Proteica , Movimento Celular , Humanos , Integrinas/metabolismo , Células K562 , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
Chromatin regulators (CRs) can dynamically modulate chromatin architecture to epigenetically regulate gene expression in response to intrinsic and extrinsic signalling cues. Somatic alterations or misexpression of CRs might reprogram the epigenomic landscape of chromatin, which in turn lead to a wide range of common diseases, notably cancer. Here, we present CR2Cancer, a comprehensive annotation and visualization database for CRs in human cancer constructed by high throughput data analysis and literature mining. We collected and integrated genomic, transcriptomic, proteomic, clinical and functional information for over 400 CRs across multiple cancer types. We also built diverse types of CR-associated relations, including cancer type dependent (CR-target and miRNA-CR) and independent (protein-protein interaction and drug-target) ones. Furthermore, we manually curated around 6000 items of aberrant molecular alterations and interactions of CRs in cancer development from 5007 publications. CR2Cancer provides a user-friendly web interface to conveniently browse, search and download data of interest. We believe that this database would become a valuable resource for cancer epigenetics investigation and potential clinical application. CR2Cancer is freely available at http://cis.hku.hk/CR2Cancer.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Bases de Dados Factuais , Enzimas/fisiologia , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Metilação de DNA/genética , Coleta de Dados , Mineração de Dados , Bases de Dados Genéticas , Bases de Dados de Proteínas , Enzimas/genética , Previsões , Dosagem de Genes , Ensaios de Triagem em Larga Escala , Código das Histonas/genética , Humanos , Armazenamento e Recuperação da Informação , Anotação de Sequência Molecular , Domínios Proteicos , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Especificidade por Substrato , Interface Usuário-ComputadorRESUMO
Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.