RESUMO
Integrins are cell adhesion molecules that play important roles in many biological processes including hemostasis, immune responses, development, and cancer. Their adhesiveness is dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces outside-in signals from the extracellular domain to the cytoplasm. Advances in the past several years have shed light on structural basis for integrin regulation and signaling, especially how the large-scale reorientations of the ectodomain are related to the inter-domain and intra-domain shape shifting that changes ligand-binding affinity. Experiments have also shown how the conformational changes of the ectodomain are linked to changes in the α- and ß-subunit transmembrane and cytoplasmic domains.
Assuntos
Integrinas/química , Humanos , Integrinas/metabolismo , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αß transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αß legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and ß-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling.
Assuntos
Integrinas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Subunidades Proteicas/fisiologia , Transdução de Sinais/fisiologia , Adesão Celular , Células HEK293 , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , TransfecçãoRESUMO
We have developed dissociable antibody microarray (DAMA) staining technology that provides a new approach to the global analysis of protein subcellular localization (SCL) in fixed cells. We have developed and optimized this technology for protein SCL profiling, generated ChipView, a program for management and analysis of molecular image database, and utilized the technique to identify proteins with unique SCL in breast cancer cell lines. We compared the SCL profiles of 325 proteins among nine different breast cell lines, and have identified one protein, Cyclin B1, with distinctively different SCLs between normal and cancer cell lines. With classic individual immunostaining, Cyclin B1 was confirmed to localize to the cytoplasm of seven breast cancer cell lines and in both cytoplasm and nuclei of two normal breast cell lines, and to have higher expression levels in the cancer cell lines tested.
Assuntos
Neoplasias da Mama/química , Análise Serial de Proteínas/métodos , Proteoma/análise , Anticorpos/imunologia , Linhagem Celular Tumoral , Ciclina B1/análise , Humanos , Frações Subcelulares/químicaRESUMO
In order to express and purify the catalytic domain of SHP-1/SHP-2 (named as D1C and D2C respectively) and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine (pY) as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant (K(m)) was 2.04 mmol catalytic constant (K(cat)) was 44.98 s(-1), specific constant (K(cat)/K(m)) was 22.05 L/(mmol x s); the molecular weigh of D2C was 35.3 kD, and its Michaelis constant (K(m)) was 2.47 mmol, catalytic constant (K(cat)) was 27.45 s(-1), specific constant (K(cat)/K(m)) was L/(mmol x s). The enzyme activity of D1C is stronger than that of D2C.
Assuntos
Domínio Catalítico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Plasmídeos , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Dissociable antibody microarray (DAMA) staining is a technology that combines protein microarrays with traditional immunostaining techniques. It can simultaneously determine the expression and subcellular location of hundreds of proteins in cultured cells and tissue samples. We developed this technology and demonstrated its application in identifying potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among three normal breast cell lines and seven breast cancer cell lines and identified 10 differentially expressed proteins by the data analysis program DAMAPEP (DAMA protein expression profiling). Among those proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2 were confirmed by Western blot and statistical analysis to have higher expression levels in breast cancer cells than in normal breast cells. These proteins could be potential biomarkers for the diagnosis of breast cancer.