RESUMEN
In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.
Asunto(s)
Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/metabolismo , Cromatografía en Gel , Dicroismo Circular , Proteína Adaptadora GRB7/genética , Humanos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Fosforilación , Multimerización de Proteína/genética , Multimerización de Proteína/fisiologíaRESUMEN
We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J. Mol. Graph., 1990, 8(1), 52-56) falling closer to expected values for well-refined structures.
Asunto(s)
Proteína Adaptadora GRB7/química , Conformación Proteica , Receptor ErbB-2/química , Receptores de Péptidos/química , Dominios Homologos src , Sitios de Unión , Cristalografía por Rayos X , Humanos , Ligandos , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Multimerización de Proteína , Soluciones , Agua/químicaRESUMEN
Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer properties, however, poor pharmacological properties have limited the use of this compound. We have modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a promising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as research tools, with activities that provide impetus for further development as anti-tumor agents.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Cisteamina/análogos & derivados , Cinesinas/antagonistas & inhibidores , Compuestos de Tritilo/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cisteamina/síntesis química , Cisteamina/química , Cisteamina/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Embrión no Mamífero/efectos de los fármacos , Células HeLa , Humanos , Modelos Moleculares , Estructura Molecular , Erizos de Mar/efectos de los fármacos , Erizos de Mar/embriología , Estereoisomerismo , Relación Estructura-Actividad , Compuestos de Tritilo/síntesis química , Compuestos de Tritilo/químicaRESUMEN
A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.
Asunto(s)
Membrana Celular/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Compuestos de Piridinio/química , Compuestos de Piridinio/metabolismo , Permeabilidad de la Membrana Celular , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Compuestos de Piridinio/síntesis química , Piridonas/síntesis química , Piridonas/químicaRESUMEN
Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7-mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been fully determined. In this study, we report that Grb7 interacts with Hax-1, a cytoskeletal-associated protein found overexpressed in metastatic tumors and cancer cell lines. Additionally, in yeast 2-hybrid assays, we show that the interaction is specific to the Grb7-RA and -PH domains. We have also demonstrated that full-length Grb7 and Hax-1 interact in mammalian cells and that Grb7 is tyrosine phosphorylated. Isothermal titration calorimetry measurements demonstrate the Grb7-RA-PH domains bind to the Grb7-SH2 domain with micromolar affinity, suggesting full-length Grb7 can exist in a head-to-tail conformational state that could serve a self-regulatory function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Células HeLa , Humanos , Inmunoprecipitación , Fosforilación , Fosfotirosina , Unión Proteica , Relación Estructura-Actividad , Termodinámica , Transfección , Dominios Homologos srcRESUMEN
Grb7 is a multidomain intracellular signaling protein that links activated tyrosine kinases with downstream signaling targets. Best known for its regulatory role in cell migration and tumor metastasis, Grb7 also regulates inflammation by coupling NF-kappaB-inducing kinase with erbB/EGFR family receptors. The "adaptor" role of Grb7 in these processes depends upon binding to membrane-associated tyrosine kinases through its C-terminal SH2 domain. The Grb7-SH2 domain shares structural and functional similarity with the SH2 domain of Grb2, a constituent of the MAP kinase pathway. Both domains show unusual affinity for cyclic (beta-turn) ligands. The Grb2-SH2 domain also shows distinctive self-association behavior, forming intertwined ("swapped") dimers. While Grb7 and its SH2 domain are each known to dimerize, the mechanisms and functional significance of this self-association are incompletely understood. Additional residues in the Grb7-SH2 domain effectively lengthen its "EF loop" and render the domain a good candidate for swapped dimerization, through exchange of a C-terminal helix. We propose the existence of a swapped dimeric form of the Grb7-SH2 domain and offer a structural model derived through novel application of nuclear magnetic resonance-derived restraints.
Asunto(s)
Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/metabolismo , Inflamación/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Dominios Homologos src , Progresión de la Enfermedad , Humanos , Inflamación/patología , Relación Estructura-ActividadRESUMEN
OBJECT: Investigators in experimental and clinical studies have used the intrathecal route to deliver drugs to prevent or treat vasospasm. However, a clot near an artery or arteries after subarachnoid hemorrhage (SAH) may hamper distribution and limit the effects of intrathecally delivered compounds. In a primate model of right middle cerebral artery (MCA) SAH, the authors examined the distribution of Isovue-M 300 and 3% Evans blue after infusion into the cisterna magna CSF. METHODS: Ten cynomolgus monkeys were assigned to SAH and sham SAH surgery groups (5 in each group). Monkeys received CSF injections as long as 28 days after SAH and were killed 3 hours after the contrast/Evans blue injection. The authors assessed the distribution of contrast material on serial CT within 2 hours after contrast injection and during autopsy within 3 hours after Evans blue staining. RESULTS: Computed tomography cisternographies showed no contrast in the vicinity of the right MCA (p < 0.05 compared with left); the distribution of contrast surrounding the entire right cerebral hemisphere was substantially reduced. Postmortem analysis demonstrated much less Evans blue staining of the right hemisphere surface compared with the left. Furthermore, the Evans blue dye did not penetrate into the right sylvian fissure, which occurred surrounding the left MCA. The authors observed the same pattern of changes and differences in contrast distribution between SAH and sham SAH animals and between the right and the left hemispheres on Days 1, 3, 7, 14, 21, and 28 after SAH. CONCLUSIONS: Intrathecal drug distribution is substantially limited by SAH. Thus, when using intrathecal drug delivery after SAH, vasoactive drugs are unlikely to reach the arteries that are at the highest risk of delayed cerebral vasospasm.
Asunto(s)
Preparaciones Farmacéuticas/líquido cefalorraquídeo , Hemorragia Subaracnoidea/líquido cefalorraquídeo , Animales , Cisterna Magna/metabolismo , Colorantes/farmacocinética , Medios de Contraste/farmacocinética , Azul de Evans/farmacocinética , Procesamiento de Imagen Asistido por Computador , Infarto de la Arteria Cerebral Media/patología , Inyecciones Espinales , Yopamidol/farmacocinética , Macaca fascicularis , Preparaciones Farmacéuticas/metabolismo , Hemorragia Subaracnoidea/metabolismo , Terapia Trombolítica , Tomografía Computarizada por Rayos X , Vasoespasmo Intracraneal/fisiopatologíaRESUMEN
Grb7 is an adaptor molecule that can mediate signal transduction from multiple cell surface receptors to various downstream signaling pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase (RTK), erbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, though the participants in these pathways have not been determined. In this study, we report that Grb7 interacts with four and half lim domains isoform 2 (FHL2), a transcription regulator with an important role in oncogenesis, including breast cancer. Additionally, in yeast 2-hybrid (Y2H) assays, we show that the interaction is specific to the Grb7 RA and PH domains. We have also demonstrated that full-length (FL) Grb7 and FHL2 interact in mammalian cells and that Grb7 must be tyrosine phosphorylated for this interaction to occur. Immunofluorescent microscopy demonstrates possible co-localization of Grb7 and FHL2. A model with supporting NMR evidence of Grb7 autoinhibition is proposed.
Asunto(s)
Movimiento Celular/fisiología , Proteína Adaptadora GRB7/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Musculares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Sitios de Unión , Western Blotting , Técnica del Anticuerpo Fluorescente , Proteína Adaptadora GRB7/genética , Células HeLa , Proteínas de Homeodominio/genética , Humanos , Inmunoprecipitación , Proteínas con Homeodominio LIM , Modelos Moleculares , Proteínas Musculares/genética , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Tirosina/metabolismoAsunto(s)
Cirugía General/ética , Cuidados Paliativos/ética , Rol del Médico , Cuidado Terminal/ética , Directivas Anticipadas/ética , Anciano , Técnicas de Apoyo para la Decisión , Ética Médica , Femenino , Cuidados Paliativos al Final de la Vida/ética , Humanos , Masculino , Inutilidad Médica/ética , Persona de Mediana Edad , Neoplasias/cirugía , Relaciones Médico-Paciente/éticaAsunto(s)
Ansiedad , Astenia , Depresión , Cuidados Paliativos , Enfermo Terminal/psicología , Antidepresivos/uso terapéutico , Ansiedad/diagnóstico , Ansiedad/terapia , Astenia/diagnóstico , Astenia/terapia , Depresión/diagnóstico , Depresión/terapia , Humanos , Neoplasias/psicología , Neoplasias/terapia , Cuidado TerminalRESUMEN
PURPOSE: To explore the relationship between findings at thin-section computed tomography (CT) and pulmonary function tests in lymphangioleiomyomatosis (LAM) and to evaluate the influence of pleurodesis on this relation and the effectiveness of quantitative versus qualitative CT in the assessment of disease severity. MATERIALS AND METHODS: Thirty-seven patients with LAM (17 with pleurodesis) underwent CT and pulmonary function tests. The severity of pulmonary cystic involvement was graded qualitatively by two independent readers and measured quantitatively at CT with a thresholding technique. Relationships between findings at CT and pulmonary function tests and the influence of pleurodesis on these findings were assessed with regression analysis and analysis of covariance. RESULTS: Qualitative ratings had good agreement between observers (kappa = 0.75). Quantitative CT had good repeatability and showed significant correlation with the percent predicted forced expiratory volume in 1 second (FEV(1)%) (r = 0.67, P <.001), percent predicted diffusing capacity of lung for carbon monoxide (DLCO%) (r = 0.48, P <.005), percent predicted ratio of residual volume to total lung capacity (RV/TLC%) (r = -0.65, P <.001), and percent predicted TLC (r = 0.34, P <.04). Quantitative CT results were somewhat better than qualitative CT results. The standard error of the FEV(1)% for the quantitative CT was about 85% of that for the qualitative CT. Pleurodesis had no statistically significant effect on the slope of the regression line between quantitative CT findings, FEV(1)%, and DLCO% (corrected for alveolar volume). The slope between quantitative CT and RV/TLC% was significantly (P =.044) more negative in patients with pleurodesis. CONCLUSION: Qualitative and quantitative CT findings correlate with pulmonary dysfunction over a wide range of disease severity in patients with LAM. Pleurodesis influences the relationship between CT measurements and pulmonary function test results.