Characterization and Analysis of Platelet Septins

这个建议的目的是了解血小板septins的结构-功能关系和分子病理学，一个家庭的细胞质蛋白参与细胞的动态膜运动发生的事件。Septin家族最初在酵母中发现，从酵母延伸到人类，并与胞质分裂到囊泡运输等事件相关。 在初步研究中，我们描述了一种原型人septin，称为CDCrel-1，在脑，心脏和巨核细胞中表达。 神经生物学实验室的工作将CDCrel-1与神经元的胞吐复合物联系起来，暗示CDCrel-1调节神经递质的释放。 我们已经研究了具有针对性缺失CDCrel-1的鼠同源物的小鼠集落。 最引人注目的是，与野生型同窝出生的动物相比，CDCrel-1缺失动物的血小板聚集并释放14 C-5-羟色胺以响应阈下水平的激动剂。 因此，建立了CDCrel-1在调节血小板释放反应中的体内作用。 所有septins含有一个保守的中央核心结构域，两侧是每个蛋白质特有的末端。相对于CDCrel-1，提出研究以检查核心结构域中GTP和PtdIns（4，5）P2结合基序的体内调节作用（目的1和2），并检查由NH 2和COOH末端提供的功能特异性（目的3）。 这些目标将通过异源细胞表达和septin表达的转基因模型的综合方法来实现。初步研究还确定了其他血小板septins。 我们建议检查与CDCrel-1相互作用的未表征的血小板隔蛋白的作用（目的4），并定义血小板隔蛋白的完整库（目的5）。 这些研究将提供有关调节血小板分泌的分子事件的新信息，并与控制止血和血栓形成的机制直接相关。 这些研究也将提供新的见解血小板分泌障碍的原因和巨核细胞/血小板生物学的其他方面，其中主动膜运动是至关重要的。 我们的研究结果将与任何数量的疾病过程中控制分泌是相关的。 检查隔蛋白功能的血小板模型利用了分泌对于血管损伤部位的血小板应答的根本重要性。

The objectives of this proposal are to understand the structure-function relationships and molecular pathology of platelet septins, a family of cytoplasmic proteins involved in cellular events where dynamic membrane movements occur. Originally identified in yeast, the septin family extends from yeast to humans and has been associated with events ranging from cytokinesis to vesicle trafficking. In Preliminary Studies we characterize a prototypic human septin, termed CDCrel-1, expressed in brain, heart and megakaryocytes. Work from neurobiology labs has linked CDCrel-1 to the exocytic complex of neurons with implications that CDCrel-1 regulates neurotransmitter release. We have studied a mouse colony with a targeted deletion of the murine homologue to CDCrel-1. Most strikingly, platelets from CDCrel-1null animals aggregate and release 14C-serotonin in response to subthreshold levels of agonist compared to their wild-type littermates. Thus, an in vivo role for CDCrel-1 in regulating the platelet release reaction is established. All septins contain a conserved central core domain flanked by terminal ends unique to each protein. Relative to CDCrel-1, studies are proposed to examine the in vivo regulatory role of GTP and PtdIns(4,5)P2 binding motifs in the core domain (Aims 1 and 2) and examine the functional specificity provided by the NH2 and COOH termini (Aim 3). These aims will be achieved via an integrated approach of heterologous cell expression and transgenic models of septin expression. Preliminary studies have also identified additional platelet septins. We propose to examine the role of an uncharacterized platelet septin that interacts with CDCrel-1 (Aim 4) and define the complete repertoire of platelet septins (Aim 5). These studies will provide new information on the molecular events regulating platelet secretion and are directly relevant to mechanisms controlling hemostasis and thrombosis. These studies will also provide new insights on the causes of platelet secretion disorders and other aspects of megakaryocyte/platelet biology where active membrane movement is critical. Our results will be relevant to any number of disease processes where controlled secretion is relevant. A platelet model to examine septin function exploits the fundamental importance of secretion for the platelet response at sites of vascular injury.