Characterization and Analysis of Platelet Septins

这个建议的目的是了解血小板间隔蛋白的结构-功能关系和分子病理学，这是一类参与细胞活动的细胞质蛋白，其中发生动态的膜运动。最初在酵母中发现的Septin家族从酵母延伸到人类，并与从胞质分裂到囊泡运输的一系列事件有关。在初步研究中，我们描述了一种原型的人类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与神经元的胞外复合体联系起来，暗示CDCrel-1调节神经递质的释放。我们已经研究了一个靶向删除CDCrel-1的小鼠同源物的小鼠群体。最引人注目的是，与野生型相比，CDCrel-1缺失动物的血小板在阈值以下的激动剂水平下聚集并释放14C-5-羟色胺。因此，CDCrel-1在体内调节血小板释放反应的作用被确立。所有的间隔蛋白都含有一个保守的中心核心区，两侧是每个蛋白质所特有的末端。关于CDCrel-1，有人建议研究GTP和PtdIns(4，5)P2结合基序在核心域(AIMS 1和2)中的调节作用，并研究NH2和COOH末端提供的功能特异性(AIM 3)。这些目标将通过异源细胞表达的综合方法和Septin表达的转基因模型来实现。初步研究还发现了更多的血小板隔素。我们建议研究一个未知的与CDCrel-1相互作用的血小板间隔蛋白的作用(目标4)，并确定完整的血小板间隔蛋白谱系(目标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。这些研究还将对血小板分泌紊乱的原因以及巨核细胞/血小板生物学的其他方面提供新的见解，在这些方面，主动的膜运动是关键。我们的结果将与任何数量的疾病过程相关，在这些过程中，控制分泌是相关的。一种检测间隔蛋白功能的血小板模型利用了分泌对血管损伤部位的血小板反应的基本重要性。

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.