Characterization and Analysis of Platelet Septins

本提案的目的是了解血小板septin的结构-功能关系和分子病理学，血小板septin是细胞质蛋白家族，参与动态膜运动发生的细胞事件。septin家族最初是在酵母中发现的，从酵母延伸到人类，并与从细胞质分裂到囊泡运输等一系列事件有关。在初步研究中，我们描述了一种原型人类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调节神经递质释放。我们研究了靶向缺失小鼠CDCrel-1同源物的小鼠群体。最引人注目的是，与野生型相比，cdcrel -1缺失动物的血小板聚集并释放14c - 5 -羟色胺，以响应亚阈值水平的激动剂。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septin都包含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，我们提出研究GTP和PtdIns(4,5)P2结合基序在核心结构域的体内调节作用（Aims 1和2），并研究NH2和COOH末端提供的功能特异性（Aim 3）。这些目标将通过异种细胞表达和septin表达的转基因模型的综合方法来实现。初步研究还发现了其他血小板septin。我们建议研究一种与CDCrel-1相互作用的未表征的血小板隔离素的作用（目的4），并定义血小板隔离素的完整库（目的5）。这些研究将为血小板分泌调节分子事件提供新的信息，并与止血和血栓形成的控制机制直接相关。这些研究还将为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提供新的见解，其中活性膜运动至关重要。我们的结果将与控制分泌相关的任何数量的疾病过程相关。一种检测septin功能的血小板模型利用了血管损伤部位血小板反应分泌的基本重要性。

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.