Target discovery in platelets by in situ proteome reactivity profiling

描述(由申请人提供)： 当循环血小板接触细胞外基质蛋白和可溶性激动剂时，会产生细胞内信号，导致黏附受体的快速激活，如整合素Alphallb-beta3、血小板聚集、颗粒内容物的分泌和促凝活性的增加。这些变化不仅有助于正常止血，还有助于血栓事件，如心肌梗死和中风。血小板是无核的，因此不适用于直接的分子途径，从而限制了我们对这些依赖于激活的信号通路的特定组成部分的了解。Parise实验室已经熟练地使用巨核细胞中的敲除和过表达方法来剖析与血小板功能相关的信号通路。巨核细胞是血小板的前体。在这里，我们建议与斯克里普斯研究所的本杰明·克拉瓦特博士合作，他开发了一种受天然产品启发的新型螺环氧化物探针库，该探针库以前曾用于鉴定转化细胞中的重要酶。来自该文库的探针与基于细胞的筛选中的靶酶共价结合，从而影响特定的细胞读数。通过使用一种名为原位蛋白质组图谱的新技术，共价结合的探针随后被标记上生物素和荧光标记，通过“点击化学”，从而有助于通过质谱学快速识别靶标。然而，这个库从未被应用于血小板。因此，我们建议1)通过使用螺环氧化物探针库筛选人血小板并进行原位蛋白质组反应性分析来识别必要的血小板功能所需的分子靶点，以及2)通过独立的计数器筛选来验证巨核细胞和血小板中的分子靶点，包括过度表达和敲除，以及可能的情况下，候选靶点的药理抑制和酶活性分析。该文库基于血小板的筛选和蛋白质组反应性分析，再加上调节巨核细胞中蛋白质表达水平的能力，为快速识别和验证调节血小板功能的新治疗靶点提供了血小板领域的强大方法组合。

DESCRIPTION (provided by applicant): Circulating platelets generate intracellular signals when exposed to extracellular matrix proteins and soluble agonists, resulting in rapid activation of adhesion receptors such as integrin alphallb-beta3, platelet aggregation, secretion of granular contents and increased procoagulant activity. These changes contribute not only to normal hemostasis but also to thrombotic events such as myocardial infarction and stroke. Platelets are anucleate and therefore not amenable to direct molecular approaches, thus limiting our understanding of the specific components of these activation-dependent signaling pathways. The Parise laboratory has become proficient in the use of knockdown and overexpression approaches in megakaryocytes, which are platelet precursors, to dissect signaling pathways relevant to platelet function. Here we propose to join forces with Dr. Benjamin Cravatt of The Scripps Research Institute, who has developed a novel natural products inspired spiroepoxide probe library that has been used previously to identify enzymes of importance in transformed cells. Probes from this library bind covalently to target enzymes in cell-based screens, thus affecting specific cellular readouts. By use of new technology termed in situ proteome profiling, covalently bound probes are then labeled with a biotin and fluorescent tag by use of "click chemistry", thus facilitating rapid identification of targets via mass spectrometry. However, this library has never been applied to platelets. Therefore, we propose to 1) identify molecular targets necessary for essential platelet functions by screening human platelets with the spiroepoxide probe library and performing in situ proteome reactivity profiling, and 2) validate molecular targets in megakaryocytes and platelets by use of independent counter screens involving overexpression and knock down, as well as pharmacological inhibition and enzyme activity assays, when possible, of candidate targets. Platelet-based screening and proteome reactivity profiling with this library, together with the ability to modulate protein expression levels in megakaryocytes provides a powerful combination of approaches in the platelet field for rapidly identifying and verifying new therapeutic targets for modulating platelet function.