Chemical Genetic and Proteomic Analysis of PTP1B

描述（由申请人提供）： 蛋白酪氨酸磷酸酶1B（PTP 1B）是胰岛素信号传导的主要负调节因子，也是治疗2型糖尿病、肥胖和其他相关代谢综合征的新靶点。然而，除了在胰岛素信号传导中的作用之外，PTP 1B还涉及几个其他生理过程，包括瘦素和整合素介导的途径。此外，由于PTP 1B缺失和PTP 1B敲除小鼠显示的对饮食诱导的肥胖的抵抗的意外表型，观察到的对胰岛素作用的组织特异性效应的分子基础还没有很好地理解。因为PTPIB可能是多种信号通路的调节剂，并且它可以增强和拮抗细胞事件，所以重要的是建立PTPIB在这些过程中的生理相关性。这是开发基于PTP 1B的2型糖尿病和肥胖症疗法的重要先决条件。本研究的目标是：1）利用化学遗传学和相互作用蛋白质组学方法进一步确定PTP 1B在细胞信号传导中的功能作用; 2）开发新的基于活性的PTP探针，用于在整个蛋白质组中全面分析PTP活性。具体而言，我们将采用我们实验室开发的小分子、有效和选择性PTPIB抑制剂来描述PTP 1B在胰岛素、瘦素和整合素信号传导中的生理作用。我们将应用高亲和力PTPIB底物捕获突变体结合质谱快速分离，鉴定和表征PTP 1B的生理底物。这将有助于阐明这种酶的功能，以及PTP 1B的分配到一个特定的信号通路。最后，我们将开发一种基于活性的蛋白质组学技术，利用PTP特异性探针在正常生理和病理条件下询问PTP在整个蛋白质组中的功能，并研究PTP活性的补偿性变化对PTP 1B缺失的反应。 我们相信，化学遗传学与蛋白质组学的融合将为PTPIB在人类生理和发病机制中的分子理解提供最直接的途径。

DESCRIPTION (provided by applicant): Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of insulin signaling and a novel therapeutic target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. However, besides a role in insulin signaling, PTP1B is also implicated in several other physiological processes including leptin and integrin mediated pathways. In addition, the molecular basis for the observed tissue-specific effects on insulin action due to PTP1B deletion and the unexpected phenotype of resistance to diet-induced obesity displayed by the PTP1B knockout mice is not well understood. Because PTPIB may be a regulator of multiple signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance of PTPIB in these processes. This is an important prerequisite for the development of PTP 1B-based therapeutics for type 2 diabetes and obesity. The goals of this proposal are 1) to further define the functional role of PTP1B in cellular signaling using chemical genetics and interaction proteomic approaches, and 2) to develop novel activity-based PTP probes for global analysis of PTP activity in the whole proteome. Specifically, we will employ small molecule, potent and selective PTPIB inhibitors developed in our laboratory to delineate the physiological roles of PTP1B in insulin, leptin, and integrin signaling. We will apply a high-affinity PTPIB substrate-trapping mutant in combination with mass spectrometry for rapid isolation, identification, and characterization of physiological substrates of PTP1B. This will help elucidate the function of this enzyme as well as assignment of PTP1B to a specific signaling pathway. Finally, we will develop an activity-based proteomic technology that utilizes PTP specific probes to interrogate the function of the PTPs in the whole proteome both in normal physiology and in pathological conditions and to study compensatory changes in PTP activity in response to PTP1B deletion. We believe that the fusion of chemical genetics with proteomics will provide the most direct path to the molecular understanding of PTPIB in human physiology and pathogenesis.