The Hydroxylprolylproteome

描述（由申请人提供）：翻译后修饰在许多信号转导途径中发挥重要作用，我们尚未充分认识到这些修饰对细胞功能的影响程度。最近的研究强调了一个独特的修饰，脯氨酰羟基化，在缺氧反应的关键作用。在本申请中，我们建议开发和采用新的捕获试剂，这种翻译后修饰。 在后生动物中低氧反应的主要转录调节因子是低氧诱导因子（HIF），其由和亚基组成。氧调节HIF周转的关键事件是HIF的翻译后修饰-通过位点特异性脯氨酰羟基化，这允许von Hippel Lindau（VHL）肿瘤抑制蛋白（E3泛素连接酶复合物的一种组分）识别，选择性靶向羟基化HIF-降解。这反过来又提出了一个更大的问题，即脯氨酰羟基化是否可能在缺氧反应中发挥更广泛的作用。解决这个问题的一个主要限制是缺乏合适的试剂，可以特异性地识别脯氨酰羟基化的蛋白质。我们建议使用VHL作为引入定点突变的支架来开发新型的羟基脯氨酰捕获试剂。这些突变将保留VHL的羟脯氨酰结合口袋，目的是延长对其他含羟脯氨酰蛋白的反应性。基于VHL的羟基脯氨酰捕获试剂将用于免疫沉淀蛋白质，并通过质谱法获得其身份和脯氨酰羟基化位点。采用细胞培养和小鼠组织提取物的研究将用于表征这些脯氨酰羟基化事件的意义。将开发以高通量方式评估脯氨酰羟基化变化的平台。我们的合作者Stephen Master博士将专注于这些高通量研究和质谱分析。这些研究将挑战目前的范式，即脯氨酰羟基化在缺氧反应中的作用仅限于HIF。此外，他们将提供评估的羟基蛋白质组，这是相当重要的缺氧的疾病，包括心肌梗死，脑血管疾病和癌症的作用的动态变化的基础。 公共卫生相关性：本项目旨在开发新型捕获试剂，用于翻译后修饰脯氨酰羟基化，该修饰在缺氧诱导因子的氧调节周转中起核心作用。长期的目标将是采用这些试剂来确定脯氨酰羟基化调节细胞缺氧反应的程度，并表征响应于不同氧浓度的羟基脯氨酰蛋白质组的变化。这些研究将对理解心脏病发作、中风和癌症等疾病产生影响，因为它们的特征是缺氧。

DESCRIPTION (provided by applicant): Posttranslational modifications play important roles in many signal transduction pathways, and we have yet to realize the full extent to which these modifications impact on cell function. Recent studies have highlighted a key role for a distinctive modification, prolyl hydroxylation, in the hypoxic response. In this application, we propose to develop and employ novel capture reagents for this posttranslational modification. The master transcriptional regulator of the hypoxic response in metazoans is Hypoxia Inducible Factor (HIF), which consists of an and a subunit. The key event in oxygen-regulated HIF turnover is posttranslational modification of HIF- by site-specific prolyl hydroxylation, which allows recognition by the von Hippel Lindau (VHL) tumor suppressor protein, a component of an E3 ubiquitin ligase complex that selectively targets hydroxylated HIF- for degradation. This, in turn, raises the larger question of whether prolyl hydroxylation may play a broader role in the hypoxic response. A major limitation to addressing this question is the lack of suitable reagents that can specifically recognize prolyl hydroxylated proteins. We propose developing novel hydroxylprolyl capture reagents using VHL as a scaffold for introducing site- directed mutations. These mutations will preserve the hydroxylprolyl binding pocket of VHL with the intention of extending reactivity to other hydroxylprolyl-containing proteins. The VHL-based hydroxylprolyl capture reagents will be employed to immunoprecipitate proteins, and their identity and sites of prolyl hydroxylation will be obtained by mass spectrometry. Studies employing cell culture and mouse tissue extracts will be employed to characterize the significance of these prolyl hydroxylation events. Platforms for assessing changes in prolyl hydroxylation in a high throughput manner will be developed. Our collaborator, Dr. Stephen Master, will focus on these high throughput studies and the mass spectrometry analyses. These studies will challenge the current paradigm that the role of prolyl hydroxylation in hypoxic responses is limited to HIF. Moreover, they will provide the basis for assessing dynamic changes in the hydroxylproteome, which is of considerable significance given the role of hypoxia in diseases that include myocardial infarction, cerebrovascular disease, and cancer. PUBLIC HEALTH RELEVANCE: This project seeks to develop novel capture reagents for a posttranslational modification, prolyl hydroxylation, that plays a central role in the oxygen-regulated turnover of Hypoxia Inducible Factor. The long term goal will be to employ these reagents to determine the extent to which prolyl hydroxylation regulates the cellular hypoxic response, and characterize changes in the hydroxylprolylproteome in response to differing oxygen concentrations. These studies will have implications for understanding diseases such as heart attacks, stroke, and cancer, because they are characterized by hypoxia.