Defining roles of nitroTyrosine in desease via genetic code expansion

活性氮在80多种人类疾病中的作用，包括动脉粥样硬化，癌症， 神经退行性变和中风是很好地证明了生物标志物3-硝基酪氨酸的积累 （硝基Tyr）。NitroTyr并不像预期的那样随机分布在蛋白质组中，而是在蛋白质组中发现。 特定蛋白质上的特定酪氨酸针对这些观察结果，PI极大地推进了这一领域 通过开发遗传密码扩展（GCE）技术，使得能够将硝基Tyr位点特异性地掺入到 在细菌和哺乳动物细胞中的重组蛋白。使用这些工具的协作工作现已牢固地 确定硝基酪氨酸蛋白是肌萎缩侧索硬化症、动脉粥样硬化和 癌症，支持我们的核心假设，即硝基酪氨酸修饰的蛋白质是人类疾病的关键参与者， 了解它们的积累和去除的基础，以及它们在病理学中的机械作用， 将为治疗干预带来新的机会。进一步的支撑来自于突破 发现了一种肿瘤抑制剂- 酪氨酸磷酸酶受体T（PTPRTD 2）是一种酪氨酸磷酸酶，当敲除时会促进癌症 增长这颠覆了硝基酪氨酸蛋白质是压力不受调节的副产品的范式， 一项新的研究策略可能会加速进展。而不是识别特定的疾病， 相关的硝基酪氨酸修饰的蛋白质一次一个，假设这种酶代表一种新的 参与调节硝基酪氨酸的影响的酶家族，表征这些酶和 它们的底物应该加速生理学相关硝基Tyr修饰的鉴定，并且还提供了 确定其影响的新途径。这将通过追求两个目标来实现，包括：（1）界定 酶底物范围和对底物识别至关重要的结构-功能关系，以及（2） 将酶及其底物转化为陷阱和抑制剂， 酶/底物对，并帮助研究其在细胞中的生理/病理影响。前期工作 证明其可行性的研究已经确定了另外两种功能改变的酶底物， 在特定位点的硝化作用下所提出的工作，以确定什么样的硝基酪氨酸蛋白质是底物的酶将 也有助于解决为什么硝化蛋白质在疾病中积累，对于每一个发现的病例， 一个酶/硝基酪氨酸-底物对有助于病理学的发展，该过程的映射将打开 开辟了一条治疗干预的新途径。作为（i）现有硝基Tyr GCE技术的开发商，（ii） 作为酶学家和（iii）非天然蛋白质设施的代理主任，PI非常有资格领导这项工作 所有需要的设施都有。此外，主要合作者已经参与进来， 结构生物学和细胞生物学方面的专业知识，以满足拟议工作的广度。

The role of reactive nitrogen species in over eighty human diseases including atherosclerosis, cancer, neurodegeneration, and stroke is well demonstrated by the accumulation of the biomarker 3-nitrotyrosine (nitroTyr). NitroTyr is not randomly distributed across the proteome as might be expected, but rather is found on specific tyrosines on specific proteins. In response to these observations, the PI has greatly advanced this field by developing genetic code expansion (GCE) technologies enabling site-specific incorporation of nitroTyr into recombinant proteins in bacteria and mammalian cells. Collaborative work using these tools has now firmly established that nitroTyr-proteins are causative agents in amyotrophic lateral sclerosis, atherosclerosis, and cancer, supporting our central hypothesis that nitroTyr-modified proteins are key players in human disease and that understanding the basis for their accumulation and removal, as well as their mechanistic roles in pathology will lead to new opportunities for therapeutic intervention. Further support comes from the breakthrough discovery of a denitrase enzyme that is a tumor suppressor: the “D2” pseudo-phosphatase domain of the protein tyrosine phosphatase receptor T (PTPRTD2) is a tyrosine denitrase that when knocked out promotes cancer growth. This upends the paradigm that nitroTyr-proteins are an unregulated by-product of stress and makes possible a new research strategy that should accelerate progress. Instead of identifying specific diseases and associated nitroTyr modified proteins one at a time, under the hypothesis that this denitrase represents a new enzyme family involved in regulating the impact of nitroTyr, characterizing these denitrases and the breadth of their substrates should speed the identification of physiologically relevant nitroTyr modifications and also provide new avenues to define their impact. This will be done through pursuing two aims that encompass: (1) defining the denitrase substrate scope and the structure-function relationships critical for substrate recognition, and (2) converting denitrases and their substrates into traps and inhibitors which will be used to identify denitrase/substrate pairs and aid studies of their physiological/pathological impacts in cells. Preliminary work demonstrating feasibility has already identified two additional denitrase substrates, which have altered function upon site-specific nitration. The proposed work to define what nitroTyr proteins are substrates of denitrases will also help resolve why nitrated proteins accumulate in disease, and for every case in which it is discovered that a denitrase/nitroTyr-substrate pair contribute to pathology development, the mapping of that process will open up a new avenue for therapeutic intervention. As (i) the developer of existing nitroTyr GCE technologies, (ii) an enzymologist and (iii) acting director of the Unnatural Protein Facility, the PI is superbly qualified to lead this work and all needed facilities are available. Furthermore, key collaborators are already engaged who bring the expertise in structural biology and cell biology needed for the breadth of work proposed.