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通过生物标记物3-硝基酪氨酸的积累得到很好的证明 (硝基酪氨酸)。NitroTyr并不像预期的那样随机分布在蛋白质组中，而是在 特定蛋白质上的特定酪氨酸。作为对这些观察的回应，PI极大地推进了这一领域 通过开发遗传密码扩展(GCE)技术，使硝基酪氨酸能够在 细菌和哺乳动物细胞中的重组蛋白质。使用这些工具的协作工作现在已经牢牢地 证实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，支持我们的中心假设，即硝基酪氨酸修饰的蛋白质是人类疾病和 了解它们的积累和清除的基础，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。进一步的支持来自于这一突破 发现一种具有肿瘤抑制作用的脱氮酶：蛋白质的“D2”假磷酸酶结构域 酪氨酸磷酸酶受体T(PTPRTD2)是一种酪氨酸脱氮酶，当被敲除时会促进癌症的发生 成长。这颠覆了硝基酪氨酸蛋白是压力的不受调控的副产品的范式，并使 可能会有一个新的研究战略，应该会加快进展。而不是识别特定的疾病和 相关的硝基酪氨酸修饰蛋白，一次一个，假设这种反硝酸酶代表一个新的 参与调节硝基酪氨酸影响的酶家族，表征这些脱氮酶和 它们的底物应该加快生理上相关的硝基酪氨酸修饰的鉴定，并还提供 定义其影响的新途径。这将通过追求两个目标来实现，包括：(1)界定 底物识别关键的反硝酶底物范围和结构-功能关系，以及(2) 将脱氮酶及其底物转化为陷阱和抑制剂，将用于鉴定 反硝酸酶/底物对，并有助于研究它们在细胞中的生理/病理影响。前期工作 证明可行性已经确定了另外两种脱硝酶底物，它们改变了功能 根据特定部位的硝化作用。关于确定哪些硝基酪氨酸蛋白是脱氮酶的底物的拟议工作将 也有助于解决为什么硝化蛋白在疾病中积累，以及每一个被发现的病例 反硝酸酶/硝基酪氨酸底物对有助于病理发展，这一过程的图谱将打开 开辟了一条治疗干预的新途径。作为(I)现有硝化酪氨酸GCE技术的开发商，(Ii)和 作为一名酶学家和(三)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 而且所有需要的设施都是可用的。此外，主要合作伙伴已经接洽，他们带来了 建议的工作范围需要结构生物学和细胞生物学方面的专业知识。

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.