Defining roles of nitroTyrosine in desease via genetic code expansion

通过遗传密码扩展定义硝基酪氨酸在疾病中的作用

基本信息

批准号：
10641726
负责人：
RYAN A MEHL
金额：
$ 28.84万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-05 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10641726
关键词：
3-nitrotyrosine ALS pathology Acceleration Alzheimer&apos s Disease Amino Acids Amyotrophic Lateral Sclerosis Animal Model Applied Genetics Arthritis Atherosclerosis Back Bacteria Biological Assay Biological Markers Cell Culture Techniques Cells Cellular biology Collaborations Colon Carcinoma Development Disease Enzymatic Biochemistry Enzymes Escherichia coli Eukaryotic Cell Evaluation Excision Family Genetic Code Growth Hand In Vitro Infection Inflammation Knock-out Lead Libraries Literature MAPK3 gene Malignant Neoplasms Mammalian Cell Maps Methods Modification Mus Nerve Degeneration Oxidative Stress Parkinson Disease Pathologic Pathology Phosphoric Monoester Hydrolases Physiological Physiology Post-Translational Protein Processing Process Property Protein Tyrosine Phosphatase Proteins Proteome Qualifying Reactive Nitrogen Species Recombinant Proteins Regulation Reporting Research Role Sepsis Severities Signal Transduction Site Specificity Speed Stress Stroke Structural Models Structure-Activity Relationship Substrate Interaction Substrate Specificity Suicide Technology Tertiary Protein Structure Therapeutic Intervention Time Tissues Tumor Suppressor Proteins Tyrosine Universities Work Xenograft procedure chronic pain human disease improved in vivo inhibitor insight lung injury mouse model nitration organ transplant rejection paxillin receptor response structural biology success tool tumor growth

项目摘要

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.

活性氮在八十多种人类疾病中的作用，包括动脉粥样硬化、癌症、生物标志物 3-硝基酪氨酸的积累充分证明了神经退行性疾病和中风（硝基酪氨酸）。硝基酪氨酸并不像预期的那样随机分布在蛋白质组中，而是在根据这些观察结果，PI 极大地推进了这一领域的发展。通过开发遗传密码扩展（GCE）技术，能够将硝基酪氨酸定点整合到使用这些工具在细菌和哺乳动物细胞中进行重组蛋白的合作现已牢固。确定硝基酪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，即硝基酪氨酸修饰蛋白是人类疾病和疾病的关键参与者了解它们积累和清除的基础，以及它们在病理学中的机制作用突破将为治疗干预带来新的机会。发现一种肿瘤抑制因子的脱硝酶：蛋白质的“D2”假磷酸酶结构域酪氨酸磷酸酶受体 T (PTPRTD2) 是一种酪氨酸脱硝酶，敲除后会促进癌症这颠覆了硝基酪蛋白是压力不受调节的副产品的范式。可能会出现一种新的研究策略，该策略应该加速进展，而不是识别特定的疾病和疾病。相关的硝基酪氨酸每次修饰一个蛋白质，假设这种脱硝酶代表了一种新的参与调节硝基酪氨酸影响的酶家族，描述了这些脱硝酶的特征以及它们的底物应加速生理相关硝基酪氨酸修饰的鉴定，并提供这将通过追求两个目标来实现，其中包括：(1) 定义其影响。脱硝酶底物关系范围和对底物识别至关重要的结构功能，以及 (2) 将脱硝酶及其底物转化为陷阱和抑制剂，用于识别脱硝酶/底物对并帮助研究它们对细胞的生理/病理影响。证明可行性已经确定了两种额外的脱硝酶底物，它们具有功能确定哪些硝基酪蛋白是脱硝酶底物的拟议工作将基于位点特异性硝化。还有助于解决硝化蛋白在疾病中积聚的原因，并且对于每一个发现的案例脱硝酶/硝基酪氨酸底物有助于病理学发展，该过程的图谱将开放作为 (i) 现有 nitroTyr GCE 技术的开发者，(ii) 开辟治疗干预的新途径。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此外，主要合作者已经参与进来，带来了所需的设施。拟议工作范围所需的结构生物学和细胞生物学专业知识。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Doping of Green Fluorescent Protein into Superfluid Helium Droplets: Size and Velocity of Doped Droplets.

将绿色荧光蛋白掺杂到超流氦液滴中：掺杂液滴的大小和速度。

DOI：
发表时间：
2017-09-14
期刊：
影响因子：
0
作者：
Alghamdi, Maha;Zhang, Jie;Oswalt, Andrew;Porter, Joseph J;Mehl, Ryan A;Kong, Wei
通讯作者：
Kong, Wei

Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(Tyr) Suppressor.

通过工程化 tRNA(Tyr) 抑制剂改善非规范氨基酸的掺入。

DOI：
发表时间：
2016-01-26
期刊：
影响因子：
2.9
作者：
Rauch, Benjamin J;Porter, Joseph J;Mehl, Ryan A;Perona, John J
通讯作者：
Perona, John J

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications.

遗传密码扩展：了解氧化应激蛋白修饰的生理后果的强大工具。

DOI：
发表时间：
2018
期刊：
影响因子：
0
作者：
Porter, Joseph J;Mehl, Ryan A
通讯作者：
Mehl, Ryan A

Unraveling the effects of peroxiredoxin 2 nitration; role of C-terminal tyrosine 193.

揭示过氧化还原蛋白 2 硝化的影响；

DOI：
发表时间：
2019
期刊：
影响因子：
7.4
作者：
Randall, Lía M;Dalla Rizza, Joaquín;Parsonage, Derek;Santos, Javier;Mehl, Ryan A;Lowther, W Todd;Poole, Leslie B;Denicola, Ana
通讯作者：
Denicola, Ana

Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins.

创建针对含有 3-硝基酪氨酸的蛋白质的选择性纳米抗体。

DOI：
发表时间：
2022
期刊：
影响因子：
0
作者：
Van Fossen, Elise M;Grutzius, Sonia;Ruby, Carl E;Mourich, Dan V;Cebra, Chris;Bracha, Shay;Karplus, P Andrew;Cooley, Richard B;Mehl, Ryan A
通讯作者：
Mehl, Ryan A