Structural systems biology of microenvironmental oxidative stress and synthetic biology intervention

微环境氧化应激的结构系统生物学与合成生物学干预

• 批准号: 10715112
• 负责人: ROGER LARKEN CHANG
• 金额: $ 42万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715112
• 关键词: 3-Dimensional; Aging; Bacteria; Binding; Biochemical Pathway; Biological Assay; Cells; Cellular Stress; Conserved Sequence; Data Analyses; Development; Diagnosis; Diagnostic; Disease; Engineering; Environmental Impact; Escherichia coli; Foundations; Functional disorder; Future; Glyceraldehyde-3-Phosphate Dehydrogenases; Human; In Vitro; Intervention; Lead; Machine Learning; Metabolic; Metabolic dysfunction; Metabolism; Mitochondrial Proteins; Modeling; Molecular; Molecular and Cellular Biology; Organism; Outcome; Outer Mitochondrial Membrane; Oxidation-Reduction; Oxidative Stress; Phenotype; Property; Protein Engineering; Proteins; Proteome; Proteomics; Radiation Toxicity; Reactive Oxygen Species; Recombinants; Research; Resistance; Resolution; Site; Stress; Structure-Activity Relationship; Systems Biology; Technology; Testing; Theoretical model; Therapeutic; Variant; Work; biological systems; design; enzyme activity; genome-wide; human disease; interest; metabolomics; mitochondrial dysfunction; mitochondrial membrane; molecular modeling; oxidation; oxidative damage; protein function; protein structure; protein structure function; rational design; reconstruction; simulation; synthetic biology; therapeutic development

ABSTRACT I seek to characterize proteomic and fundamental molecular properties of bacteria and human cells under oxidative stress as a means to understand mechanistic underpinnings of sensitivity phenotypes. 1) Oxidative stress broadly impacts protein function, but it is very challenging to experimentally determine which protein malfunctions lead to cellular stress phenotypes. I propose a structural systems biology approach to answering these questions for induced stress in E. coli and human cells. Genome-scale metabolic network reconstruction will be integrated with solved and modeled protein structures to enable detailed models of proteomic oxidative damage and its impact on cellular metabolism, permitting stress simulations and prediction of metabolic bottlenecks. Predicted stress phenotypes will be validated by proteomics, metabolomics, and targeted in vitro enzyme activity assays under oxidative stress. This approach will reveal protein targets to inform future efforts in diagnosing and treating oxidative-stress-associated conditions including radiation toxicity, metabolic dysfunction, and aging. 2) I will develop a theoretical model of molecular sensitivity to oxidative damage of generic proteins of interest and serve for design and engineering more robust variants. Redox proteomics can identify oxidation sites at residue resolution on specific proteins or proteome-wide. Analysis of this data in the context of 3D protein structures will uncover molecular properties rendering some sites and proteins more vulnerable than others. I will validate the model in the context of mammalian glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which aggregates on the mitochondrial membrane causing dysfunction under oxidative stress. I will combine the model for molecular vulnerability to oxidation with evolutionary sequence conservation analysis to design oxidation-robust GAPDH variants. These designs will be experimentally characterized through recombinantly expressed proteins and cell-based assays for enzyme activity, oxidation states, and phenotypic outcomes under stress. Results will have implications for human diseases related to GAPDH dysfunction and will serve as a foundation for rational design of stress-resistant proteins, a significant technological advance. 3) I will investigate the functionality of specialized intrinsically disordered proteins (IDPs) for cellular protection against oxidative stress. Exploiting the model case of GAPDH oxidation again, here I will not alter GAPDH itself but introduce synthetic IDPs engineered to target the mitochondrial outer membrane or GAPDH directly through molecular interactions. Some IDPs are known to form protective barriers to reactive oxygen species (ROS) or disaggregate proteins, and I will investigate whether these can serve to protect GAPDH under stress. Designs will be tested on purified GAPDH in enzymatic activity assays and in cell-based assays for mitochondrial dysfunction and protein oxidation. This work would further the fundamental understanding of IDP function and lay groundwork for therapeutic development.

摘要 我试图在以下条件下描述细菌和人类细胞的蛋白质组和基本分子特性 氧化应激是理解敏感性表型的机制基础的一种手段。 1)氧化应激广泛地影响蛋白质的功能，但在实验上确定是非常具有挑战性的。 哪些蛋白质故障会导致细胞应激表型。我提出了一种结构系统生物学方法。 回答这些关于诱导大肠杆菌和人类细胞应激的问题。基因组规模的代谢网络 重建将与已解决的和建模的蛋白质结构相结合，以使详细的模型 蛋白质组氧化损伤及其对细胞代谢的影响，允许应激模拟和预测 新陈代谢瓶颈。预测的应激表型将通过蛋白质组学、代谢组学和 氧化应激下的体外靶向性酶活性测定。这种方法将揭示蛋白质靶标以 告知今后在诊断和治疗包括辐射在内的氧化应激相关疾病方面的努力 毒性、代谢功能障碍和衰老。 2)我将建立一个感兴趣的通用蛋白对氧化损伤的分子敏感性的理论模型 并为更强大的变种设计和工程服务。氧化还原蛋白质组学可以确定氧化位点 在特定蛋白质或蛋白质组范围内的残基解析。在3D蛋白质的背景下对这些数据进行分析 结构将揭示分子特性，使某些部位和蛋白质比其他部位更容易受到攻击。我 将在哺乳动物甘油醛-3-磷酸脱氢酶(GAPDH)的背景下验证该模型， 它聚集在线粒体膜上，导致氧化应激下的功能障碍。我会结合在一起 用进化序列守恒分析设计分子氧化易损性模型 抗氧化能力强的GAPDH变种。这些设计将通过重组的方式进行实验表征 表达蛋白和基于细胞的酶活性、氧化状态和表型结果的分析 在压力下。这一结果将对与GAPDH功能障碍相关的人类疾病产生影响，并将有助于 作为合理设计抗逆蛋白质的基础，是一项重大的技术进步。 3)我将研究专门的内在无序蛋白(Idp)对细胞保护的功能 对抗氧化应激。再次利用GAPDH氧化的模型案例，这里我不会更改GAPDH 而是引入人工合成的IDPs，直接针对线粒体外膜或GAPDH 通过分子间的相互作用。众所周知，一些国内流离失所者会对活性氧形成保护性屏障。 (ROS)或解聚蛋白质，我将研究这些是否可以在压力下保护GAPDH。 设计将在酶活性分析和基于细胞的分析中对纯化的GAPDH进行测试 线粒体功能障碍和蛋白质氧化。这项工作将进一步加深对 IdP的功能，并为治疗开发奠定基础。