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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的我试图描述细菌和人类细胞的蛋白质组学和基本分子特性氧化应激作为了解敏感性表型的机制基础的一种手段。 1）氧化应激广泛影响蛋白质功能，但通过实验确定非常具有挑战性哪些蛋白质功能失常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大肠杆菌和人类细胞中诱导应激的问题。基因组规模的代谢网络重建将与已解决和建模的蛋白质结构相结合，以实现详细的模型蛋白质组氧化损伤及其对细胞代谢的影响，允许压力模拟和预测的代谢瓶颈。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靶向体外酶活性测定。这种方法将揭示蛋白质靶标为未来诊断和治疗氧化应激相关疾病（包括辐射）的努力提供信息毒性、代谢功能障碍和衰老。 2) 我将开发一个关于目标通用蛋白质氧化损伤的分子敏感性的理论模型并用于设计和工程更强大的变体。氧化还原蛋白质组学可以识别氧化位点特定蛋白质或蛋白质组范围内的残基分辨率。在 3D 蛋白质背景下分析此数据结构将揭示分子特性，使某些位点和蛋白质比其他位点和蛋白质更容易受到攻击。我将在哺乳动物甘油醛-3-磷酸脱氢酶 (GAPDH) 的背景下验证模型，它聚集在线粒体膜上，导致氧化应激下功能障碍。我会结合通过进化序列保守分析来设计分子氧化脆弱性模型抗氧化的 GAPDH 变体。这些设计将通过重组进行实验表征表达的蛋白质和基于细胞的酶活性、氧化态和表型结果测定在压力下。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应激蛋白的基础，这是一项重大的技术进步。 3) 我将研究专门的本质无序蛋白 (IDP) 的细胞保护功能对抗氧化应激。再次利用GAPDH氧化的模型案例，这里我不会改变GAPDH 本身，但引入了人工合成的 IDP，旨在直接靶向线粒体外膜或 GAPDH 通过分子相互作用。已知一些国内流离失所者会对活性氧形成保护屏障（ROS）或分解蛋白质，我将研究这些是否可以在压力下保护 GAPDH。设计将在酶活性测定和基于细胞的测定中对纯化的 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 发挥作用并为治疗发展奠定基础。