Structural systems biology of microenvironmental oxidative stress and synthetic biology intervention

摘要 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， 氧化应激作为理解敏感性表型的机制基础的手段。 1)氧化应激广泛地影响蛋白质功能，但通过实验确定它是非常具有挑战性的 哪些蛋白质故障导致细胞应激表型。我提出了一种结构系统生物学方法 来回答这些关于E.大肠杆菌和人类细胞。基因组尺度代谢网络 重建将与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，允许模拟和预测应激 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点， 为未来诊断和治疗氧化应激相关疾病（包括放射）的努力提供信息 毒性、代谢功能障碍和衰老。 2)我将开发一个理论模型的分子敏感性的氧化损伤的通用蛋白质的利益 并用于设计和工程更坚固的变型。氧化还原蛋白质组学可以识别氧化位点， 对特定蛋白质或蛋白质组范围的残基解析。在3D蛋白质的背景下分析这些数据 结构将揭示使某些位点和蛋白质比其他位点更脆弱的分子特性。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， 其聚集在线粒体膜上，在氧化应激下引起功能障碍。我会把联合收割机 基于进化序列保守性分析的分子氧化易损性模型设计 氧化稳定的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、氧化状态和表型结果的测定 在压力下。结果将对GAPDH功能障碍相关的人类疾病产生影响， 作为合理设计抗逆蛋白的基础，这是一项重大的技术进步。 3)我将研究专门的内在无序蛋白（IDPs）的细胞保护功能 对抗氧化应激再次利用GAPDH氧化的模型情况，这里我将不改变GAPDH 但引入经工程化以直接靶向线粒体外膜或GAPDH的合成IDP 通过分子间的相互作用一些国内流离失所者被称为形成保护屏障的活性氧物种 (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。这项工作将进一步从根本上理解 IDP功能并为治疗发展奠定基础。

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.