Decoding and Rewiring Enzymatic Redox Signal Transduction Pathways

项目摘要 细胞已经进化出复杂的酶机制，帮助它们在氧化还原压力下生存。 微环境酶促氧化还原感觉、信号和响应机制对于各种不同的生物学过程是至关重要的。 从细菌、植物到人类的所有生命形式的生理过程。与其他蜂窝 在信号传导过程中，氧化还原信号传导涉及高反应性试剂，例如一氧化氮（NO）、碳 一氧化碳（CO）和活性氧物质，这引起了人们对这些试剂用于 参与其他非特异性反应。然而，这种副作用在生理条件下是不常见的。 条件表明酶促氧化还原信号转导途径的高特异性和选择性。反过来， 我们提出了以下两个相关的问题：a）是什么使氧化还原信号通路如此特异？和B）可以 我们合理和系统地重新布线氧化还原信号转导通路，以恢复/破坏细胞的氧化还原 平衡？这些问题在化学生物学和生物无机化学中很大程度上被忽视了 尽管事实上，氧化还原失衡是造成各种疾病的原因， 从神经系统疾病到癌症我们的实验室专注于这些范式转变的问题和长期目标 我们的研究计划的一部分是开发分子策略， 生物氧化还原试剂参与人类健康和疾病。在本提案中，我们关注DosS-DosR 分枝杆菌中的酶信号通路，其在其微环境中感知NO/CO，并向细胞 进入非复制的休眠状态DosS-DosR系统有三个组成部分-血红素铁 一个与NO/CO结合的传感结构域，一个与NO/CO结合的锌依赖性信号激酶结构域， 事件和与DNA结合并开启休眠的响应域。利用我们在以下领域的综合专长 金属蛋白结构-功能，蛋白质工程，酶学和光谱学，我们将设计诱变， 基于金属取代和肽模拟的重新连接DosS-DosR感觉、信号和响应的方法 域.在我们实验室进行的概念验证研究已经证明了DoS的成功重新布线- DosR氧化还原传感功能通过结构指导的合理蛋白质设计。未来的工作将揭开分子 氧化还原重新布线的机制及其对细胞生理学和表型反应的影响。我们的发现， 将不仅提供对细胞氧化还原感测/信号/响应机制的基本理解，而且 为今后治疗和预防氧化还原相关疾病的方法提供信息。 健康相关性：维持正常的细胞内氧化还原状态对于调节生理功能至关重要。 应答这种状态的任何失衡都会导致各种急性和慢性退行性疾病， 癌症、心血管和神经系统疾病。我们的研究计划旨在设计分子方法 重新连接细胞的能力，感知和信号的氧化还原变化，在他们的环境。这些方法可以 用于恢复患病状态下的细胞氧化还原稳态。

PROJECT SUMMARY Cells have evolved intricate enzymatic machineries that help them exist and survive redox stresses in their microenvironment. Enzymatic redox sense, signal, and response mechanisms are critical for a diverse set of physiological processes in all forms of life ranging from bacteria and plants to humans. Unlike other cellular signaling processes, redox signaling involves highly reactive reagents such as nitric oxide (NO), carbon monoxide (CO), and reactive oxygen species that raise concerns regarding the potential of these reagents for participation in other non-specific reactions. Yet, such side reactions are uncommon under physiological conditions suggesting high specificity and selectivity of enzymatic redox signal transduction pathways. In turn, we ask the following two pertinent questions: a) What makes redox signaling pathways so specific? and b) Can we rationally and systematically rewire redox signal transduction pathways to re-instate/disrupt cellular redox balance? These questions have been largely overlooked from the chemical biology and bioinorganic chemistry perspective, despite the fact that redox imbalances are responsible for a variety of diseases ranging from neurological disorders to cancer. Our lab focuses on these paradigm shifting questions and the long-term goal of our research program is to develop molecular strategies that rewire sensing/signaling mechanisms of biological redox reagents involved in human health and disease. In this proposal, we focus on DosS-DosR enzymatic signaling pathway in mycobacteria that senses NO/CO in its microenvironment and signals cellular transition into a non-replicating, dormant state. The DosS-DosR system has three components – a heme iron sensing domain that binds to NO/CO, a zinc-dependent signaling kinase domain that communicates the binding event and a response domain that binds to DNA and turns on dormancy. Using our combined expertise in metalloprotein structure-function, protein engineering, enzymology and spectroscopy, we will devise mutagenic, metal-substitution and peptidomimetic-based approaches to rewire DosS-DosR sense, signal and response domains. Proof-of-concept studies conducted in our laboratory have demonstrated successful rewiring of DosS- DosR redox sensing function via structure-guided rational protein design. Future work will unravel the molecular mechanisms of redox rewiring and its implications on cellular physiology and phenotypic responses. Our findings, will not only provide a fundamental understanding of cellular redox sense/signal/response mechanisms, but also inform future methodologies for treatment and prevention of redox-related diseases. Health Relevance: Maintenance of a normal intracellular redox status is crucial for regulating physiological responses. Any imbalance in this status results in a variety of acute and chronic degenerative diseases such as cancer, cardiovascular and neurological disorders. Our research program aims to design molecular approaches that rewires the ability of cells to sense and signal redox changes in their environment. These approaches could be applied to re-instate cellular redox homeostasis in diseased states.