Structural and Functional Studies of the Regulatory Mechanism of Ubiquitin Enzymes in Stress Response and Gene Expression

细胞有复杂的机制来对它们所处的生理条件的变化做出反应，例如氧气或营养缺乏或供应过剩，电解质失衡，以及偏离正常体温。为了最大限度地在这些压力下存活，细胞使用不同的分子机制来调整其内部生物过程。在快速防御中，执行细胞功能的蛋白质在某些残基被修饰以改变其构象和相互作用伙伴，从而使它们被激活、失活或标记为降解。在更长的时间尺度上，基因的表达谱从根本上改变了，以使细胞适应变化的环境。 泛素化是一种将一种叫做泛素的小调节蛋白标记到底物蛋白上的可逆的动态过程，在应激反应和伴随基因调控的许多方面发挥着关键作用。泛素特异性蛋白水解酶9X(USP9X)是一种很大的酶，它能从泛素化的底物中去除泛素标签并控制它们的功能。参与应激反应的三种底物蛋白是ASK1，一种调节氧化应激信号转导的蛋白；SOX2，一种DNA结合蛋白，调节细胞的多能性并控制许多其他基因的表达；以及MCL1，决定细胞在应激反应中的生死。 我们将使用各种生物物理和生化技术来评估蛋白质的构象、活性和相互作用，包括X射线结晶学、酶分析、热变化测量，以了解USP9X如何在原子水平上与其底物相互作用，USP9X不同部分的分子内相互作用如何调节USP9X泛素标签的去除，以及其他修饰，如底物蛋白质的磷酸化和甲基化，如何影响它们与USP9X的相互作用。 应激反应是细胞适应内外部挑战的一种保守机制。从这项研究中获得的关于应激反应的分子机制的知识可以外推到其他生物，如动物和植物。USP9X是一种进化上保守的多功能蛋白质，对发育和应激反应至关重要，但人们对其功能的分子细节知之甚少。这项拟议的研究结果将为理解泛素酶如何调控细胞应激反应和基因调控建立一个原型模型。更好地理解泛素化的调节机制以及合理地调节它们的能力对于细胞重新编程和药物发现领域是重要的。

Cells have sophisticated mechanisms to respond to changes in the physiological conditions they live in, such as oxygen or nutrient deprivation or over-supply, imbalances of electrolytes, and deviation from normal body temperatures. To maximize survival under these stresses, cells use different molecular mechanisms to adjust their internal biological processes. In rapid defences, the proteins that carry out cellular functions are modified at certain residues to switch their conformation and interaction partners so that they are activated, deactivated, or tagged for degradation. Over a longer time scale, the expression profile of genes is fundamentally altered to adapt the cells to a changed environment. Ubiquitination, a reversible, dynamic process of tagging a small regulatory protein called ubiquitin to a substrate protein, plays a critical role in many aspects of stress response and collateral gene regulation. Ubiquitin-specific protease 9X (USP9X) is a very large enzyme that removes the ubiquitin tag from ubiquitinated substrates and controls their functions. Three such substrate proteins involved in stress response are ASK1, a kinase that regulates oxidative stress signal transduction; SOX2, a DNA-binding protein that regulates the pluripotency of cells and controls the expression of many other genes; and MCL1, which determines the life and death of cells in response to stress. We will use various biophysical and biochemical techniques that assess the conformation, activity, and interaction of the proteins, including X-ray crystallography, enzymatic assays, thermal change measurement, to find out how USP9X interacts with its substrate at the atomic level, how its removal of the ubiquitin tags is regulated by the intra-molecular interactions of different parts of USP9X, and how other modifications, such as phosphorylation and methylation of the substrate proteins, affect their interaction with USP9X. Stress response is a conserved mechanism for cells to adapt to internal and external challenges. Knowledge of the molecular mechanisms of stress response obtained from this study can be extrapolated to other organisms, such as animals and plants. USP9X is an evolutionarily conserved, multi-functional protein that is crucial for development and stress response, yet not much is known about the molecular details of its functions. The outcome of the proposed research will establish a prototypical model for understanding how ubiquitin enzymes regulate cell stress response and gene regulation. A better understanding of the regulatory mechanisms of ubiquitination and the ability to rationally modulate them is important for the fields of cell reprogramming and drug discovery.