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

泛素酶在应激反应和基因表达中的调节机制的结构和功能研究

• 批准号: RGPIN-2017-06520
• 负责人: Tong, Yufeng
• 金额: $ 3.79万
• 依托单位: University of Windsor
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=762119
• 关键词: Structural; Functional; Studies; Regulatory; Mechanism

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.

细胞具有复杂的机制来应对其所生活的生理状况的变化，例如氧或营养剥夺或过度供应，电解质失衡以及与正常体温的偏离。为了在这些应力下最大化生存，细胞使用不同的分子机制来调整其内部生物学过程。在快速防御中，执行细胞功能的蛋白质在某些残基上进行了修改，以切换其构象和相互作用伴侣，以使它们被激活，停用或标记为降解。在较长的时间尺度上，基因的表达曲线从根本上改变了，以使细胞适应改变的环境。 泛素化是将一种称为泛素的小调节蛋白标记为底物蛋白的小调节蛋白的动态过程，在压力反应和侧支基因调节的许多方面都起着至关重要的作用。泛素特异性蛋白酶9X（USP9X）是一种非常大的酶，可从泛素化底物中去除泛素标签并控制其功能。参与应力反应的三个这样的底物蛋白是Ask1，这是一种调节氧化应激信号转导的激酶。 Sox2是一种调节细胞多能性并控制许多其他基因表达的DNA结合蛋白。和MCL1，它决定了响应压力的细胞生命和死亡。我们将使用各种生物物理和生化技术评估蛋白质的构象，活性，活性和相互作用，包括X射线晶体学测定，酶促测定，热变化测量值，以与其在ISP9X相互作用的标签相互作用。 USP9X不同部分的分子内相互作用，以及其他修饰（例如底物蛋白的磷酸化和甲基化）如何影响它们与USP9X的相互作用。压力反应是细胞适应内部和外部挑战的保守机制。可以将从这项研究获得的压力反应的分子机制知识推断到其他生物，例如动物和植物。 USP9X是一种进化保守的多功能蛋白，对于发育和应激反应至关重要，但对其功能的分子细节知之甚少。拟议研究的结果将建立一个原型模型，以了解泛素酶如何调节细胞应激反应和基因调节。更好地理解泛素化的调节机制以及合理调节它们的能力对于细胞重编程和药物发现的领域很重要。