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

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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.