Function of SMYD lysine methyltransferases in stress responses and proteostasis - PROJECT SUMMARY The deterioration of protein homeostasis is a signature of aging cells and underlies the etiology of numerous aging-related diseases. Proteostasis is normally maintained through a well-coordinated network of factors that include protein synthesis regulators, molecular chaperones that promote proper folding, quality control and proteolytic degradation machinery. This proteostasis network serves to prevent the accumulation of misfolded, non-functional, and aggregated proteins, which is particularly critical during stress and as cells age. Proteins which function within the proteostasis network are highly-regulated by different mechanisms including their post- translational modification. The modification lysine methylation has been identified on many factors that contribute to proteostasis, though the molecular role for methylation within the proteostasis network remains poorly-defined. The conserved SMYD family of lysine methyltransferases are known to methylate factors important to proteome integrity, such as chaperones, although their function in maintaining proteostasis is not well-understood. We have investigated the function of an orphan yeast SMYD lysine methyltransferase, Set6, and determined that it is a critical regulator of the proteostasis network via its methyltransferase activity. The overarching goal of the proposed work is to identify the specific regulatory role for Set6 in proteostasis and to define its catalytic activity on factors important for proteome integrity, including the cytosolic chaperone Hsp70. To address this, we propose to use a series of molecular and cell biological assays to dissect the molecular contribution of Set6 and its catalytic activity to the proteostasis network under normal and stress conditions. We will also use quantitative proteomics, structure-function analysis, and genetic approaches to define regulatory mechanisms directing the activity of Set6 under normal and stress conditions, with a focus on its protein-protein interactions. Finally, we will define the molecular consequences of Set6-dependent methylation on Hsp70 using molecular, biochemical, and genetic approaches, and test the hypothesis that methylation by Set6 alters the Hsp70 interactome during stress. We will also investigate whether Set6 has additional substrates in the proteostasis network using biochemical, proteomic, and genetic assays. Altogether, these studies will reveal new regulatory mechanisms governing the proteostasis network and will advance our understanding of the roles for SMYD enzymes in proteome integrity. This work will also provide insight into aging-related pathologies characterized by misfolded or aggregated proteins and may uncover new mechanisms that can be targeted therapeutically to promote healthy proteostasis as cells age and prevent age-dependent diseases.
