Function of SMYD lysine methyltransferases in stress responses and proteostasis

项目摘要 蛋白质稳态的恶化是衰老细胞的标志，并且是许多疾病的病因学基础。 与衰老有关的疾病。蛋白质稳态通常通过一个协调良好的因子网络来维持， 包括蛋白质合成调节剂、促进正确折叠分子伴侣、质量控制 蛋白水解降解机制。这种蛋白质稳态网络用于防止错误折叠的， 非功能性和聚集的蛋白质，这在压力和细胞老化期间特别重要。蛋白 其在蛋白质稳态网络中的功能受到不同机制的高度调节，包括它们的后 翻译修饰修饰赖氨酸甲基化已被确定在许多因素，有助于 蛋白质稳态，虽然甲基化的蛋白质稳态网络中的分子作用仍然不明确。 已知保守的赖氨酸甲基转移酶SMYD家族甲基化蛋白质组重要因子 完整性，如分子伴侣，尽管它们在维持蛋白质稳态中的功能还不清楚。我们 研究了孤儿酵母SMYD赖氨酸甲基转移酶Set 6的功能，并确定它 是通过其甲基转移酶活性的蛋白质稳态网络的关键调节剂。的首要目标 建议的工作是确定Set 6在蛋白质稳定中的特定调节作用，并确定其催化活性 对蛋白质组完整性重要的因素，包括胞质分子伴侣Hsp 70。为了解决这个问题，我们 建议使用一系列分子和细胞生物学测定来剖析Set 6的分子贡献， 其在正常和应激条件下对蛋白质稳态网络的催化活性。我们还将使用定量 蛋白质组学、结构-功能分析和遗传学方法来确定指导基因表达的调控机制。 Set 6在正常和应激条件下的活性，重点是其蛋白质-蛋白质相互作用。最后我们 将使用分子，生物化学， 和遗传学方法，并测试Set 6甲基化改变Hsp 70相互作用组的假设， 应力我们还将研究Set 6在蛋白质稳态网络中是否有额外的底物， 生物化学、蛋白质组学和遗传分析。总之，这些研究将揭示新的调控机制， 管理蛋白质稳态网络，并将促进我们对SMYD酶在蛋白质稳态中的作用的理解。 蛋白质组完整性这项工作也将提供洞察衰老相关的病理特点是错误折叠的， 或聚集的蛋白质，并可能揭示新的机制，可以靶向治疗，以促进 随着细胞衰老，蛋白质代谢保持健康，并预防年龄依赖性疾病。

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.