Function of SMYD lysine methyltransferases in stress responses and proteostasis

项目总结 蛋白质稳态的恶化是细胞老化的标志，也是许多 与衰老相关的疾病。蛋白质平衡通常通过一个协调良好的因素网络来维持，这些因素 包括蛋白质合成调节剂、促进适当折叠分子伴侣、质量控制和 蛋白质降解机械。该蛋白质平衡网络用于防止错误折叠的堆积， 无功能的和聚集的蛋白质，这在应激和细胞老化时特别关键。蛋白质 它们在蛋白平衡网络中的功能受到不同机制的高度调控，包括它们的后 翻译修饰。修饰的赖氨酸甲基化已经在许多因素上被确定。 蛋白平衡，尽管甲基化在蛋白平衡网络中的分子作用仍不明确。 保守的SMYD赖氨酸甲基转移酶家族是已知的对蛋白质组重要的甲基化因子 完整性，如伴侣，尽管它们在维持蛋白稳定方面的功能还不是很清楚。我们 研究了一种孤儿酵母SMYD赖氨酸甲基转移酶Set6的功能，并确定了它 是蛋白质平衡网络的关键调节者，通过其甲基转移酶的活性。的首要目标是 建议的工作是确定Set6在蛋白平衡中的特定调节作用，并确定其催化活性 关于蛋白质组完整性的重要因素，包括胞质伴侣热休克蛋白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方法来剖析Set6和Set6的分子贡献 在正常和应激条件下，其对蛋白质平衡网络的催化活性。我们还将使用量化 蛋白质组学、结构-功能分析和遗传方法，以确定指导 Set6在正常和应激条件下的活性，重点是它的蛋白质-蛋白质相互作用。最后，我们 将使用分子、生物化学、 和遗传方法，并测试假设，即Set6甲基化改变Hsp70相互作用体在 压力。我们还将调查Set6是否在蛋白质平衡网络中具有额外的底物 生化、蛋白质组学和遗传分析。总之，这些研究将揭示新的监管机制 控制蛋白平衡网络，并将促进我们对SMYD酶在 蛋白质组的完整性。这项工作还将提供对与衰老相关的病理的洞察，其特征是折叠错误 或聚集蛋白，并可能发现新的机制，可以靶向治疗促进 随着细胞老化，健康的蛋白质平衡，并预防年龄依赖的疾病。

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.