Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio 43210, United States.
J Proteome Res. 2020 Apr 3;19(4):1663-1673. doi: 10.1021/acs.jproteome.9b00843. Epub 2020 Mar 4.
Lysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by lysine acetyltransferases is still unclear in most cases. Hat1 is a lysine acetyltransferase originally identified based on its ability to acetylate histones. Using an unbiased proteomics approach, we have determined how loss of Hat1 affects the mammalian acetylome. Hat1 and Hat1 mouse embryonic fibroblast cell lines were grown in both glucose- and galactose-containing media, as Hat1 is required for growth on galactose, and Hat1 cells exhibit defects in mitochondrial function. Following trypsin digestion of whole cell extracts, acetylated peptides were enriched by acetyllysine affinity purification, and acetylated peptides were identified and analyzed by label-free quantitation. Comparison of the acetylome from Hat1 cells grown on galactose and glucose demonstrated that there are large carbon source-dependent changes in the mammalian acetylome where the acetylation of enzymes involved in glycolysis were the most affected. Comparisons of the acetylomes from Hat1 and Hat1 cells identified 65 proteins whose acetylation decreased by at least 2.5-fold in cells lacking Hat1. In Hat1 cells, acetylation of the autoregulatory loop of CBP (CREB-binding protein) was the most highly affected, decreasing by up to 20-fold. In addition to the proteins involved in chromatin structure, Hat1-dependent acetylation was also found in a number of transcriptional regulators, including p53 and mitochondrial proteins. Hat1 mitochondrial localization suggests that it may be directly involved in the acetylation of mitochondrial proteins. Data are available via ProteomeXchange with identifier PXD017362.
赖氨酸乙酰化已成为最重要的翻译后修饰之一，调节着不同的生物过程。然而，在大多数情况下，赖氨酸乙酰转移酶对其的调节作用还不清楚。Hat1 最初是根据其乙酰化组蛋白的能力被鉴定为一种赖氨酸乙酰转移酶。我们采用一种无偏蛋白质组学方法，确定了 Hat1 缺失如何影响哺乳动物乙酰组。我们在含有葡萄糖和半乳糖的培养基中培养 Hat1 和 Hat1 鼠胚胎成纤维细胞系，因为 Hat1 是在半乳糖中生长所必需的，而 Hat1 细胞在线粒体功能方面存在缺陷。在整个细胞提取物中用胰蛋白酶消化后，通过乙酰赖氨酸亲和纯化富集乙酰化肽，通过无标记定量法鉴定和分析乙酰化肽。将在半乳糖和葡萄糖上生长的 Hat1 细胞的乙酰组进行比较，结果表明哺乳动物乙酰组有很大的碳源依赖性变化，其中参与糖酵解的酶的乙酰化受影响最大。将 Hat1 和 Hat1 细胞的乙酰组进行比较，鉴定出 65 种蛋白质，其乙酰化程度在缺乏 Hat1 的细胞中至少降低了 2.5 倍。在 Hat1 细胞中，CBP（CREB 结合蛋白）的自调节环的乙酰化受影响最大，降低了 20 倍。除了参与染色质结构的蛋白质外，Hat1 依赖性乙酰化还存在于许多转录调节剂中，包括 p53 和线粒体蛋白质。Hat1 的线粒体定位表明它可能直接参与线粒体蛋白质的乙酰化。数据可通过 ProteomeXchange 以标识符 PXD017362 获得。
