Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions

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（lysine acetylation）已成为目前最重要的翻译后修饰（post-translational modifications）之一，可调控多种生物学过程。然而在多数情况下，赖氨酸乙酰转移酶（lysine acetyltransferases）对其的调控机制仍未阐明。Hat1是一类最初通过其组蛋白乙酰化活性被鉴定得到的赖氨酸乙酰转移酶。本研究采用无偏倚蛋白质组学（proteomics）方法，解析了Hat1缺失对哺乳动物乙酰化组（acetylome）的影响。我们分别在含葡萄糖（glucose）与半乳糖（galactose）的培养基中培养Hat1野生型（Hat1+/+）与Hat1敲除（Hat1–/–）小鼠胚胎成纤维细胞（mouse embryonic fibroblast）系，鉴于Hat1是细胞在半乳糖培养基中生长的必需因子，且Hat1–/–细胞存在线粒体功能缺陷。对全细胞提取物进行胰蛋白酶消化（trypsin digestion）后，通过乙酰赖氨酸亲和纯化（acetyllysine affinity purification）富集乙酰化肽段，并采用无标记定量（label-free quantitation）技术对鉴定得到的乙酰化肽段进行分析。对比在半乳糖与葡萄糖培养基中培养的Hat1+/+细胞的乙酰化组，发现哺乳动物乙酰化组存在显著的碳源依赖性变化，其中参与糖酵解（glycolysis）的酶的乙酰化水平受影响最为显著。对比Hat1+/+与Hat1–/–细胞的乙酰化组，本研究共鉴定出65种蛋白质，其乙酰化水平在Hat1缺失细胞中至少下降2.5倍。在Hat1–/–细胞中，CREB结合蛋白（CREB-binding protein，CBP）的自调控环路的乙酰化受影响程度最高，下调幅度可达20倍。除参与染色质结构（chromatin structure）调控的蛋白质外，本研究还在多种转录调控因子（包括p53）以及线粒体蛋白质中发现了Hat1依赖性的乙酰化修饰。Hat1的线粒体定位提示其可能直接参与线粒体蛋白质的乙酰化过程。相关数据集可通过蛋白质组交换库（ProteomeXchange）获取，标识符为PXD017362。