Lysine acetylation is a posttranslational modification that occurs on thousands of human proteins, most of which are cytoplasmic. Acetylated proteins are involved in numerous cellular processes and human diseases. Therefore, how the acetylation/deacetylation cycle is regulated is an important question. Eleven metal‐dependent lysine deacetylases (KDACs) have been identified in human cells. These enzymes, along with the sirtuins, are collectively responsible for reversing lysine acetylation. Despite several large‐scale studies which have characterized the acetylome, relatively few of the specific acetylated residues have been matched to a proposed KDAC for deacetylation. To understand the function of lysine acetylation, and its association with diseases, specific KDAC‐substrate pairs must be identified. Identifying specific substrates of a KDAC is complicated both by the complexity of assaying relevant activity and by the non‐catalytic interactions of KDACs with cellular proteins. Here, we discuss in vitro and cell‐based experimental strategies used to identify KDAC‐substrate pairs and evaluate each for the purpose of directly identifying non‐histone substrates of metal‐dependent KDACs. We propose criteria for a combination of reproducible experimental approaches that are necessary to establish a direct enzymatic relationship. This critical analysis of the literature identifies 108 proposed non‐histone substrate‐KDAC pairs for which direct experimental evidence has been reported. Of these, five pairs can be considered well‐established, while another thirteen pairs have both cell‐based and in vitro evidence but lack independent replication and/or sufficient cell‐based evidence. We present a path forward for evaluating the remaining substrate leads and reliably identifying novel KDAC substrates.

中文翻译：

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金属依赖性赖氨酸脱乙酰酶的非组蛋白人类底物的批判性审查

赖氨酸乙酰化是一种翻译后修饰，发生在数千种人类蛋白质上，其中大部分是细胞质的。乙酰化蛋白质参与许多细胞过程和人类疾病。因此，如何调控乙酰化/去乙酰化循环是一个重要的问题。已在人类细胞中鉴定出 11 种金属依赖性赖氨酸脱乙酰酶 (KDAC)。这些酶与沉默调节蛋白一起共同负责逆转赖氨酸乙酰化。尽管有几项大规模研究对乙酰化组进行了表征，但相对较少的特定乙酰化残基与提议的 KDAC 相匹配以进行脱乙酰化。要了解赖氨酸乙酰化的功能及其与疾病的关联，必须确定特定的 KDAC-底物对。由于检测相关活性的复杂性以及 KDAC 与细胞蛋白质的非催化相互作用，识别 KDAC 的特定底物很复杂。在这里，我们讨论了用于识别 KDAC 底物对的体外和基于细胞的实验策略，并评估每种策略以直接识别金属依赖性 KDAC 的非组蛋白底物。我们提出了建立直接酶促关系所必需的可重复实验方法组合的标准。这种对文献的批判性分析确定了 108 个提议的非组蛋白底物-KDAC 对，已报告了直接的实验证据。其中，五对可以被认为是成熟的，而另外 13 对同时具有基于细胞和体外的证据，但缺乏独立复制和/或足够的基于细胞的证据。我们提出了评估剩余基板引线和可靠识别新型 KDAC 基板的前进道路。