Department of Chemistry, Xavier University of Louisiana, New Orleans, LA, USA.
FASEB J. 2020 Oct;34(10):13140-13155. doi: 10.1096/fj.202001301RR. Epub 2020 Aug 30.
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.
赖氨酸乙酰化是一种发生在数千个人类蛋白质上的翻译后修饰，其中大多数位于细胞质中。乙酰化蛋白质参与许多细胞过程和人类疾病。因此，乙酰化/去乙酰化循环如何被调控是一个重要的问题。人类细胞中已经鉴定出 11 种金属依赖性赖氨酸去乙酰化酶（KDACs）。这些酶与 Sirtuins 一起，共同负责逆转赖氨酸乙酰化。尽管已经有几项大规模的研究对乙酰组进行了特征描述，但只有相对较少的特定乙酰化残基与提议的去乙酰化 KDAC 相对应。为了理解赖氨酸乙酰化及其与疾病的关联，必须鉴定特定的 KDAC-底物对。鉴定 KDAC 的特定底物既受到相关活性测定的复杂性的影响，也受到 KDAC 与细胞蛋白的非催化相互作用的影响。在这里，我们讨论了用于鉴定 KDAC-底物对的体外和基于细胞的实验策略，并评估了每种策略的目的，即直接鉴定金属依赖性 KDAC 的非组蛋白底物。我们提出了一个组合标准，该标准需要可重复的实验方法，以建立直接的酶促关系。对文献的批判性分析确定了 108 对被提议的非组蛋白底物-KDAC 对，其中已有直接实验证据报道。在这些中，有 5 对可以被认为是成熟的，而另外 13 对既有基于细胞的证据，也有体外证据，但缺乏独立的重复和/或足够的基于细胞的证据。我们提出了一个评估剩余底物线索并可靠地鉴定新的 KDAC 底物的前进道路。
