Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5).
Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5).
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DOI：
10.1007/978-1-4939-3667-0_5
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Cristea IM
中科院分区：
文献类型：
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作者：
Guise AJ;Cristea IM
组蛋白去乙酰化酶5（HDAC 5）是组蛋白去乙酰化酶IIa类家族的一员，具有核质穿梭功能，是一种重要的转录调节因子。它的失调与人类的主要疾病有关，包括心脏病和肿瘤。在这一章中，我们描述了几个实验方法，已被证明有效的研究HDAC 5的功能和监管特点。我们目前的方法，用于评估的亚细胞定位，蛋白质相互作用，翻译后修饰（PTM），和活动的HDAC 5从调查的立场无论是内源性蛋白质或标记的蛋白质形式在人类细胞。具体而言，鉴于HDAC 5调节的核心在于其动态定位，相互作用和PTM，我们提出了用于评估HDAC 5在固定和活细胞中的定位，用于分离含HDAC 5的蛋白质复合物以鉴定其相互作用和修饰，以及用于确定这些PTM如何映射到预测的HDAC 5结构基序的方法。最后，我们提供了研究HDAC 5功能的方法的例子，重点是它在细胞周期进程中的调节。这些方法可以很容易地适用于其他HDAC或非HDAC蛋白的研究。单独地，这些技术捕获HDAC 5功能的时间和空间快照;然而，这些方法一起为研究HDAC 5在与健康和疾病相关的不同细胞环境中的调节和调节作用提供了强大的工具。
As a member of the class IIa family of histone deacetylases, the histone deacetylase 5 (HDAC5) is known to undergo nuclear–cytoplasmic shuttling and to be a critical transcriptional regulator. Its misregulation has been linked to prominent human diseases, including cardiac diseases and tumorigenesis. In this chapter, we describe several experimental methods that have proven effective for studying the functions and regulatory features of HDAC5. We present methods for assessing the subcellular localization, protein interactions, posttranslational modifications (PTMs), and activity of HDAC5 from the standpoint of investigating either the endogenous protein or tagged protein forms in human cells. Specifically, given that at the heart of HDAC5 regulation lie its dynamic localization, interactions, and PTMs, we present methods for assessing HDAC5 localization in fixed and live cells, for isolating HDAC5-containing protein complexes to identify its interactions and modifications, and for determining how these PTMs map to predicted HDAC5 structural motifs. Lastly, we provide examples of approaches for studying HDAC5 functions with a focus on its regulation during cell-cycle progression. These methods can readily be adapted for the study of other HDACs or non-HDAC-proteins of interest. Individually, these techniques capture temporal and spatial snapshots of HDAC5 functions; yet together, these approaches provide powerful tools for investigating both the regulation and regulatory roles of HDAC5 in different cell contexts relevant to health and disease.