Activity-based probes for proteomic profiling of histone deacetylase complexes
Activity-based probes for proteomic profiling of histone deacetylase complexes
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DOI：
10.1073/pnas.0608659104
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发表时间：
2007-01-23
影响因子：
11.1
通讯作者：
Cravatt, Benjamin F.
中科院分区：
文献类型：
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作者：
Salisbury, Cleo M.;Cravatt, Benjamin F.
组蛋白脱乙酰酶（HDAC）是基因表达的关键调节因子，其需要组装成较大的蛋白质复合物以获得活性。了解相关蛋白如何调节HDAC功能的努力将受益于评估天然生物系统中HDAC活性的新技术。在这里，我们描述了一种活性定点化学探针，用于分析天然蛋白质组和活细胞中的HDAC。该探针，命名为SAHA-BPyne，含有一般HDAC抑制剂辛二酰苯胺异羟肟酸（SAHA）的结构元件，以及二苯甲酮和炔部分，以分别实现HDAC的共价修饰和富集。I类和11类HDAC都被鉴定为蛋白质组中SAHA-BPyne的特异性靶标。有趣的是，即使在探针标记的蛋白质组变性后，SAHA-BPyne也富集了多种HDAC相关蛋白。这些数据表明，某些HDAC相关蛋白质被SAHA-BPyne直接修饰，使它们靠近HDAC活性位点，在那里它们将被引发以调节底物识别和活性。我们进一步表明，SAHA-BPyne可用于测量人类疾病模型中HDAC含量和复杂组装的差异。因此，这种化学蛋白质组学探针应该证明对HDAC的活性状态和调节其功能的结合蛋白的分析是有价值的。
Histone deacetylases (HDACs) are key regulators of gene expression that require assembly into larger protein complexes for activity. Efforts to understand how associated proteins modulate the function of HDACs would benefit from new technologies that evaluate HDAC activity in native biological systems. Here, we describe an active site-directed chemical probe for profiling HDACs in native proteomes and live cells. This probe, designated SAHA-BPyne, contains structural elements of the general HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), as well as benzophenone and alkyne moieties to effect covalent modification and enrichment of HDACs, respectively. Both class I and 11 HDACs were identified as specific targets of SAHA-BPyne in proteomes. Interestingly, multiple HDAC-associated proteins were also enriched by SAHA-BPyne, even after denaturation of probe-labeled proteomes. These data indicate that certain HDAC-associated proteins are directly modified by SAHA-BPyne, placing them in close proximity to HDAC active sites where they would be primed to regulate substrate recognition and activity. We further show that SAHA-BPyne can be used to measure differences in HDAC content and complex assembly in human disease models. This chemical proteomics probe should thus prove valuable for profiling both the activity state of HDACs and the binding proteins that regulate their function.