利用定量相互作用蛋白质组学阐明核小体重塑与去乙酰化酶复合物的稳定性、动力学和结构。

Towards elucidating the stability, dynamics and architecture of the nucleosome remodeling and deacetylase complex by using quantitative interaction proteomics.

作者信息

Kloet Susan L, Baymaz H Irem, Makowski Matthew, Groenewold Vincent, Jansen Pascal W T C, Berendsen Madeleine, Niazi Hassin, Kops Geert J, Vermeulen Michiel

机构信息

Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, the Netherlands.

出版信息

FEBS J. 2015 May;282(9):1774-85. doi: 10.1111/febs.12972. Epub 2014 Sep 11.

DOI:10.1111/febs.12972

PMID:25123934

Abstract

UNLABELLED

The nucleosome remodeling and deacetylase (NuRD) complex is an evolutionarily conserved chromatin-associated protein complex. Although the subunit composition of the mammalian complex is fairly well characterized, less is known about the stability and dynamics of these interactions. Furthermore, detailed information regarding protein-protein interaction surfaces within the complex is still largely lacking. Here, we show that the NuRD complex interacts with a number of substoichiometric zinc finger-containing proteins. Some of these interactions are salt-sensitive (ZNF512B and SALL4), whereas others (ZMYND8) are not. The stoichiometry of the core subunits is not affected by high salt concentrations, indicating that the core complex is stabilized by hydrophobic interactions. Interestingly, the RBBP4 and RBBP7 proteins are sensitive to high nonionic detergent concentrations during affinity purification. In a subunit exchange assay with stable isotope labeling by amino acids in cell culture (SILAC)-treated nuclear extracts, RBBP4 and RBBP7 were identified as dynamic core subunits of the NuRD complex, consistent with their proposed role as histone chaperones. Finally, using cross-linking MS, we have uncovered novel features of NuRD molecular architecture that complement our affinity purification-MS/MS data. Altogether, these findings extend our understanding of MBD3-NuRD structure and stability.

STRUCTURED DIGITAL ABSTRACT

MBD3 physically interacts with ZNF512B, HDAC1, ZMYND8, GATAD2B, SALL4, GATAD2A, ZNF592, MTA3, ZNF687, CDK2AP1, CHD3, ZNF532, HDAC2, MTA2, CHD4, MTA1, KPNA2, CHD5, RBBP4 and RBBP7 by pull down (View interaction) CDK2AP1 physically interacts with MBD3, MTA3, HDAC2, GATAD2A, CHD4, CDK2AP1, MTA2, HDAC1, MTA1, CHD3, GATAD2B, MBD2, RBBP4 and RBBP7 by pull down (View interaction) MBD3 physically interacts with MTA2, MTA3, RBBP4, RBBP7, HDAC2, HDAC1, CHD4, CHD3 and MTA1 by cross-linking study (View interaction).

摘要

未标记

核小体重塑与去乙酰化酶（NuRD）复合物是一种进化上保守的染色质相关蛋白复合物。尽管哺乳动物复合物的亚基组成已得到相当充分的表征，但对这些相互作用的稳定性和动力学了解较少。此外，关于复合物内蛋白质 - 蛋白质相互作用表面的详细信息仍然非常缺乏。在这里，我们表明NuRD复合物与许多亚化学计量的含锌指蛋白相互作用。其中一些相互作用对盐敏感（ZNF512B和SALL4），而其他一些（ZMYND8）则不敏感。核心亚基的化学计量不受高盐浓度影响，表明核心复合物通过疏水相互作用得以稳定。有趣的是，在亲和纯化过程中，RBBP4和RBBP7蛋白对高浓度非离子去污剂敏感。在细胞培养中用氨基酸稳定同位素标记（SILAC）处理的核提取物进行的亚基交换试验中，RBBP4和RBBP7被鉴定为NuRD复合物的动态核心亚基，这与其作为组蛋白伴侣的假定作用一致。最后，使用交联质谱，我们发现了NuRD分子结构的新特征，这些特征补充了我们的亲和纯化 - MS/MS数据。总之，这些发现扩展了我们对MBD3 - NuRD结构和稳定性的理解。

结构化数字摘要

通过下拉实验，MBD3与ZNF512B、HDAC1、ZMYND8、GATAD2B、SALL4、GATAD2A、ZNF592、MTA3、ZNF687、CDK2AP1、CHD3、ZNF532、HDAC2、MTA2、CHD4、MTA1、KPNA2、CHD5、RBBP4和RBBP7发生物理相互作用（查看相互作用）；通过下拉实验，CDK2AP1与MBD3、MTA3、HDAC2、GATAD2A、CHD4、CDK2AP1、MTA2、HDAC1、MTA1、CHD3、GATAD2B、MBD2、RBBP4和RBBP7发生物理相互作用（查看相互作用）；通过交联研究，MBD3与MTA2、MTA3、RBBP4、RBBP7、HDAC2、HDAC1、CHD4、CHD3和MTA1发生物理相互作用（查看相互作用）。