Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase
Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase
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DOI：
10.1074/jbc.m115.636894
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发表时间：
2015-04-10
影响因子：
4.8
通讯作者：
Shechter, David
中科院分区：
文献类型：
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作者：
Burgos, Emmanuel S.;Wilczek, Carola;Shechter, David
蛋白质精氨酸甲基转移酶PRMT 5与WD重复蛋白MEP 50（也称为Wdr 77或雄激素共激活因子p44）在脊椎动物中以异二聚体的四聚体复合。假设MEP 50是蛋白质底物募集到PRMT 5的催化结构域所需的。在这里，我们证明了交叉二聚体MEP 50与其同源PRMT 5分子配对，以促进组蛋白甲基化。我们采用定性甲基化测定和一种新的超灵敏连续测定来测量酶动力学。我们证明，无论是全长人类PRMT 5，也没有非洲爪蟾PRMT 5催化结构域具有可观的蛋白质甲基转移酶活性。我们发现，组蛋白H4和H3与组蛋白H2 A（1-20）和H4（1-20）肽相比更有效地结合PRMT 5-MEP 50。组蛋白结合通过组蛋白折叠相互作用介导，如通过竞争实验和高密度组蛋白肽阵列相互作用研究所确定的。核小体不是PRMT 5-MEP 50的底物，这与通过H3-H4的组蛋白折叠的主要相互作用模式一致，被核小体中的DNA掩盖。MEP 50插入环上的保守精氨酸（Arg-42）的突变通过增加与秀丽隐杆线虫PRMT 5相当的组蛋白底物K-m来损害PRMT 5-MEP 50酶促效率。我们发现，PRMT 5-MEP 50喜欢未甲基化的底物，与二甲基化的分布模型一致，并建议单和二甲基精氨酸修饰的蛋白质的离散生物学作用。我们提出了一个模型，其中MEP 50和PRMT 5同时从事蛋白质底物，定向其靶向精氨酸的催化位点。
The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K-m comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.