Burgos Emmanuel S, Wilczek Carola, Onikubo Takashi, Bonanno Jeffrey B, Jansong Janina, Reimer Ulf, Shechter David
From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and.
JPT Peptide Technologies GmbH, Volmerstrasse 5, 12489 Berlin, Germany.
J Biol Chem. 2015 Apr 10;290(15):9674-89. doi: 10.1074/jbc.M115.636894. Epub 2015 Feb 24.
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 Km, 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.
在脊椎动物中，蛋白质精氨酸甲基转移酶PRMT5与WD重复蛋白MEP50（也称为Wdr77或雄激素共激活因子p44）以异二聚体四聚体的形式复合。据推测，MEP50是将蛋白质底物募集到PRMT5催化结构域所必需的。在此，我们证明交叉二聚体MEP50与其同源PRMT5分子配对以促进组蛋白甲基化。我们采用定性甲基化测定法和一种新型超灵敏连续测定法来测量酶动力学。我们证明全长人PRMT5和非洲爪蟾PRMT5催化结构域均没有明显的蛋白质甲基转移酶活性。我们表明，与组蛋白H2A(1 - 20)和H4(1 - 20)肽相比，组蛋白H4和H3与PRMT5 - MEP50的结合更有效。通过竞争实验和高密度组蛋白肽阵列相互作用研究确定，组蛋白结合是通过组蛋白折叠相互作用介导的。核小体不是PRMT5 - MEP50的底物，这与通过H3 - H4组蛋白折叠的主要相互作用模式一致，该模式在核小体中被DNA掩盖。MEP50插入环上保守精氨酸（Arg - 42）的突变通过增加其组蛋白底物Km来损害PRMT5 - MEP50的酶促效率，这与秀丽隐杆线虫PRMT5相当。我们表明PRMT5 - MEP50更喜欢未甲基化的底物，这与二甲基化的分布模型一致，并暗示单甲基化和二甲基化精氨酸修饰的蛋白质具有不同的生物学作用。我们提出了一个模型，其中MEP50和PRMT5同时与蛋白质底物结合，将其靶向精氨酸定位到催化位点。
