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首页> 外文期刊>Biochemistry >Sequence specificity and role of proximal amino acids of the histone H3 tail on catalysis of murine G9a lysine 9 histone H3 methyltransferase
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Sequence specificity and role of proximal amino acids of the histone H3 tail on catalysis of murine G9a lysine 9 histone H3 methyltransferase

机译：在鼠G9A赖氨酸9种组蛋白H3甲基转移酶的催化下组蛋白H3尾部近端氨基酸的序列特异性和作用

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摘要

The activity of recombinant murine G9a toward lysine 9 of histone H3 was investigated. GST fusion proteins containing various lengths of the histone H3 amino-terminal tail were used as substrates in the presence of recombinant G9a enzyme and AdoMet cosubstrate. The minimal substrate methylated by G9a contained seven amino acids (TARKSTG) of the histone H3 tail. Furthermore, mutational analysis of the minimal substrate was performed to identify the amino acids essential for G9a-mediated methylation. All amino acids except Thr-11 were indispensable for the methylation reaction. Steady-state kinetic analysis of the wild-type and histone H3 point mutants, lysine 4 changed to alanine (K4A) or lysine 27 changed to alanine (K27A), with purified G9a revealed similar catalytic efficiency but a reduction in turnover number (k(cat)) from 78 to 58 h(-1). G9a methylated synthetic peptide substrates containing the first 13 amino acids of histone H3 efficiently, although methylation, acetylation, or mutation of proximal Lys-4 amino acids reduced Lys-9 methylation. The k(cat) for wild-type peptide substrate vs Lys-4 acetyl- or trimethyl-modified peptide were 88 and 32 h-1, respectively, and the K-m for the peptides varied from 0.6 to 2.2 mu M, resulting in a large difference (15-91) in catalytic efficiency. Ser-10 or Thr-11 phosphorylation resulted in poor methylation by G9a. Immunoprecipitation of unmodified and Ser-10 and Thr-11 phosphorylated histone H3 displayed mostly Lys-4 dimethylation. Dimethylated Lys-9 was reduced in Set-10 and Thr-11 immunoprecipitated phosphorylated histones as compared to nonphosphorylated H3. In an immunocytochemical assay, GFP fusion SUV39H1 or G9a did not colocalize with phosphorylated histone H3. Thus, Set-10/Thr-11 phosphorylation impairs Lys-9 methylation. These data suggest that the sequence context of the modified residue affects G9a activity and the modification in the proximal amino acids influences methylation.
机译：研究了对组蛋白H3的重组鼠G9a的活性鼠麦氨酸9。在重组G9A酶和ADOSTumssstration的存在下，使用含有各种长度的组蛋白H3氨基末端尾部的GST融合蛋白。 G9A甲基化的最小基材含有七种氨基酸（Tarkstg）的组蛋白H3尾。此外，进行最小基质的突变分析以鉴定对G9A介导的甲基化必需的氨基酸。除了Thr-11外的所有氨基酸对于甲基化反应是必不可少的。野生型和组蛋白H3点突变体的稳态动力学分析，将赖氨酸4变为丙氨酸（K4a）或赖氨酸27变为丙氨酸（K27a），纯化的G9a显示出类似的催化效率，但营业率下降（K（猫））从78到58小时（-1）。 G9A甲基化的合成肽底物，含有组蛋白H3的前13个氨基酸，尽管近端Lys-4氨基酸的甲基化，乙酰化或突变降低了Lys-9甲基化。野生型肽基材的K（猫）与Lys-4乙酰基或三甲基改性肽分别为88和32h-1，并且肽的Km变化0.6至2.2μm，导致大差异（15-91）催化效率。 Ser-10或Thr-11磷酸化导致G9A的甲基化差。免疫沉淀未修饰的和Ser-10和Thr-11磷酸化组蛋白H3主要显示为Lys-4二甲基化。与非磷酸化H3相比，在设定-10和Thr-11免疫沉淀的磷酸化组蛋白中减少了二甲基化液体-9。在免疫细胞化学测定中，GFP融合SUV39H1或G9A与磷酸化组蛋白H3没有脱钙。因此，SET-10 / TH11磷酸化损害Lys-9甲基化。这些数据表明，改性残基的序列上下文影响G9A活性和近端氨基酸中的改性影响甲基化。

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