Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP

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• 作者：Aravind Basavapathruni ; Jodi Gureasko ; Margaret Porter Scott ; William Hermans ; Adarsh Godbole ; Peter A. Leland ; P. Ann Boriack-Sjodin ; Tim J. Wigle ; Robert A. Copeland ; Thomas V. Riera
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：March 22, 2016
• 年：2016
• 卷：55
• 期：11
• 页码：1645-1651
• 全文大小：305K
• ISSN：1520-4995

文摘

The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of k_cat as the substrate K_M values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners.