In many cases the meaning of these studies is restricted by the very fact tha

Examining crosstalk between methylation and other posttranslational modifications can also be benefited from using well-defined homogenous proteins as PMT substrates. With an N terminal H3 peptide and its posttranslationally modified versions as substrates, the Pradhan laboratory analyzed how Thr11 ALK Inhibitor phosphorylation and Ser10 phosphorylation influence G9a catalyzed H3K9 methylation. 73 The kinetic analysis showed that S10 phosphorylation decreased kcat and Km of the methylation for over 10 fold when comparing to only 2 fold loss of kcat/Km by phosphorylation. Yamagata et. al. demonstrated that PRMT1 methylates FOXO1 at R248 and R250. 9 The 2 methylations inhibited Aktmediated phosphorylation of S253, but the S253 phosphorylation doesnt inhibit the methylation of R248/R250. Upon reviewing this work as well as other crosstalk involved with RXRXXS/T pattern, Rust and Thompson offered twelve proteins including B Raf, EZH2 and FOXG1 as highly probable PRMT1 substrates. 74 This prediction is likely to be examined easily after getting the corresponding peptides. The Zheng laboratory recently reported an approach utilizing a fluorescent peptide as Inguinal canal a chemical probe to study the transient kinetics of PMT catalysis. 75,76 In Zhengs work, Leu10 of the H4 N final peptide was replaced with a fluorescein moiety. The resultant fluorescent H4 peptide showed similar kinetics to indigenous H4 peptide as a substrate. The fluorescein labeled peptide displayed multiple cycle kinetics upon binding PRMT1, as shown by change. After dissecting the kinetics, the authors figured PRMT1 catalyzes H4 methylation via a multiple step process including an extremely fast substrate binding step, then a modestly fast development of the ternary PRMT1 SAM substrate complex, GW0742 and finally the rate limiting methylation. 75 This demonstrates an elegant using substrate type chemical probes to characterize PMTs. Proteins or protein complexes as PMT substrates The target specificity of PMTs can be changed considerably depending on the nature of these substrates. For instance, NSD2 methylates H3K36 if nucleosomes are supplied as substrates but acts on H4K44 if histone octamers while the substrates. 77 In these cases, fulllength proteins or protein complexes are more appropriate as in vitro substrates of PMTs. As substrates of CARM1 and PRMT1, p300 using in vitro reconstituted chromatin layouts, the Roeder laboratory was able to examine the p53 dependent cross-talk between the three activators. 78 The authors showed that PRMT1 involved H4R3 methylation, p300 involved CARM1 and H3/H4 acetylation involved H3R2/17/26 methylation may appear in a sequentially stimulated approach. Daujat et. al. showed the same cross-talk on the pS2 promoter, where CBP mediated H3K14/18 acetylation stimulates the connection of CARM1 with chromatin and the resultant H3R17 methylation.