Current Protocols in Chemical Biology

Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York.
Current protocols in chemical biology 03/2013; 5(1):67-88. DOI: 10.1002/9780470559277.ch120241
Source: PubMed


Enzymatic transmethylation from the cofactor S-adenosyl-L-methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne-containing SAM mimic, Se-adenosyl-L-selenomethionine (ProSeAM), and a cleavable azido-azo-biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM. The article continues with the enrichment of the ProSeAM-labeled proteins with the azido-azo biotin probe as a pulldown reagent and the subsequent reductive elution with sodium dithionate for proteomic analysis. The protocols provided here were formulated for ProSeAM as a profiling reagent but can be applied to other terminal-alkyne-containing SAM analog cofactors. Curr. Protoc. Chem. Biol. 5:67-88 © 2013 by John Wiley & Sons, Inc.

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    ABSTRACT: Human protein methyltransferases (PMTs) play essential roles through methylating histone and nonhistone targets. It is very challenging to profile global methylation (or methylome) in the context of relevant cellular settings. Unlike other posttranslational modifications such as lysine acetylation or Ser/Thr/Tyr/His phosphorylation, methylation of lysine or arginine does not significantly alter its physical properties (e.g. charge and size) and therefore may not be probed readily by conventional biological tools such antibodies. It is also not trivial to assign unambiguously dynamic methylation events to specific PMTs given their potential redundancy. This review focuses on the decade-long progress in developing complementary chemical tools to elucidate targets of designated PMTs. One of such efforts was to develop the Bioorthogonal Profiling of Protein Methylation (BPPM) technology in vitro and inside living cells.
    No preview · Article · Sep 2013 · Current opinion in chemical biology
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    ABSTRACT: Protein arginine N-methyltransferase 3 (PRMT3) belongs to the family of type I PRMTs and harbors the activity to use S-adenosyl-l-methionine (SAM) as a methyl-donor cofactor for protein arginine labeling. However, PRMT3's functions remain elusive with the lacked knowledge of its target scope in cellular settings. Inspired by the emerging Bioorthogonal Profiling of Protein Methylation (BPPM) using engineered methyltransferases and SAM analogues for target identification, the current work documents the endeavor to systematically explore the SAM-binding pocket of PRMT3 and identify suitable PRMT3 variants for BPPM. The M233G single point mutation transforms PRMT3 into a promiscuous alkyltransferase using sp(2)-β-sulfonium-containing SAM analogues as cofactor surrogates. Here the conserved methionine was defined as a hot spot that can be engineered alone or in combination with nearby residues to render cofactor promiscuity of multiple type I PRMTs. With this promiscuous variant and the matched 4-propargyloxy-but-2-enyl (Pob)-SAM analogue as the BPPM reagents, more than 80 novel proteins were readily uncovered as potential targets of PRMT3 in the cellular context. Subsequent target validation and functional analysis correlated the PRMT3 methylation to several biological processes such as cytoskeleton dynamics, whose roles might be compensated by other PRMTs. These BPPM-revealed substrates are primarily localized but not restricted in cytoplasm, the preferred site of PRMT3. The broad localization pattern may implicate the diverse roles of PRMT3 in the cellular setting. The revelation of PRMT3 targets and the transformative character of BPPM for other PRMTs present unprecedented pathways toward elucidating physiological and pathological roles of diverse PRMTs.
    No preview · Article · Dec 2013 · ACS Chemical Biology
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    ABSTRACT: Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-L-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAM) into the SAM analogues with engineered MATs. Here we describe a general, sensitive activity assay for engineered MATs by converting the reaction products into S-alkyl-thioadenosines, followed by HPLC/MS/MS quantification. With this assay, 40 human MAT mutants were evaluated against seven SAAM as potential substrates. The structure-activity-relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower Km value in contrast to native MATs), the gained activity towards the bulky SAAM stems from their ability to maintain the desired linear SN2 transition state (reflected by higher kcat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAM.
    No preview · Article · Dec 2013 · Analytical Biochemistry