Bioorthogonal Profiling of Protein Methylation Using Azido Derivative of S-Adenosyl-l-methionine

Abstract

Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally-engineered PMTs to label substrates of the corresponding native PMTs. Here 4-azido-but-2-enyl derivative of S-adenosyl-L-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor-enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e. g. EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the non-histone targets of EuHMT1/2. Comparison of EuHMT1/2’s methylomes indicates that the two human PMTs, though similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification.

Defining efficient enzyme–cofactor pairs for bioorthogonal profiling of protein methylation

“…Multiple SAM analogs have been reported as active cofactors for native and engineered PMTs (10,13,(23)(24)(25)(26). Most of these compounds contain a characteristic sulfonium-β-sp 2 carbon to promote the enzymatic transalkylation reaction (10).…”

“…These cofactor surrogates can therefore be used for target identification (10) when coupled with copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC/"Click Chemistry") (11,12). The utility of terminal alkyne/azide-containing SAM analogs was further expanded by the bioorthogonal profiling of protein methylation (BPPM) technology (13), in which PMTs are engineered to accommodate bulky SAM analogs (the "bumphole" approach as applied for kinases) (14) for substrate labeling and subsequent target identification (SI Appendix, Fig. S1).…”

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

“…For example, incorporation of different transferrable amine moieties ( 21 ,4, 18 22 ,4, 19 23 ,4, 19b Table S2) can be followed by a coupling reaction with the N ‐hydroxysuccinimide (NHS) ester of an amine‐reactive probe 18. In addition, several examples exist on the application of AdoMet analogues with a transferrable terminal alkyne ( 5 ,20 Scheme 6; 20 ,20g,20h 24 ,20b,20c,20g and 25 ,4, 19b Table S2) or azide ( 26 g ,20, 21 27 ) 4. The transferred alkynes and azides can be further functionalized by using the biocompatible and highly‐efficient azide–alkyne cycloaddition reaction (one of the “click” series of reactions).…”

“…The focus of their work has been the human protein MTases G9a (also known as EuHMT2), GLP1 (also known as EuHMT1), and PRMT1 (for a complete overview, see Table 2). Enzyme‐mediated transalkylation reactions using an azido‐modified AdoMet analogue with engineered G9a and GLP121, 60 and an alkyne‐modified AdoMet analogue with engineered G9a20c and PRMT1 have been demonstrated 20g. Additionally, a selenium‐based SAM analogue with an alkyne linker was effectively employed in transalkyation reactions directed and catalyzed by the native protein MTases GLP1, G9a and SUV39H2 28, 60, 61…”

“…Target proteins are then coupled to a clickable dye, for example, dibenzylcyclooctyne‐coupled dyes, and identified by mass spectrometry (MS) analysis or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE; Figure 3). 21, 60, 61 The approach was used for detecting the substrates of the human protein MTase PRMT3, which preferably resides within the cytoplasm 20a. Interestingly, while the protein MTase localizes in the cytoplasm, 23 % of the modifications were found in the nucleus, thus suggesting a broader role for PRMT3.…”