Current chemical biology approaches to interrogate protein methyltransferases

Abstract

Protein methyltransferases (PMTs) play various physiological and pathological roles through methylating histone and nonhistone targets. However, most PMTs including more than 60 human PMTs remain to be fully characterized. The current approaches to elucidate the functions of PMTs have been diversified by many emerging chemical biology technologies. This review focuses on progress in these aspects and is organized into four discussion modules (assays, substrates, cofactors, and inhibitors) that are important to elucidate biological functions of PMTs. These modules are expected to provide general guidance and present emerging methods for researchers to select and combine suitable PMT-activity assays, well-defined substrates, novel SAM surrogates, and PMT inhibitors to interrogate PMTs.

Figure 1

Reactions catalyzed by transferase enzymes and protein methyltransferases (PMTs). A transferase enzyme (X-transferase) has the ability to transfer a functional moiety (X) from a cofactor or coenzyme (A–X) to its substrates (B) and generate modified products (B–X) and a cofactor metabolite (A). In the case of PMTs, the functional moiety, the cofactor, substrates, products and the byproduct are a methyl group, S-adenosylmethionine (SAM), specific Lys/Arg side chains of proteins, methylated products and S-adenosylhomocysteine, respectively.

Figure 2

Human PMTs. The human genome encodes > 60 PMTs, which diverge into protein lysine methyltransferases (PKMTs, > 50) and protein arginine methyltransferases (PRMTs, around 9). PKMTs can mono-/di-/tri-methylate their substrates in a processive or distributive manner. According to the three forms of arginine methylation products (MMA, ADMA and SDMA for monomethylarginine, asymmetric dimethylarginine and symmetric dimethylarginine, respectively), PRMTs can be categorized into three subtypes: Type I (MMA-then-ADMA products for PRMT1, 2, 3, 4, 6, 8), Type II (MMA-then-SDMA products for PRMT5, 7) and Type III (MMA product for certain targets of PRMT7). The methylation pattern of PRMT9 remains to be characterized unambiguously.

Figure 3

Schematic presentation of current PMT-activity assays and potential interfering factors. The current PMT-activity assays mainly rely on quantification of methylated protein products or the byproduct SAH. Methylated peptide/protein products can be quantified directly by radiometric and top-down mass spectrometric methods (Methods 1,2). The digested products of peptide/protein can be quantified by middle-down/shot-gun mass spectrometric or electrophoresis methods (Method 3). In contrast, the byproduct SAH can be quantified directly by anti-SAH antibody or MS (Methods 1,2) or indirectly by various colorimetric assays (Methods 4–7) with coupling enzymes (Pathways a–g): MTAN (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase), LuxS (S-ribosylhomocysteine lyase), SAH hydrolyase, adenosine kinase, adenine deaminase, xanthine oxidase, APRT (adenine phosphoribosyl transferase) or PPDK (pyruvate orthophosphate dikinase). SAM can spontaneously decompose into SAH, adenine and MTA (Pathways h, i, j).

Figure 4

Emerging SPA-based and antibody-based homogenous PMT-activity assays for HTS. The principles of SPA, AlphaScreen/LISA, LANCE Ultra and LanthaScreen are briefly described below. In the reported SPA-based PMT-activity assay, a biotinylated peptide substrate was radiolabeled by PMT with [Me-³H]-labeled-SAM and then immobilized to streptavidin-conjugated SPA beads. The proximity between the β-particles from the immobilized peptide and SPA-coated scintillation fluid led to light emission. For the reported AlphaScreen and AlphaLISA PMT-activity assays,^, the methylated, biotin-tagged peptide product caused the proximity between streptavidin-coated donor beads and anti-methyllysine antibody-conjugated acceptor beads (or primary antibody-immunized secondary-antibody-conjugated acceptor beads). Exciting the donor beads at 680 nm led to emitting singlet oxygen (¹O₂), which excited the acceptor beads to generate emission at 520–620 nm (AlphaScreen) or at 615 nm (AlphaLISA). For the reported LANCE Ultra and LanthaScreen PMT-activity assays,^, europium/terbium-labeled anti-methyllysine antibodies were used as FRET donors and streptavidin-conjugated Ulight (LANCE Ultra) or GFP-fused protein (LanthaScreen) as FRET acceptors. Exciting the donors at 320 nm or 340 nm led to FRET-mediated light emission of the acceptors.

Figure 5

Well-defined homogenous peptides and proteins as PMT substrates. PMT-substrate specificity can be affected by amino acid sequences that are adjacent to or remote to methylation sites. In a similar manner, PMT-substrate specificity can be affected by other posttranslational modifications or allosteric factors that are adjacent to or remote to methylation sites. The crosstalk can only be examined with well-defined peptide or protein substrates.

Figure 6

Emerging chemical biology approaches to prepare PMT protein substrates containing well-defined posttranslational modifications: (a) chemical conjugation, (b) nonsense-suppression mutagenesis (NSM) and (c) chemical ligation. These approaches alone or in combination have been applied to prepare histones containing mono/di/trimethyllysine, acetyllysine, ubiquitin or their mimics. A utilization of these approaches to prepare K120-ubiquitinated H2B is described as well.

Figure 7

Substrate candidates that can be used to identify novel PMT targets. The conventional approach relies on laborious screening of <100 individual peptides to identify novel PMT substrates. The peptide array approach allows several hundred distinct peptides to be examined in a single run. Split-pool peptide library allows several thousand candidates to be examined in a single run. The protein array approach maintains comparable capability of the split-pool library but present structurally-relevant proteins as substrate candidates.

Figure 8

Bioorthogonal profiling approach to label PMT substrates. Native PMTs use SAM as the cofactor to methylate their targets. In contrast, PMTs can be engineered to accommodate SAM derivatives as cofactors and label their substrates with distinct chemical groups. Since bulky SAM analogues are inert to native PMTs, the resultant distinctly-modified substrates can be assigned to a single, designated PMT.

Figure 9

SAM analogues as cofactor surrogates or chemical probes for PMTs. Clockwise: N⁶-benzyl SAM analogue as allele-specific cofactor and inhibitor of Rmt1; 2′,3′-dibenzyl SAM analogue as an allele-specific cofactor of vSET; sulfonium-alkyl SAM as cofactor surrogates and allele-specific chemical probes; 5′-N-iodoethyl/5′-aziridine SAM analogues as precursors of bisubstrate inhibitors of PRMT1 and DOT1L.

Figure 10

Representative inhibitors of PMTs. SAH and sinefungin are the best characterized pan-inhibitors of PMTs. Peptidic inhibitors were designed on the basis of the sequenence of PMT substrates with their Arg residue conjugated with some moiety of the SAM or replaced with a functional group. Allantodapsone is a potential PRMT1 inhibitor with IC₅₀ = 1300 nM. Compounds 1 and 2 are so far the most potent CARM1-selective inhibitors. EPZ004777 and UNC0639 are so far the most potent and best-characterized inhibitors of DOT1L and G9a/GLP, respectively. AZ505 is so far the most potent SMYD2 inhibitor.