Methylation of alkyl thiols is a biotransformation pathway designed to reduce thiol reactivity and potential toxicity, yet the gene and protein responsible for human alkyl thiol methyltransferase (TMT) activity remain unknown. Here we demonstrate with a range of experimental approaches using cell lines, in vitro systems, and recombinantly expressed enzyme, that human methyltransferase-like protein 7B (METTL7B) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to hydrogen sulfide (H2S) and other exogenous thiol small molecules. METTL7B gene modulation experiments, including knockdown in HepG2 cells and overexpression in HeLa cells, directly alter the methylation of the drug captopril, a historic probe substrate for TMT activity. Furthermore, recombinantly expressed and purified wild-type METTL7B methylates several thiol compounds, including H2S, 7α-thiospironolactone, l-penicillamine, and captopril, in a time- and concentration-dependent manner. Typical for AdoMet-dependent small molecule methyltransferases, S-adenosyl-l-homocysteine (AdoHcy) inhibited METTL7B activity in a competitive fashion. Similarly, mutating a conserved aspartate residue, proposed to anchor AdoMet into the active site, to an alanine (D98A) abolished methylation activity. Endogenous thiols such as glutathione and cysteine, or classic substrates for other known small molecule S-, N-, and O-methyltransferases, were not substrates for METTL7B. Our results confirm, for the first time, that METTL7B, a gene implicated in multiple disease states including rheumatoid arthritis and breast cancer, encodes a protein that methylates small molecule alkyl thiols. Identifying the catalytic function of METTL7B will enable future pharmacological research in disease pathophysiology where altered METTL7B expression and, potentially H2S levels, can disrupt cell growth and redox state.

S-Methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine dependent membrane-associated phase II enzyme known as thiol methyltransferase (TMT). TMT has a broad substrate specificity and methylates the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine pro-drugs, clopidogrel, and prasugrel. Despite TMT's role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol-methyltransferase. METTL7B is an endoplasmic-reticulum-associated protein with similar biochemical properties and substrate specificity to TMT. Yet, the historic TMT inhibitor, 2,3-dichloro-a-methylbenzylamine (DCMB), has no effect on the activity of METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, also acts as a thiol-methyltransferase. METTL7A exhibits similar biochemical properties to TMT, including inhibition by DCMB (IC50 1.2 uM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7a-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which we have renamed TMT1A1 and TMT1B1, respectively, that are responsible for TMT activity in liver microsomes.

S-Methylation of drugs containing thiol-moieties often alters their activity and results in detoxification.Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative Sadenosyl-L-methionine (SAM) dependent membrane-associated enzyme referred to as thiol methyltransferase (TMT). This putative TMT appeared to have a broad substrate specificity and methylated the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine pro-drugs, clopidogrel, and prasugrel. Despite TMT's role in the Smethylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown.We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol-methyltransferase.METTL7B is an endoplasmic-reticulum-associated protein with similar biochemical properties and substrate specificity to the putative TMT. Yet, the historic TMT inhibitor, 2,3-dichloro-αmethylbenzylamine (DCMB), did not inhibit METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, is also a SAM dependent thiol-methyltransferase. METTL7A exhibits similar biochemical properties to METTL7B and putative TMT, including inhibition by DCMB (IC 50 1.17 µM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which are now renamed TMT1A and TMT1B, respectively, that are responsible for TMT activity in human liver microsomes.

No abstract

Paragraph/Abstract: 27Methyltransferase-like protein 7B (METTL7B) is implicated in tumor growth and 28 progression while gene expression is upregulated in several different disease states such as 29 72 including hydrogen sulfide, captopril, 7α-thiospironolactone, D-and L-penicillamine, and the 73 active metabolites of prasugrel, and ziprasidone (1, 2,(23)(24)(25)(26)(27). However, despite numerous 74 attempts, researchers have not successfully identified the TMT gene or protein (28-31). 75Our preliminary approach to identify TMT expanded on earlier research which attempted 76 to purify TMT from rat liver microsomes using a number of chromatographic steps (28, 31). 77After significant increases in TMT specific activity, preliminary non-targeted proteomic 78 experiments were conducted to identify potential methyltransferase proteins in the TMT-active 79 fractions. The major candidate protein in active fractions was identified as rat METTL7B which 80 was also localized to the endoplasmic reticulum (Extended Data Table 1). Rat and human 81 METTL7B share 83% sequence homology, which suggests a conserved function. Subsequent 82 experiments modulating the expression of human METTL7B in two cell lines also altered 83 captopril methylation, a known TMT substrate. Once identified, we cloned, recombinantly84 expressed, and purified human full length METTL7B in E. coli and conducted small molecule 85 substrate screening with the purified protein. The activity screens confirmed that METTL7B 86 specifically catalyzes SAM-dependent methylation of aliphatic thiol compounds, including 87 hydrogen sulfide, in a time and protein concentration dependent manner. No methylation was 88 observed with classic probe substrates of other known small molecule S-, N-, and O-89 methyltransferases(32-36) or endogenous thiols such as cysteine or glutathione.90 91 92 93 109 B) Methylation of captopril activity significantly decreased in HepG2 cells treated with METTL7B siRNA compared 110 to control cells. C) HeLa cells treated with a METTL7B overexpression plasmid showed ~ 1,000-fold increase in 111 METT7B gene expression compared to control cells transfected with an empty expression vector. D) HeLa cells 112transfected with the METTL7B expression vector showed a 10-fold increase in captopril methylation activity 113 compared to negative control cells. E) FLAG-tagged METTL7B expression is only observed in cells treated with 114 the METTL7B overexpression plasmid compared to controls (Lanes 2 and 1 respectively). ß-actin was used as a 115 loading control. All data is presented as the mean ± standard deviation. Individual data points from two (A, C, and 116 D) or three (B) experiments are plotted. Significance was determined using unpaired two-tailed t test.