HumanMETTL7BEncodes an Alkyl Thiol Methyltransferase that Methylates Hydrogen Sulfide

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.

Bioinformatic analysis of methyltransferase-like protein family reveals clinical and functional outcomes in human cancer

“…In lung cancer, overexpression of METTL7B signi cantly in uenced tumor growth in vivo and in vitro 49 . A recent study deposited in BioRxiv claims that METTL7B is an alkyl thiol methyltransferase that methylates hydrogen sul de residues and has potential to alter the redox state and growth cycle of cells 57 .…”