Methanethiol: A Scent Mark of Dysregulated Sulfur Metabolism in Cancer.

In order to cope with increased demands for energy and metabolites as well as to enhance stress resilience, tumor cells develop various metabolic adaptations, representing a hallmark of cancer. In this regard, the dysregulation of sulfur metabolism that may result in elevated levels of volatile sulfur compounds (VSCs) in body fluids, breath, and/or excretions of cancer patients has recently gained attention. Besides hydrogen sulfide (H2S), methanethiol is the predominant cancer-associated VSC and has been proposed as a promising biomarker for non-invasive cancer diagnosis. Gut bacteria are the major exogenous source of exposure to this foul-smelling toxic gas, with methanethiol-producing strains such as Fusobacterium nucleatum highly abundant in the gut microbiome of colorectal carcinoma (CRC) patients. Physiologically, methanethiol becomes rapidly degraded through the methanethiol oxidase (MTO) activity of selenium-binding protein 1 (SELENBP1). However, SELENBP1, which is considered a tumor suppressor, is often downregulated in tumor tissues, and this has been epidemiologically linked to poor clinical outcomes. In addition to impaired removal, an increase in methanethiol levels may derive from non-enzymatic reactions, such as a Maillard reaction between glucose and methionine, two metabolites enriched in cancer cells. High methionine concentrations in cancer cells may also result in enzymatic methanethiol production in mitochondria. Moreover, enzymatic endogenous methanethiol production may occur through methyltransferase-like protein 7B (METTL7B), which is present at elevated levels in some cancers, including CRC and hepatocellular carcinoma (HCC). In conclusion, methanethiol contributes to the scent of cancer as part of the cancer-associated signature combination of volatile organic compounds (VOCs) that are increasingly being exploited for non-invasive early cancer diagnosis.

Selenium-binding protein 1 (SELENBP1) is a copper-dependent thiol oxidase.

Selenium-binding protein 1 (SELENBP1) was reported to act as a methanethiol oxidase (MTO) in humans, catalyzing the conversion of methanethiol to hydrogen peroxide, hydrogen sulfide and formaldehyde. Here, we identify copper ions as essential to this novel MTO activity. Site-directed mutagenesis of putative copper-binding sites in human SELENBP1 produced as recombinant protein in E. coli resulted in loss of its enzymatic function. On the other hand, the eponymous binding of selenium (as selenite) was no requirement for MTO activity and only moderately increased SELENBP1-catalyzed oxidation of methanethiol. Furthermore, SEMO-1, the SELENBP1 ortholog recently identified in the nematode C. elegans, also requires copper ions, and MTO activity was enhanced or abrogated, respectively, if worms were grown in the presence of cupric chloride or of a Cu chelator. In addition to methanethiol, we identified novel substrates of SELENBP1 from the group of volatile sulfur compounds, ranging from ethanethiol to 1-pentanethiol as well as 2-propene-1-thiol. Gut microbiome-derived methanethiol as well as food-derived volatile sulfur compounds (VSCs) account for malodors that may contribute to extraoral halitosis in humans, if not metabolized properly. As SELENBP1 is particularly abundant in tissues exposed to VSCs, such as colon, liver, and lung, it appears to contribute to copper-dependent VSC degradation.

Altered Capacity for H2S Production during the Spontaneous Differentiation of Caco-2 Cells to Colonocytes Due to Reciprocal Regulation of CBS and SELENBP1.

Hydrogen sulfide (H2S) has been proposed to promote tumor growth. Elevated H2S levels have been detected in human colorectal cancer (CRC) biopsies, resulting from the selective upregulation of cystathionine ß-synthase (CBS). In contrast, the recently identified novel H2S-generating enzyme, selenium-binding protein 1 (SELENBP1), is largely suppressed in tumors. Here, we provide the first comparative analysis of the four human H2S-producing enzymes and the key H2S-catabolizing enzyme, sulfide:quinone oxidoreductase (SQOR), in Caco-2 human colorectal adenocarcinoma cells. The gene expression pattern of proliferating Caco-2 cells parallels that of CRC, while confluent cells undergo spontaneous differentiation to a colonocyte-like phenotype. SELENBP1 and SQOR were strongly upregulated during spontaneous differentiation, whereas CBS was downregulated. Cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase remained unaffected. Terminally differentiated cells showed an enhanced capacity to produce H2S from methanethiol and homocysteine. Differentiation induced by exposure to butyrate also resulted in the upregulation of SELENBP1, accompanied by increased SELENBP1 promoter activity. In contrast to spontaneous differentiation, however, butyrate did not cause downregulation of CBS. In summary, SELENBP1 and CBS are reciprocally regulated during the spontaneous differentiation of Caco-2 cells, thus paralleling their opposing regulation in CRC. Butyrate exposure, while imitating some aspects of spontaneous differentiation, does not elicit the same expression patterns of genes encoding H2S-modulating enzymes.

SEMO-1, a novel methanethiol oxidase in Caenorhabditis elegans, is a pro-aging factor conferring selective stress resistance.

Methanethiol is a toxic gas produced through bacterial degradation of sulfur-containing amino acids. Applying a novel enzymatic assay, we here identified a methanethiol oxidase (MTO) that catalyzes the degradation of methanethiol in the nematode Caenorhabditis elegans (C. elegans). The corresponding protein, Y37A1B.5, previously characterized as a C. elegans ortholog of human selenium-binding protein 1 (SELENBP1), was renamed SEMO-1 (SELENBP1 ortholog with methanethiol oxidase activity). Worms rendered deficient in SEMO-1 not only showed decreased hydrogen sulfide production from methanethiol catabolism but they were also more resistant to oxidative stress and had an elevated life span. In contrast, resistance to selenite was significantly lowered in SEMO-1-deficient worms. Naturally occurring mutations of human SELENBP1 were introduced to recombinant SEMO-1 through site-directed mutagenesis and resulted in loss of its MTO activity, indicating a similar enzymatic mechanism for SELENBP1 and SEMO-1. In summary, SEMO-1 confers resistance to toxic selenite and the ability to metabolize toxic methanethiol. These beneficial effects might be a trade-off for its negative impact on C. elegans life span.

A coupled enzyme assay for detection of selenium-binding protein 1 (SELENBP1) methanethiol oxidase (MTO) activity in mature enterocytes.

Methanethiol, a gas with the characteristic smell of rotten cabbage, is a product of microbial methionine degradation. In the human body, methanethiol originates primarily from bacteria residing in the lumen of the large intestine. Selenium-binding protein 1 (SELENBP1), a marker protein of mature enterocytes, has recently been identified as a methanethiol oxidase (MTO). It catalyzes the conversion of methanethiol to hydrogen sulfide (H2S), hydrogen peroxide (H2O2) and formaldehyde. Here, human Caco-2 intestinal epithelial cells were subjected to enterocyte-like differentiation, followed by analysis of SELENBP1 levels and MTO activity. To that end, we established a novel coupled assay to assess MTO activity mimicking the proximity of microbiome and intestinal epithelial cells in vivo. The assay is based on in situ-generation of methanethiol as catalyzed by a bacterial recombinant l-methionine gamma-lyase (MGL), followed by detection of H2S and H2O2. Applying this assay, we verified the loss and impairment of MTO function in SELENBP1 variants (His329Tyr; Gly225Trp) previously identified in individuals with familial extraoral halitosis. MTO activity was strongly enhanced in Caco-2 cells upon enterocyte differentiation, in parallel with increased SELENBP1 levels. This suggests that mature enterocytes located at the tip of colonic crypts are capable of eliminating microbiome-derived methanethiol.