Regulation and Functional Role of H₂S-Related Enzymes MPST and TST In Vitro

Abstract

Hydrogen sulphide (H₂S) is a signalling molecule involved in cellular metabolism, redox homeostasis, and stress responses. It is produced enzymatically by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MPST), while its mitochondrial catabolism is regulated by sulfide:quinone oxidoreductase (SQOR), persulfide dioxygenase (ETHE1), and thiosulfate sulfurtransferase (TST). Although MPST and TST contribute to maintaining H₂S homeostasis, their precise regulatory functions in hepatic metabolism and stress adaptation remain insufficiently characterised. This study hypothesised that MPST and TST play key regulatory roles in liver sulphur metabolism and cellular adaptation to oxidative and metabolic stress. The research aimed to investigate their gene regulation, expression patterns, and functional impact in liver-derived models, with a focus on detoxification, H₂S generation, and cellular stress responses. An integrated approach was taken, combining bioinformatics and experimental methods. Promoter and enhancer analyses were used to identify transcriptional regulatory elements and transcription factor binding sites. The effects of metabolic stressors (including free fatty acids, altered glucose concentrations, and xenobiotic receptor agonists (PPARα, PXR, AhR, CAR)) on MPST and TST expression were evaluated in Huh7, HepG2, and McA-RH7777 cells. Stable MPST- and TST overexpressing Huh7 cell lines were generated via plasmid-based transfection and G418 selection to assess their roles in cell viability, proliferation, and stress responses. Protein expression and subcellular localisation were confirmed by Western blot and fluorescence microscopy. Enzyme activity and H₂S production were quantified using fluorescent probes (AzMC, SF7-AM, SSP4), while pharmacological inhibition was I used to probe functional relevance. Cell viability and proliferation assays were conducted under various stress and chemotherapy conditions. The results showed that MPST and TST are highly expressed in hepatic tissue. The expression of both genes is downregulated in response to xenobiotic receptor activation in Huh7 cells. Functional assays revealed that MPST activity was more consistently reflected by sulfane sulfur levels than by H₂S production in Huh7 cells overexpressing MPST. Overexpression of MPST and TST did not significantly alter proliferation in Huh7 cells or confer resistance to doxorubicin-induced cytotoxicity, suggesting limited involvement in chemoresistance under the tested conditions. In conclusion, this work provides new insight into the molecular regulation and functional significance of MPST and TST in liver sulphur metabolism. These findings suggest the potential influence of dietary factors on enzyme gene expression and highlight sulfane sulphur as a potentially reliable biomarker of MPST activity. Further studies are needed to explore their broader involvement in redox regulation, apoptosis, and liver disease progression.