Biosynthesis and Reactivity of Cysteine Persulfides in Signaling

Hydrogen sulfide (H₂S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H₂S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys–SSH) from cystine and H₂S from cysteine and/or homocysteine. Recently, Cys–SSH was proposed as the primary product of the transsulfuration pathway with H₂S representing a decomposition product of Cys–SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys–SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H₂S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H₂S rather than Cys–SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys–SSH product is short and decomposition leads to a mixture of polysulfides (Cys–S–(S)_<i>n</i>–S–Cys). These in vitro data, together with the intrinsic reactivity of Cys–SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions

Cytochrome <i>c</i> Reduction by H₂S Potentiates Sulfide Signaling

Hydrogen sulfide (H₂S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome <i>c</i> (Cyt C) by H₂S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H₂S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H₂S oxidized. H₂S increases O₂ consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H₂S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling

DataSheet_1_Redox integration of signaling and metabolism in a head and neck cancer model of radiation resistance using COSMRO.docx

Redox metabolism is increasingly investigated in cancer as driving regulator of tumor progression, response to therapies and long-term patients’ quality of life. Well-established cancer therapies, such as radiotherapy, either directly impact redox metabolism or have redox-dependent mechanisms of action defining their clinical efficacy. However, the ability to integrate redox information across signaling and metabolic networks to facilitate discovery and broader investigation of redox-regulated pathways in cancer remains a key unmet need limiting the advancement of new cancer therapies. To overcome this challenge, we developed a new constraint-based computational method (COSMro) and applied it to a Head and Neck Squamous Cell Cancer (HNSCC) model of radiation resistance. This novel integrative approach identified enhanced capacity for H2S production in radiation resistant cells and extracted a key relationship between intracellular redox state and cholesterol metabolism; experimental validation of this relationship highlights the importance of redox state in cellular metabolism and response to radiation.</p

DataSheet_2_Redox integration of signaling and metabolism in a head and neck cancer model of radiation resistance using COSMRO.xlsx

Redox metabolism is increasingly investigated in cancer as driving regulator of tumor progression, response to therapies and long-term patients’ quality of life. Well-established cancer therapies, such as radiotherapy, either directly impact redox metabolism or have redox-dependent mechanisms of action defining their clinical efficacy. However, the ability to integrate redox information across signaling and metabolic networks to facilitate discovery and broader investigation of redox-regulated pathways in cancer remains a key unmet need limiting the advancement of new cancer therapies. To overcome this challenge, we developed a new constraint-based computational method (COSMro) and applied it to a Head and Neck Squamous Cell Cancer (HNSCC) model of radiation resistance. This novel integrative approach identified enhanced capacity for H2S production in radiation resistant cells and extracted a key relationship between intracellular redox state and cholesterol metabolism; experimental validation of this relationship highlights the importance of redox state in cellular metabolism and response to radiation.</p