Organization of the Human Mitochondrial Hydrogen Sulfide Oxidation Pathway

Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.     

DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.     

Polymorphic Variants of Human Rhodanese Exhibit Differences in Thermal Stability and Sulfur Transfer Kinetics

Rhodanese is a component of the mitochondrial H₂S oxidation pathway. Rhodanese catalyzes the transfer of sulfane sulfur from glutathione persulfide (GSSH) to sulfite generating thiosulfate and from thiosulfate to cyanide generating thiocyanate. Two polymorphic variations have been identified in the rhodanese coding sequence in the French Caucasian population. The first, 306A→C, has an allelic frequency of 1% and results in an E102D substitution in the encoded protein. The second polymorphism, 853C→G, has an allelic frequency of 5% and leads to a P285A substitution. In this study, we have examined differences in the stability between wild-type rhodanese and the E102D and P285A variants and in the kinetics of the sulfur transfer reactions. The Asp-102 and Ala-285 variants are more stable than wild-type rhodanese and exhibit k_cat/K_m,CN values that are 17- and 1.6-fold higher, respectively. All three rhodanese forms preferentially catalyze sulfur transfer from GSSH to sulfite, generating thiosulfate and glutathione. The k_cat/K_m,sulfite values for the variants in the sulfur transfer reaction from GSSH to sulfite were 1.6- (Asp-102) and 4-fold (Ala-285) lower than for wild-type rhodanese, whereas the k_cat/K_m,GSSH values were similar for all three enzymes. Thiosulfate-dependent H₂S production in murine liver lysate is low, consistent with a role for rhodanese in sulfide oxidation. Our studies show that polymorphic variations that are distant from the active site differentially modulate the sulfurtransferase activity of human rhodanese to cyanide versus sulfite and might be important in differences in susceptibility to diseases where rhodanese dysfunction has been implicated, e.g. inflammatory bowel diseases.     

Nutritional modulation of gene expression and homocysteine utilization by vitamin B12

Vitamins B12, B6, and folic acid converge at the homocysteine metabolic junction where they support the activities of two key enzymes involved in intracellular homocysteine management, methionine synthase (MS) and cystathionine beta-synthase. The molecular mechanism for the regulation of homocysteine metabolism by B12 supplementation has been investigated in this study. B12 supplementation does not alter mRNA or protein turnover rates but induces translational up-regulation of MS by shifting the mRNA from the ribonucleoprotein to the polysome pool. The B12-responsive element has been localized by deletion analysis using a reporter gene assay to a 70-bp region located at the 3' end of the 5'-untranslated region of the MS mRNA. The cellular consequence of the B12 response is a 2- and 3.5-fold increase in the flux of homocysteine through the MS-dependent transmethylation pathway in HepG2 and 293 cells, respectively. It is speculated that B12-induced up-regulation of MS may have evolved as an adaptive strategy for rapidly sequestering an essential and rare nutrient whose availability may have been limited in the evolutionary history of mammals, a problem that is exacerbated by the absence of this vitamin from the plant kingdom.     

Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.     

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO^•), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O₂ to Fe(III)-CBS, forming superoxide radical anion (O₂^˙̄). In this study, we describe the kinetics of nitrite (NO₂⁻) reduction by Fe(II)-CBS to form Fe(II)NO^•-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO^•-CBS by O₂ showed complex kinetic behavior and led to peroxynitrite (ONOO⁻) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO^• and peroxynitrite.     

Hemoglobin in Chironomus ramosus (Insecta, Diptera): an electrophoretic study of polymorphism, developmental sequence and interspecific relationship

The larvae of the dipteran insect, Chironomus ramosus, found in Shillong, India, contain eleven (11) hemoglobin (Hb) components of which three are monomers (CI, CIV and CVI) and seven are dimers (CIII, CV, CVII, CVIII, CIX, CX and CXI), while one (CII) exists in both monomeric and dimeric states.Four monomeric components were isolated, purified and partially characterized. The N-terminal amino acids were determined and showed glycine for CI and leucine for the other components (CII, CIV and CVI).Three hemoglobin components were found to be present in all stages of larval development, except the first instar larvae. Some Hb components were synthesized in a particular instar, as revealed by electrophoretic appearance.Electrophoretic mobilities of seven components and N-terminal amino acid residues of two components of Hb were similar in both Chironomus ramosus and Chironomus thummi thummi.