hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Kinetic and anion inhibition studies of a β-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis

Several β-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C₄ plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO₂ hydrase catalytic activity (k_cat of 1.2 × 10⁵ and k_cat/K_m of 7.5 × 10⁶ M^?1 × s^?1) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (K_Is in the range of 4–60 μM). Such inhibitors may be used as tools to better understand the role of various β-CA isoforms in photosynthesis.     

The extremo-α-carbonic anhydrase (CA) from Sulfurihydrogenibium azorense,the fastest CA known,is highly activated by amino acids and amines

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the extremophilic bacterium Sulfurihydrogenibium azorense (SazCA) was recently shown to be the fastest CA known. Here we investigated this enzyme for its activation with a series of amino acids and amines. The best SazCA activators were d-Phe, l-DOPA, l- and d-Trp, dopamine and serotonin, which showed activation constants in the range of 3–23 nM. l- and d-His, l-Phe, l-Tyr, 2-pyridyl-methylamine and L-adrenaline were also effective activators (K_As in the range of 62–90 nM), whereas d-Dopa, d-Tyr and several heterocyclic amines showed activity in the micromolar range. The good thermal stability, robustness, very high catalytic activity and propensity to be activated by simple amino acids and amines, make SazCA a very interesting candidate for biomimetic CO₂ capture processes.     

Carbonic anhydrase inhibitors. Benzenesulfonamides incorporating cyanoacrylamide moieties strongly inhibit Saccharomyces cerevisiae β-carbonic anhydrase

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogs) were assayed as inhibitors of the β-carbonic anhydrase (CA, EC 4.2.1.1) from Saccharomyces cerevisiae, ScCA. Some of these compounds were low nanomolar or subnanomolar ScCA inhibitors and showed selectivity ratios in the range of 4.91–69.86 for inhibiting the yeast enzyme over the offtarget human (h) isoforms hCA I and of 6.46–13.52 for inhibiting ScCA over hCA II. The model organism S. cerevisiae and this particular enzyme may be useful for detecting antifungals with a novel mechanism of action compared to the classical azole drugs to which significant drug resistance emerged. Indeed, some of these sulfonamides inhibited the growth of the yeast with CC₅₀-s in the range of 0.73–6.54 μM.     

A highly catalytically active γ-carbonic anhydrase from the pathogenic anaerobe Porphyromonas gingivalis and its inhibition profile with anions and small molecules

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the γ-class are present in archaea, bacteria and plants but, except the Methanosarcina thermophila enzymes CAM and CAMH, they were poorly characterized so far. Here we report a new such enzyme (PgiCA), the γ-CA from the oral cavity pathogenic bacterium Porphyromonas gingivalis, the main causative agent of periodontitis. PgiCA showed a good catalytic activity for the CO₂ hydration reaction, comparable to that of the human (h) isoform hCA I. Inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate were effective PgiCA inhibitors with inhibition constants in the range of 41–97 μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, and phenylboronic acid, with K_Is of 4.0–9.8 μM. The role of this enzyme as a possible virulence factor of P. gingivalis is poorly understood at the moment but its good catalytic activity and the possibility to be inhibited by a large number of compounds may lead to interesting developments in the field.     

Distinct cutaneous bacterial assemblages in a sampling of South American Amerindians and US residents

The human skin harbors complex bacterial communities. Prior studies showing high inter-individual variation focused on subjects from developed countries. We therefore compared cutaneous bacterial communities of Amerindians in the Venezuelan Amazon with subjects in the United States. Forearm skin specimens were studied from healthy Amerindians in Platanillal village in Amazonas State, and from healthy persons in New York and Colorado. All skin sampling used similar swab/buffer techniques. Multiplexed V2-targeted 16S rRNA gene pyrosequencing yielded high quality sequences from 112 samples. The results show 20 phyla, with three (Proteobacteria, Firmicutes, Actinobacteria) predominating. US residents and Venezuelan Amerindians had significantly different forearm skin bacterial community compositions, with United States dominated by Propionibacterium. Among the Amerindians, there was a deep split based on bacterial community membership, with 30 and 42 samples, respectively, falling into each of the two groups, not associated with age, gender, or body mass index. One Amerindian group had diversity similar to the United States, but was dominated by Staphylococcus rather than Propionibacterium. The other Amerindian group was significantly more diverse and even than the US or the other Amerindian group, and featured a broad range of Proteobacteria. The results provide evidence that ethnicity, lifestyle and/or geography are associated with the structure of human cutaneous bacterial communities.     

Optimization-driven identification of genetic perturbations accelerates the convergence of model parameters in ensemble modeling of metabolic networks

The ensemble modeling (EM) approach has shown promise in capturing kinetic and regulatory effects in the modeling of metabolic networks. Efficacy of the EM procedure relies on the identification of model parameterizations that adequately describe all observed metabolic phenotypes upon perturbation. In this study, we propose an optimization-based algorithm for the systematic identification of genetic/enzyme perturbations to maximally reduce the number of models retained in the ensemble after each round of model screening. The key premise here is to design perturbations that will maximally scatter the predicted steady-state fluxes over the ensemble parameterizations. We demonstrate the applicability of this procedure for an Escherichia coli metabolic model of central metabolism by successively identifying single, double, and triple enzyme perturbations that cause the maximum degree of flux separation between models in the ensemble. Results revealed that optimal perturbations are not always located close to reaction(s) whose fluxes are measured, especially when multiple perturbations are considered. In addition, there appears to be a maximum number of simultaneous perturbations beyond which no appreciable increase in the divergence of flux predictions is achieved. Overall, this study provides a systematic way of optimally designing genetic perturbations for populating the ensemble of models with relevant model parameterizations.     

Bovine Lactoferrin Decreases Cholera-Toxin-Induced Intestinal Fluid Accumulation in Mice by Ganglioside Interaction

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.