José S. Rodríguez-Zavala

Inhibition of the mitochondrial calcium uniporter by the oxo-bridged dinuclear ruthenium amine complex (Ru360) prevents from irreversible injury in postischemic rat heart.

Mitochondrial calcium overload has been implicated in the irreversible damage of reperfused heart. Accordingly, we studied the effect of an oxygen‐bridged dinuclear ruthenium amine complex (Ru360), which is a selective and potent mitochondrial calcium uniporter blocker, on mitochondrial dysfunction and on the matrix free‐calcium concentration in mitochondria isolated from reperfused rat hearts. The perfusion of Ru360 maintained oxidative phosphorylation and prevented opening of the mitochondrial permeability transition pore in mitochondria isolated from reperfused hearts. We found that Ru360 perfusion only partially inhibited the mitochondrial calcium uniporter, maintaining the mitochondrial matrix free‐calcium concentration at basal levels, despite high concentrations of cytosolic calcium. Additionally, we observed that perfused Ru360 neither inhibited Ca2+ cycling in the sarcoplasmic reticulum nor blocked ryanodine receptors, implying that the inhibition of ryanodine receptors cannot explain the protective effect of Ru360 in isolated hearts. We conclude that the maintenance of postischemic myocardial function correlates with an incomplete inhibition of the mitochondrial calcium uniporter. Thus, the chemical inhibition by this molecule could be an approach used to prevent heart injury during reperfusion.

Differences in susceptibility to inactivation of human aldehyde dehydrogenases by lipid peroxidation byproducts.

Aldehyde dehydrogenases (ALDHs) are involved in the detoxification of aldehydes generated as byproducts of lipid peroxidation. In this work, it was determined that, among the three most studied human ALDH isoforms, ALDH2 showed the highest catalytic efficiency for oxidation of acrolein, 4-hydroxy-2-nonenal (4-HNE), and malondialdehyde. ALDH1A1 also exhibited significant activity with these substrates, whereas ALDH3A1 only showed activity with 4-HNE. ALDH2 was also the most sensitive isoform to irreversible inactivation by these compounds. Remarkably, ALDH3A1 was insensitive to these aldehydes even at concentrations as high as 20 mM. Formation of adducts of ALDH1A1 and ALDH2 with acrolein increased their K(d) values for NAD(+) by 2- and 3-fold, respectively. NADH exerted a higher protection than propionaldehyde to the inactivation by acrolein, and this protection was additive. These results suggested that both binding sites, those for aldehyde and NAD(+) in ALDH2, are targets for the inactivation by lipid peroxidation products. Thus, with the advantage of being relatively inactivation-insensitive, ALDH1A1 and ALDH3A1 may be actively participating in the detoxification of these aldehydes in the cells.

A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related α-proteobacteria

The F1FO and F1‐ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome‐reconstituted holoenzyme preserves its tight coupling between F1 and FO sectors, as evidenced by its high sensitivity to the FO inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N‐terminal sequencing of the band profile in SDS‐PAGE of the F1 and F1FO preparations showed a novel 11‐kDa protein in addition to the 5 canonical α, β, γ, δ, and ε subunits present in all known F1‐ATPase complexes. BN‐PAGE followed by 2D‐SDS‐PAGE confirmed the presence of this 11‐kDa protein bound to the native F1FO‐ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and ε subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F1FO and soluble F1‐ATPase complexes. The 11‐kDa protein, but not the ε subunit, inhibited the F1FO and F1‐ATPase activities of P. denitrificans. The 11‐kDa protein was also found in Rhodobacter sphaeroides associated to its native F1FO‐ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11‐kDa protein on the F1FO‐ATPase nanomotor of P. denitrificans and closely related α‐proteobacteria.—Morales‐Ríos, E., de la Rosa‐Morales, F., Mendoza‐Hernández, G., Rodríguez‐Zavala, J. S., Celis, H., Zarco‐Zavala, M., García‐Trejo, J. J. A novel 11‐kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related α‐proteobacteria. FASEB J. 24, 599–608 (2010). www.fasebj.org

Phytochelatin–cadmium–sulfide high‐molecular‐mass complexes of Euglena gracilis

High‐molecular‐mass PC complexes (PC‐HMWCs) constituted by phytochelatins (PCs), cadmium and sulfide are synthesized by several organisms after exposure to cadmium. In this study, PC‐HMWCs were isolated from photoheterotrophic Euglena gracilis and purified to homogeneity, resulting in compounds of molecular mass 50–380 kDa depending on the CdCl2 and sulfate concentrations in the culture medium. In contrast with plants and some yeasts, PC‐HMWCs from E. gracilis mainly comprise (57–75%) monothiol molecules (Cys, γ‐glutamylcysteine, GSH) and, to a lesser extent (25–43%), PCs. A similar acid‐soluble thiol compound composition was found in whole cell extracts. The –SH/Cd2+and S2–/Cd2+ ratios found in purified PC‐HMWCs were 1.5 and 1.8, respectively; the (–SH + S2–)/Cd2+ ratio was 3.2. PC‐HMWCs of molecular mass 60 and 100 kDa were also localized inside Percoll‐purified chloroplasts, in which cadmium and PCs were mainly compartmentalized. Cadmium and sulfur‐rich clusters with similar sulfur/cadmium stoichiometries to those of the purified PC‐HMWCs were detected in the chloroplast and throughout the cell by energy dispersive microanalysis and atomic resolution electron microscopy. The presence of PC‐HMWCs in primitive photosynthetic eukaryotes such as the protist, E. gracilis, suggests that their function as the final cadmium‐storage‐inactivation process is widespread. Their particular intracellular localization suggests that chloroplasts may play a major role in the cadmium‐resistance mechanism in organisms lacking a plant‐like vacuole.

Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde–alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica

The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O2 exposure (IC50 0.034 mm), whereas short‐term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe2+. CoA and acetyl‐CoA prevented, whereas pyruvate exacerbated, inactivation induced by short‐term saturating O2 exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe2+‐reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe–S clusters. After O2 exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde–alcohol dehydrogenase (EhADH2), whereas acetyl‐CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress.

Molecular mechanisms of resistance to heavy metals in the protist Euglena gracilis.

The biochemical mechanisms of resistance to several heavy metals, which are associated with their accumulation (binding by high-affinity chelating molecules such as thiol-compounds together with their compartmentalization into organelles), are analyzed for the photosynthetic, free-living protist Euglena gracilis. The complete understanding of these mechanisms may facilitate the rational design of strategies for bioremediation of heavy metal polluted water and soil systems.

Characterization of an Aldehyde Dehydrogenase from Euglena gracilis

ABSTRACT. The free‐living protist Euglena gracilis showed an enhanced growth when cultured in the dark with high concentrations of ethanol as carbon source. In a medium containing glutamate/malate plus 1% ethanol, E. gracilis reached a density of 3 × 107 cells/ml after 100 h of culture, which was 5 times higher than that attained with glutamate/malate or ethanol separately. This observation suggested the involvement of a highly active aldehyde dehydrogenase in the metabolism of ethanol. Purification of the E. gracilis aldehyde dehydrogenase from the mitochondrial fraction by affinity chromatography yielded an enrichment of 34 times and recovery of 33% of the total mitochondrial activity. SDS‐PAGE and molecular exclusion chromatography revealed a native tetrameric protein of 160 kDa. Kinetic analysis showed Km values of 5 and 50 μM for propionaldehyde and NAD+, respectively, and a Vm value of 1,300 nmol (min × mg protein)−1. NAD+ and NADH stimulated the esterase activity of the purified aldehyde dehydrogenase. The present data indicated that the E. gracilis aldehyde dehydrogenase has kinetic and structural properties similar to those of human aldehyde dehydrogenases class 1 and 2.

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4-3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7-13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6-6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.

Malfunctioning of the Iron-Sulfur Cluster Assembly Machinery in Saccharomyces cerevisiae Produces Oxidative Stress via an Iron-Dependent Mechanism, Causing Dysfunction in Respiratory Complexes

Biogenesis and recycling of iron–sulfur (Fe–S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe–S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe–S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe–S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

A CRAC-like motif in BAX sequence: relationship with protein insertion and pore activity in liposomes.

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.