Yulia I. Deryabina

Mitochondrial Calcium Transport Systems: Properties, Regulation, and Taxonomic Features

Currently available information on properties and regulation of mitochondrial Ca2+ transporting systems in eukaryotic cells is summarized. We describe in detail kinetic properties and effects of inhibitors and modulators on the energy-dependent Ca2+ uptake through the Ca2+ uniporter, as well as on Na+-dependent and Na+-independent pathways for Ca2+ release in mammalian mitochondria. Special emphasis is placed on Ca2+ transport systems (for ion uptake and release) in mitochondria of higher plants, algae, and yeasts. Potential physiological implications of mitochondrial Ca2+ fluxes (influx and efflux), e.g., regulation of activity of Ca2+-dependent enzymes of the Krebs cycle, maintaining of cellular Ca2+ homeostasis, and engagement in pathophysiological processes, are discussed.

ω-Amidase: an underappreciated, but important enzyme in L-glutamine and L-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases

In mammals, two major routes exist for the metabolic conversion of l-glutamine to α-ketoglutarate. The most widely studied pathway involves the hydrolysis of l-glutamine to l-glutamate catalyzed by glutaminases, followed by the conversion of l-glutamate to α-ketoglutarate by the action of an l-glutamate-linked aminotransferase or via the glutamate dehydrogenase reaction. However, another major pathway exists in mammals for the conversion of l-glutamine to α-ketoglutarate (the glutaminase II pathway) in which l-glutamine is first transaminated to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to α-ketoglutarate and ammonia catalyzed by an amidase known as ω-amidase. In mammals, the glutaminase II pathway is present in both cytosolic and mitochondrial compartments and is most prominent in liver and kidney. Similarly, two routes exist for the conversion of l-asparagine to oxaloacetate. In the most extensively studied pathway, l-asparagine is hydrolyzed to l-aspartate by the action of asparaginase, followed by transamination of l-aspartate to oxaloacetate. However, another pathway also exists for the conversion of l-asparagine to oxaloacetate (the asparaginase II pathway). In this pathway, l-asparagine is first transaminated to α-ketosuccinamate (KSM), followed by hydrolysis of KSM to oxaloacetate by the action of ω-amidase. One advantage of both the glutaminase II and the asparaginase II pathways is that they are irreversible, and thus are important in anaplerosis by shuttling 5-C (α-ketoglutarate) and 4-C (oxaloacetate) units into the TCA cycle. In this review, we briefly mention the importance of the glutaminase II and asparaginase II pathways in microorganisms and plants. However, the major emphasis of the review is related to the importance of these pathways (especially the common enzyme component of both pathways—ω-amidase) in nitrogen and sulfur metabolism in mammals and as a source of anaplerotic carbon moieties in rapidly dividing cells. The review also discusses a potential dichotomous function of ω-amidase as having a role in tumor progression. Finally, the possible role of KGM as a biomarker for hyperammonemic diseases is discussed.

Calcium-dependent nonspecific permeability of the inner mitochondrial membrane is not induced in mitochondria of the yeast Endomyces magnusii

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and Pi, prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.

Neurospora crassa Light Signal Transduction Is Affected by ROS

In the ascomycete fungus Neurospora crassa blue-violet light controls the expression of genes responsible for differentiation of reproductive structures, synthesis of secondary metabolites, and the circadian oscillator activity. A major photoreceptor in Neurospora cells is WCC, a heterodimeric complex formed by the PAS-domain-containing polypeptides WC-1 and WC-2, the products of genes white collar-1 and white collar-2. The photosignal transduction is started by photochemical activity of an excited FAD molecule noncovalently bound by the LOV domain (a specialized variant of the PAS domain). The presence of zinc fingers (the GATA-recognizing sequences) in both WC-1 and WC-2 proteins suggests that they might function as transcription factors. However, a critical analysis of the phototransduction mechanism considers the existence of residual light responses upon absence of WCC or its homologs in fungi. The data presented point at endogenous ROS generated by a photon stimulus as an alternative input to pass on light signals to downstream targets.

Ca2+ Efflux in Mitochondria from the YeastEndomyces magnusii

Calcium release pathways in Ca2+-preloaded mitochondria from the yeastEndomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca2+ release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca2+ and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca2+ efflux set in after uptake of ∼150 nmol of Ca2+/mg of protein. The rate of this efflux was proportional to the Ca2+ load and insensitive to aeration, protonophorous uncouplers, and Na+ions. Ca2+ efflux was inhibited by La3+, Mn2+, Mg2+, tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside andt-butyl hydroperoxide were without effect. Ca2+efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca2+ efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na+-independent pathway, in many ways similar to the one in mammalian mitochondria.

Redox status of extremophilic yeast Yarrowia lipolytica during adaptation to pH-stress

In this study we investigated the activities of antioxidant enzymes (superoxide dismutases (SODs) and catalases (CATs)) and the ROS level in cells of Yarrowia lipolytica yeasts grown in a medium with different pH values (4.5, 5.5 and 9.0). It was shown that an increase in the cellular ROS level took place under both acid and alkaline conditions. The growth under extreme conditions was accompanied by a significant increase of SOD activity (by 2.5 times in the acid medium and by 4 times in the alkaline medium), but catalase activity did not change. A study of the electrophoretic profile of catalases showed the presence of three isoforms differing in inhibitor resistance. The electrophoretic profiles of SODs and their inhibitory analysis revealed there are two other isoforms, probably of mitochondrial origin, in addition to Cu and Zn SOD. The role of SOD in pH-adaptation of extremophilic Y lipolytica yeasts is discussed.

Humic Substances Enhance Growth and Respiration in the Basidiomycetes Trametes Maxima Under Carbon Limited Conditions

Humic substances (HS) represent the major reservoir of carbon (C) in ecosystems, and their turnover is crucial for understanding the global C cycle. Although basidiomycetes clearly have a role in HS degradation, much less is known about the effect of HS on fungal traits. We studied the alteration of physiological, biochemical, and morphological characteristics of Trametes maxima in the presence of HS. Both complete medium and minimal (C-limited) medium mimicking natural environmental conditions were used. Adding HS led to increased biomass yield, but under C-limited conditions the effect was more apparent. This result indicated that HS were used as an additional substrate and agreed with data showing a greater penetration of tritium-labeled HS into the cell interior under C-limited conditions. Humic substances induced ultra-structural changes in fungal cells, especially under C limitation, including reducing the thicknesses of the hyphal sheath and cell wall. In the minimal medium, cellular respiration increased nearly three-fold under HS application, while the corresponding effect in complete medium was lower. In addition, in the presence of inhibitors, HS stimulated either the cytochrome or the alternative pathway of respiration, depending on presence or absence of glucose in the medium. Our results suggest that, under conditions mimicking the natural environment, HS may play three major roles: as a surplus substrate for fungal growth, as a factor positively affecting cell morphology, and as an activator of physiological respiration.

Determination of Coenzyme A and Acetyl-Coenzyme A in Biological Samples Using HPLC with UV Detection

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) play essential roles in cell energy metabolism. Dysregulation of the biosynthesis and functioning of both compounds may contribute to various pathological conditions. We describe here a simple and sensitive HPLC-UV based method for simultaneous determination of CoA and acetyl-CoA in a variety of biological samples, including cells in culture, mouse cortex, and rat plasma, liver, kidney, and brain tissues. The limits of detection for CoA and acetyl-CoA are >10-fold lower than those obtained by previously described HPLC procedures, with coefficients of variation <1% for standard solutions, and 1–3% for deproteinized biological samples. Recovery is 95–97% for liver extracts spiked with Co-A and acetyl-CoA. Many factors may influence the tissue concentrations of CoA and acetyl-CoA (e.g., age, fed, or fasted state). Nevertheless, the values obtained by the present HPLC method for the concentration of CoA and acetyl-CoA in selected rodent tissues are in reasonable agreement with literature values. The concentrations of CoA and acetyl-CoA were found to be very low in rat plasma, but easily measurable by the present HPLC method. The method should be useful for studying cellular energy metabolism under normal and pathological conditions, and during targeted drug therapy treatment.

Identification of Proteins Involved in pH Adaptation in Extremophile Yeast Yarrowia lipolytica

Extremophile yeast Yarrowia is now commonly acknowledged as a prospective industrial microorganism and a highly promising cell model. This is due to several unique properties of this organism. First, it is able to grow rapidly on a broad range of organic substrates including waste water, oil paraffin and non-natural substances. This property, due to the presence of peroxisomes, allows the application of Y. lipolytica for waste management, water and soil bioremediation and for conversion of fossil organic compounds and pollutants to feed ingredients. Second, due to a high metabolic activity and resistance to chemical stresses, Y. lipolytica is able to produce aggressive organic compounds (e.g. succinate) at high yields. Third, Y. lipolytica provides a well-established model of a dimorphiс transition between a yeast-like state and a mycelium forming fungi. This property enables the application of Y. lipolytica as a model for drug discovery for therapeutic control of Candida albicans (the most common fungal pathogen in humans) and other pathogenic fungi. In contrast to C.albicans, Y.lipolytica is easily cultivated and has a complete sexual cycle. Thus, genetic mating analyses are readily applicable. Dimorphic transition in Y. lipolytica is also considered as the simplest model of cell differentiation in eukaryotes. Taken together, these factors provided the incentive for complete sequencing of the Y. lipolytica genome. This has been carried out by GenoLevures Consortium in France (Dujon et al, 2004). Availability of the complete genomic sequence opened access to proteomic assays to be combined with functional studies of Y. lipolytica. The proteomic approach has been successfully used by Morin et al (2007) for the identification of major proteins involved in the dimorphic transition of Y. lipolytica.

Expression system for Yarrowia lipolytica based on a promoter of the mitochondrial potential-dependent porin VDAC gene

In this study, we designed an expression system for the Y. lipolytica yeast, which can be fully efficient in media with non-standard industrial ingredients. Previously, we reported that the mitochondrial Voltage Dependent Anion Channel (VDAC) was a major protein overproduced in the Yarrowia lipolytica yeast in an alkaline (pH = 9.0) culture medium. In this study, the VDAC promoter was cloned and tested using a reporter system based on the LacZ gene. Naturally, the VDAC gene contains an intron located just within the ATG translation initiation codon. The VDAC promoter V2 variant with the intron and V3 variant without the intron were studied. The VDAC-driven clones exhibited high variability of the expression profile. Some clones were more active than the clones induced by the artificial hp4d promoter, when grown in both complete and low-cost industrial ingredient (10% fish and sunflower meal) containing media. The new expression system may greatly expand the range of recombinant producers of feed enzymes and some other types of fodder additives in the Y. lipolytica yeast, appropriate for assimilation of low-cost and non-standard raw material.