Anton Horváth | Comenius University (original) (raw)

Papers by Anton Horváth

Journal of Biological Chemistry, May 1, 1993

PubMed, Feb 1, 1994

We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphat... more We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas1p. The need for ceramide can be overcome in the mutant strain.

The EMBO Journal, Nov 1, 1997

Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the... more Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the cell surface that is used among all eukaryotes. A common core structure, EthN-P-Man 3-GlcN-PI, is synthesized by sequential transfer of sugars and ethanolamine-P to PI and is highly conserved between organisms. We have screened for natural compounds that inhibit GPIanchoring in yeast and have identified a terpenoid lactone, YW3548, that specifically blocks the addition of the third mannose to the intermediate structure Man 2-GlcN-acylPI. Consistent with the block in GPI synthesis, YW3548 prevents the incorporation of [ 3 H]myo-inositol into proteins, transport of GPIanchored proteins to the Golgi and is toxic. The compound inhibits the same step of GPI synthesis in mammalian cells, but has no significant activity in protozoa. These results suggest that despite the conserved core structure, the GPI biosynthetic machinery may be different enough between mammalian and protozoa to represent a target for anti-protozoan chemotherapy.

Biochimica et Biophysica Acta (BBA) - General Subjects

Journal of Biological Chemistry, 2021

Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total... more Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.

The EMBO Journal, 1995

The protein encoded by the yeast gene SPT14 shows high sequence similarity to the human protein, ... more The protein encoded by the yeast gene SPT14 shows high sequence similarity to the human protein, PIG-A, whose loss of activity is at the origin of the disease paroxysmal nocturnal hemoglobinuria. The symptoms of this disease are apparently due to a loss of cell surface, glycosylphosphatidylinositol (GPI)-anchored proteins. Like PIG-A mutant cells, sptl4 mutant cells are defective in GPI anchoring due to a defect in the synthesis of GIcNAc-PI, the first step of GPI synthesis. The sptl4 mutant causes several other abnormalities including transcriptional defects and a downregulation of inositolphosphoceramide synthesis. We suggest that these defects are indirect results of the loss of GPI anchoring.

Folia Microbiologica, 2002

The occurrence of GC clusters in Saccharomyces spp. and related yeasts was examined to clarify th... more The occurrence of GC clusters in Saccharomyces spp. and related yeasts was examined to clarify their association with the stability of intact mitochondrial genome. Abundance ofnonspecific or specific GC clusters in these species decreases with phylogenetic distance from S. cerevisiae. Their number but not the number of replication origins correlates with the ability to form respiration-deficient mutants induced by ethidium bromide. This effect is not associated with the nuclear background since the cybrids having identical nuclei and mitochondria from different species gave similar results. In contrast to grand genomes, the presence of GC clusters in 9-mutants does not play any role in ethidium bromide induced mtDNA loss. The most plausible explanation for mitotically lost petite mtI)NA seems to be dilution during the distribution. Mitochondrial DNA (mtDNA) from S. cerevisiae varies in size from about 74 to 85 kbp (for review see Foury et al. 1998; Dujon 1981; de Zamaroczy et al. 1985). Because structural genes occupy less then 20 % of the genome, the major part consists of introns and intergenic spacers, lntergenic spacers are composed almost exclusively of AT regions that are interrupted at irregular intervals by short stretches (30-60 bp) of GC-rich sequences. GC clusters are the main source of polymorphism and a large portion of them contain restriction sites for enzymes Hpall or Haelll. They fall into 8 separate classes with up to 30 clusters of any class scattered throughout the genome. They are optional in the coding regions of both ribosomal subunits, gene for RNA subunit of RNAase P, VAR1 and some intronic ORFs without evident impact on the expression (de Zamaroczy and Bernardi 1986; Weiller et al. 1989). A specific class of GC clusters is the about 300 bp long ori/rep sequence, with an essential role in replication of wild-type mtDNA (de Zamaroczy et al. 1984). The ori sequences are characterized by three conserved GC blocks A, t3, C, separated by AT-rich stretches. Like the process of intron homing, GC clusters are preferentially inserted in genetic crosses into recipient alleles that lack them (Butow et al. 1985). Double-strand breaks were detected at or near the boundaries of GC clusters but not in the target sequence of recipient DNA (Zinn et al. 1988). Also they, as a group, are hot spots of DNA recombination and sources of instability (Weiller et al. 1991). Similarly preferential recombination between GC clusters can occur, leading to the integration of petite genome into wild-type mtDNA (Dieckmann and Gandy 1987). The dispensable nature ofS. cerevisiae mitochondrial genome allowed an extensive genetic analysis of mtDNA. The wild-type circular mitochondrial genomes give rise, by extensive deletion, to respiration-deficient mutants. Deletion mutants in mtDNA called petite and also p-arise at high frequency as a result of action of ethidium bromide. The amount of mtDNA in cells is the same as in wild-type cells, since the resulting molecules contain tandem repeats of amplified DNA segments (for review see Dujon 1981; Pi~kur 1994). The molecular mechanism underlying the irreversible destruction of mitochondrial genomes by ethidium bromide remains unclear. This intercalating agent inhibits de novo synthesis of mtDNA and stimulates a rapid destruction of pre-existing mtDNA molecules resulting in a 100 % conversion to respiration-deficient one (Maleszka 1994; Goldring et al. 1970). The ends of the amplified units of p-mtDNA frequently coincide with GC clusters (Dujon 1981; de Zamaroczy and Bernardi 1986). Thus, illegitimate recombination between direct repeats is regarded as a preferential event generating petites. For that reason the molecular mechanism was hypothesized as a stabilization of fragile secondary structures.

Folia Microbiologica, 2000

Mitochondrial genomes o1" Saccharomyces and close relatives previously used lot transplacement of... more Mitochondrial genomes o1" Saccharomyces and close relatives previously used lot transplacement of mitochondria to S. cerevisiae were examined Tire origins of replication m mitochondrial DNA, the presence of nuclear and mitochoudrial polymorphic loci and the ability to produce mitochondrial respiration-deficient mutants were used to reclassil~ some collection yeasts and to assign others into four separate subgroups. The first included isolates identical to Saccharomyces cerevisiae (S. italicus, S. ov!formls, S. chevalieri and S capensis) which possess 5 or more replication origins The second group consists ofX paradoxus (wlr douglasit) mitochoudrial genome with the equal number of ori sequences but incompatible mitochondria. The third group represents Saccharomyces sensu stricto petite-positive species (S. carlsbergensis, S. heterogenicus. S. uvarum, S. willianus) with 1-2 origins of replication significantly different from S. cerevisiae. In addition, the locus between tRNA tMet and tRNA Pr~ is about one-half of the 1400 bp nrembers of S. cerevisiae complex. The last group includes isolates that do not belong to Saccharomyces sensu stricto group as they are petite-negative and devoid of any S cerevtszae-like replication origins.

Current Genetics, 2000

We elaborated a simple method that allows the transfer of mitochondria from collection yeasts to ... more We elaborated a simple method that allows the transfer of mitochondria from collection yeasts to Saccharomyces cerevisiae. Protoplasts prepared from dierent yeasts were fused to the protoplasts of the ade2-1, ura3-52, kar1-1, q 0 strain of S. cerevisiae and were selected for respiring cybrids on plates containing 5uoroorotic acid and a non-fermentable carbon source. The identity of putative cybrids was assessed by restriction analysis of mitochondrial DNA, pulse ®eld electrophoresis and tetrad analysis. In the comprehensive screening, only mitochondrial genomes from synonymous species (S. italicus, S. oviformis, S. capensis and S. chevalieri) exhibited complete compatibility with S. cerevisiae nuclei. The closely related S. douglasii mitochondrial genome could also partially restore respiration-de®ciency in q 0 S. cerevisiae, whereas mitochondrial genomes from phylogenetically less related species could not.

BMC Biology, 2021

Background The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, e... more Background The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. Results We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gl...

PLOS Neglected Tropical Diseases

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genu... more Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite’s replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanoso...

Parasitology, 2021

Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the ele... more Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the electron transfer from NADH to ubiquinone that is associated with proton pumping out of the matrix. In this study, we characterized NADH dehydrogenase activity in seven monoxenous trypanosomatid species: Blechomonas ayalai, Herpetomonas tarakana, Kentomonas sorsogonicus, Leptomonas seymouri, Novymonas esmeraldas, Sergeia podlipaevi and Wallacemonas raviniae. We also investigated the subunit composition of the complex I in dixenous Phytomonas serpens, in which its presence and activity have been previously documented. In addition to P. serpens, the complex I is functionally active in N. esmeraldas and S. podlipaevi. We also identified 24–32 subunits of the complex I in individual species by using mass spectrometry. Among them, for the first time, we recognized several proteins of the mitochondrial DNA origin.

Journal of Biological Chemistry, 2000

With the aim of identification of kinetoplast-encoded proteins we investigated the subunit compos... more With the aim of identification of kinetoplast-encoded proteins we investigated the subunit composition of cytochrome c oxidase (respiratory complex IV) from kinetoplast mitochondria of the trypanosomatid protozoan Leishmania tarentolae. Eleven stoichiometric subunits were visible in Coomassie-stained, two-dimensional Blue Native/Tricine-SDS electrophoretic gels. Their partial amino acid sequences indicated that these polypeptides are nuclear-encoded. The mitochondrial subunit I was detected with the polyclonal antibodies against an internal region of this polypeptide. In two-dimensional (9 versus 14%) polyacrylamide glycine-SDS gels this subunit is found as a series of spots located off the main diagonal, a property that can be explained by abnormal electrophoretic migration and aggregation. In gels loaded with high amounts of the purified, enzymatically active oxidase, the subunit I spots could be visualized by staining. The determined N-terminal amino acid sequence of the putative monomeric subunit I (MFXLCLV-CLSVS) matched with the predicted sequence, thus indicating that the corresponding kinetoplast unedited mRNA is translated into a functional protein.

Journal of Biological Chemistry, 2006

Molecular and Biochemical Parasitology, 2012

The Trypanosoma brucei cytochrome c oxidase (respiratory complex IV) is a very divergent complex ... more The Trypanosoma brucei cytochrome c oxidase (respiratory complex IV) is a very divergent complex containing a surprisingly high number of trypanosomatid-specific subunits with unknown function. To gain insight into the functional organization of this large protein complex, the expression of three novel subunits (TbCOX VII, TbCOX X and TbCOX 6080) were down-regulated by RNA interference. We demonstrate that all three subunits are important for the proper function of complex IV and the growth of the procyclic stage of T. brucei. These phenotypes were manifested by the structural instability of the complex when these indispensible subunits were repressed. Furthermore, the impairment of cytochrome c oxidase resulted in other severe mitochondrial phenotypes, such as a decreased mitochondrial membrane potential, reduced ATP production via oxidative phoshorylation and redirection of oxygen consumption to the trypanosome-specific alternative oxidase, TAO. Interestingly, the inspected subunits revealed some disparate phenotypes, particularly regarding the activity of cytochrome c reductase (respiratory complex III). While the activity of complex III was down-regulated in RNAi induced cells for TbCOX X and TbCOX 6080, the TbCOX VII silenced cell line actually exhibited higher levels of complex III activity and elevated levels of ROS formation. This result suggests that the examined subunits may have different functional roles within complex IV of T. brucei, perhaps involving the ability to communicate between sequential enzymes in the respiratory chain. In summary, by characterizing the function of three hypothetical components of complex IV, we are able to assign these proteins as genuine and indispensable subunits of the procyclic T. brucei cytochrome c oxidase, an essential component of the respiratory chain in these evolutionary ancestral and medically important parasites.

Yeast, 1994

We have developed and evaluated an easy and rapid method for extraction of proteins from yeast ce... more We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)-gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2-mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.

Experimental Parasitology, 2000

Molecular and Biochemical Parasitology, 2014

Trypanosomatids are unicellular parasites living in a wide range of host environments, which to l... more Trypanosomatids are unicellular parasites living in a wide range of host environments, which to large extent shaped their mitochondrial energy metabolism, resulting in quite large differences even among closely related flagellates. In a comparative manner, we analyzed the activities and composition of mitochondrial respiratory complexes in four species (Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and Trypanosoma brucei), which represent the main model trypanosomatids. Moreover, we measured the activity of mitochondrial glycerol-3-phosphate dehydrogenase, the overall oxygen consumption and the mitochondrial membrane potential in each species. The comparative analysis suggests an inverse relationship between the activities of respiratory complexes I and II, as well as the overall activity of the canonical complexes and glycerol-3-phosphate dehydrogenase. Our comparative analysis shows that mitochondrial functions are highly variable in these versatile parasites.