RESUMO
It has been previously demonstrated that cytoprotective activity displayed by minocycline in the case of the yeast Saccharomyces cerevisiae cells pretreated with H2O2 requires the presence of functional VDAC (YVDAC1). Thus, we decided to transform YVDAC1-depleted yeast cells (Δpor1 cells) with plasmids expressing human VDAC isoforms (HVDAC1, HVDAC2, HVDAC3) to estimate their involvement in the minocycline cytoprotective effect. We observed that only expression of HVDAC3 in Δpor1 cells provided minocycline-mediated cytoprotection against H2O2 although all human isoforms are functional in Δpor1 cells. The observation appears to be important for on-going discussion concerning VDAC isoform roles in mitochondria and cell functioning.
Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Citoproteção , Minociclina/metabolismo , Minociclina/farmacologia , Isoformas de Proteínas/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Ligação Proteica , Isoformas de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Canais de Ânion Dependentes de Voltagem/genéticaRESUMO
BACKGROUND: An ancestral trait of eukaryotic cells is the presence of mitochondria as an essential element for function and survival. Proper functioning of mitochondria depends on the import of nearly all proteins that is performed by complexes located in both mitochondrial membranes. The complexes have been proposed to contain subunits formed by proteins common to all eukaryotes and additional subunits regarded as lineage specific. Since Amoebozoa is poorly sampled for the complexes we investigated the outer membrane complexes, namely TOM, TOB/SAM and ERMES complexes, using available genome and transcriptome sequences, including transcriptomes assembled by us. RESULTS: The results indicate differences in the organization of the Amoebozoa TOM, TOB/SAM and ERMES complexes, with the TOM complex appearing to be the most diverse. This is reflected by differences in the number of involved subunits and in similarities to the cognate proteins of representatives from different supergroups of eukaryotes. CONCLUSIONS: The obtained results clearly demonstrate structural variability/diversity of these complexes in the Amoebozoa lineage and the reduction of their complexity as compared with the same complexes of model organisms.
Assuntos
Amebozoários/metabolismo , Membranas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Amebozoários/classificação , Amebozoários/genética , Proteínas de Transporte/metabolismo , Sequência Conservada , Éxons , Variação Genética , Genoma de Protozoário , Íntrons , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Filogenia , Subunidades Proteicas , Transporte Proteico , Proteínas de Protozoários/química , TranscriptomaRESUMO
Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.
Assuntos
Acanthamoeba castellanii/enzimologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Dictyostelium/enzimologia , Acanthamoeba castellanii/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Dictyostelium/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Subunidades Proteicas/química , Subunidades Proteicas/genética , Homologia de Sequência de AminoácidosRESUMO
Available data suggest that voltage-dependent anion selective channel (VDAC) constitutes an important component of a cellular regulatory mechanism based on the intracellular reduction/oxidation (redox) state. Here, using quantitative RT-PCR, we demonstrated that depletion of VDAC1 (termed here VDAC) in Saccharomyces cerevisiae cells distinctly affected levels of mRNAs encoding nuclear proteins sensitive to changes of the intracellular redox state including the nuclear transcription factors important for adaptation to the redox state and proteins involved in communication between mitochondria and the nucleus. We also revealed that the changes of the studied protein transcript levels generally correlated with changes of the intracellular redox state although VDAC appears also to affect mRNA levels by a mechanism not based on changes of the intracellular redox states. Thus, VDAC seems to be an important element of the intracellular signaling network.
