DNA Methyltransferases: From Evolution to Clinical Applications.

DNA methylation is an epigenetic mark that living beings have used in different environments. The MTases family catalyzes DNA methylation. This process is conserved from archaea to eukaryotes, from fertilization to every stage of development, and from the early stages of cancer to metastasis. The family of DNMTs has been classified into DNMT1, DNMT2, and DNMT3. Each DNMT has been duplicated or deleted, having consequences on DNMT structure and cellular function, resulting in a conserved evolutionary reaction of DNA methylation. DNMTs are conserved in the five kingdoms of life: bacteria, protists, fungi, plants, and animals. The importance of DNMTs in whether methylate or not has a historical adaptation that in mammals has been discovered in complex regulatory mechanisms to develop another padlock to genomic insurance stability. The regulatory mechanisms that control DNMTs expression are involved in a diversity of cell phenotypes and are associated with pathologies transcription deregulation. This work focused on DNA methyltransferases, their biology, functions, and new inhibitory mechanisms reported. We also discuss different approaches to inhibit DNMTs, the use of non-coding RNAs and nucleoside chemical compounds in recent studies, and their importance in biological, clinical, and industry research.

Carbon and Nitrogen Sources Have No Impact on the Organization and Composition of Ustilago maydis Respiratory Supercomplexes.

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, U. maydis mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in U. maydis under diverse energy conditions. Supercomplexes were obtained by solubilization of U. maydis mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I1:IV1), 1400 kDa (I1:III2), 1600 kDa (I1:III2:IV1), and 1800 kDa (I1:III2:IV2). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.

Las funciones metabólicas, endocrinas y reguladoras de la expresión genética del lactato / The metabolic, endocrine and regulatory functions of lactate gene expression

Resumen El lactato se considera un metabolito de desecho que se produce durante la fatiga muscular. En contraste con esta visión simplista, en este trabajo se proporcionan evidencias de las múltiples y complejas funciones de este metabolito. Se muestra que: 1) el lactato es el producto final de la glucólisis, independientemente de la concentración de oxígeno en el medio en el que se encuentren las células; 2) el lactato forma parte de 2 tipos de lanzadera, una que funciona en el espacio intermembranal de la mitocondria, y otra intercelular, que se encarga de alimentar con lactato a ciertos tipos celulares, como las neuronas o el músculo cardiaco; 3) en los espermatozoides, el lactato se transporta directamente a la matriz mitocondrial y allí se oxida para producir piruvato y NADH; 4) en el hígado, el lactato participa en la oxidación del etanol a través de la generación de peróxido de hidrógeno; 5) que dependiendo de la estirpe celular, el lactato puede funcionar como agente antiinflamatorio (endocrino) o regulador de la expresión génica.

Abstract Lactate is considered to be a waste metabolite produced during muscle fatigue. In contrast with this simplistic point of view, in this review we provide evidence of the multiple and complex functions of this metabolite. We show that: 1) lactate is the final product of the glycolysis regardless the oxygen concentration in the cell 2) lactate is part of two types of shuttle, one that functions in the intermembrane space of the mitochondrion, and another intercellular, which is responsible for feeding lactate to certain cell types, such as neurons or heart muscle, 3) in sperm, lactate is transported directly to the mitochondrial matrix and there it is oxidized to produce pyruvate and NADH, 4) in the liver, lactate participates in the oxidation of ethanol through the generation of hydrogen peroxide, 5) Depending on the cell line, lactate can function as anti-inflammatory agent (endocrine) and/or a regulator of gene expression.

Rapamycin induces morphological and physiological changes without increase in lipid content in Ustilago maydis.

The evolutionarily conserved serine/threonine kinase TOR recruits different subunits to assemble the Target of Rapamycin Complex 1 (TORC1), which is inhibited by rapamycin and regulates ribosome biogenesis, autophagy, and lipid metabolism by regulating the expression of lipogenic genes. In addition, TORC1 participates in the cell cycle, increasing the length of the G2 phase. In the present work, we investigated the effect of rapamycin on cell growth, cell morphology and neutral lipid metabolism in the phytopathogenic fungus Ustilago maydis. Inhibition of TORC1 by rapamycin induced the formation of septa that separate the nuclei that were formed after mitosis. Regarding neutral lipid metabolism, a higher accumulation of triacylglycerols was not detected, but the cells did contain large lipid bodies, which suggests that small lipid bodies became fused into big lipid droplets. Vacuoles showed a similar behavior as the lipid bodies, and double labeling with Blue-CMAC and BODIPY indicates that vacuoles and lipid bodies were independent organelles. The results suggest that TORC1 has a role in cell morphology, lipid metabolism, and vacuolar physiology in U. maydis.

Expression of alternative NADH dehydrogenases (NDH-2) in the phytopathogenic fungus Ustilago maydis.

Type 2 alternative NADH dehydrogenases (NDH-2) participate indirectly in the generation of the electrochemical proton gradient by transferring electrons from NADH and NADPH into the ubiquinone pool. Due to their structural simplicity, alternative NADH dehydrogenases have been proposed as useful tools for gene therapy of cells with defects in the respiratory complex I. In this work, we report the presence of three open reading frames, which correspond to NDH-2 genes in the genome of Ustilago maydis. These three genes were constitutively transcribed in cells cultured in YPD and minimal medium with glucose, ethanol, or lactate as carbon sources. Proteomic analysis showed that only two of the three NDH-2 were associated with isolated mitochondria in all culture media. Oxygen consumption by permeabilized cells using NADH or NADPH was different for each condition, opening the possibility of posttranslational regulation. We confirmed the presence of both external and internal NADH dehydrogenases, as well as an external NADPH dehydrogenase insensitive to calcium. Higher oxygen consumption rates were observed during the exponential growth phase, suggesting that the activity of NADH and NADPH dehydrogenases is coupled to the dynamics of cell growth.

The mitochondrial alternative oxidase Aox1 is needed to cope with respiratory stress but dispensable for pathogenic development in Ustilago maydis.

The mitochondrial alternative oxidase is an important enzyme that allows respiratory activity and the functioning of the Krebs cycle upon disturbance of the respiration chain. It works as a security valve in transferring excessive electrons to oxygen, thereby preventing potential damage by the generation of harmful radicals. A clear biological function, besides the stress response, has so far convincingly only been shown for plants that use the alternative oxidase to generate heat to distribute volatiles. In fungi it was described that the alternative oxidase is needed for pathogenicity. Here, we investigate expression and function of the alternative oxidase at different stages of the life cycle of the corn pathogen Ustilago maydis (Aox1). Interestingly, expression of Aox1 is specifically induced during the stationary phase suggesting a role at high cell density when nutrients become limiting. Studying deletion strains as well as overexpressing strains revealed that Aox1 is dispensable for normal growth, for cell morphology, for response to temperature stress as well as for filamentous growth and plant pathogenicity. However, during conditions eliciting respiratory stress yeast-like growth as well as hyphal growth is strongly affected. We conclude that Aox1 is dispensable for the normal biology of the fungus but specifically needed to cope with respiratory stress.