Coq3p relevant residues for protein activity and stability.

Coenzyme Q (CoQ) is an essential molecule that consists of a highly substituted benzene ring attached to a polyprenyl tail anchored in the inner mitochondrial membrane. CoQ transfers electrons from NADH dehydrogenase and succinate dehydrogenase complexes toward ubiquinol-cytochrome c reductase, and that allows aerobic growth of cells. In Saccharomyces cerevisiae, the synthesis of CoQ depends on fourteen proteins Coq1p-Co11p, Yah1p, Arh1p, and Hfd1p. Some of these proteins are components of CoQ synthome. Using ab initio molecular modeling and site-directed mutagenesis, we identified the functional residues of the O-methyltransferase Coq3p, which depends on S-adenosylmethionine for catalysis and is necessary for two O-methylation steps required for CoQ maturation. Conserved residues as well as those that coevolved in the protein structure were found to have important roles in respiratory growth, CoQ biosynthesis, and also in the stability of CoQ synthome proteins. Finally, a multiple sequence alignment showed that S. cerevisiae Coq3p has a 45 amino acid residues insertion that is poorly conserved or absent in oleaginous yeast, cells that can store up to 20% of their dry weight as lipids. These results point to the Coq3p structural determinants of its biological and catalytic function and could contribute to the development of lipid-producing yeast for biotechnology.

Expression of human HSP27 in yeast extends replicative lifespan and uncovers a hormetic response.

Human HSP27 is a small heat shock protein that modulates the ability of cells to respond to heat shock and oxidative stress, and also functions as a chaperone independent of ATP, participating in the proteasomal degradation of proteins. The expression of HSP27 is associated with survival in mammalian cells. In cancer cells, it confers resistance to chemotherapy; in neurons, HSP27 has a positive effect on neuronal viability in models of Alzheimer's and Parkinson's diseases. To better understand the mechanism by which HSP27 expression contributes to cell survival, we expressed human HSP27 in the budding yeast Saccharomyces cerevisiae under control of different mutant TEF promoters, that conferred nine levels of graded basal expression, and showed that replicative lifespan and proteasomal activity increase as well as the resistance to oxidative and thermal stresses. The profile of these phenotypes display a dose-response effect characteristic of hormesis, an adaptive phenomenon that is observed when cells are exposed to increasing amounts of stress or toxic substances. The hormetic response correlates with changes in expression levels of HSP27 and also with its oligomeric states when correlated to survival assays. Our results indicate that fine tuning of HSP27 concentration could be used as a strategy for cancer therapy, and also for improving neuronal survival in neurodegenerative diseases.

Simulated microgravity accelerates aging in Saccharomyces cerevisiae.

The human body experiences physiological changes under microgravity environment that phenocopy aging on Earth. These changes include early onset osteoporosis, skeletal muscle atrophy, cardiac dysfunction, and immunosenescence, and such adaptations to the space environment may pose some risk to crewed missions to Mars. To investigate the effect of microgravity on aging, many model organisms have been used such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mice. Herein we report that the budding yeast Saccharomyces cerevisiae show decreased replicative lifespan (RLS) under simulated microgravity in a clinostat. The reduction of yeast lifespan is not a result of decreased tolerance to heat shock or oxidative stress and could be overcome either by deletion of FOB1 or calorie restriction, two known interventions that extend yeast RLS. Deletion of the sirtuin gene SIR2 worsens the simulated microgravity effect on RLS, and together with the fob1Δ mutant phenotype, it suggests that simulated microgravity augments the formation of extrachromosomal rDNA circles, which accumulate in yeast during aging. We also show that the chronological lifespan in minimal medium was not changed when cells were grown in the clinostat. Our data suggest that the reduction in longevity due to simulated microgravity is conserved in yeast, worms, and flies, and these findings may have potential implications for future crewed missions in space, as well as the use of microgravity as a model for human aging.

Structural and functional mapping of Rtg2p determinants involved in retrograde signaling and aging of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging.