Mark Rinnerthaler

miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging.

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non‐coding miRNAs are potent post‐transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age‐related and senescence‐related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8+ T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8+ T cell populations from old and young donors. Using locked nucleic acid‐based miRNA microarrays, we identified four commonly regulated miRNAs, miR‐17 down‐regulated in all seven; miR‐19b and miR‐20a, down‐regulated in six models; and miR‐106a down‐regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.

Pyruvate kinase triggers a metabolic feedback loop that controls redox metabolism in respiring cells.

Summary In proliferating cells, a transition from aerobic to anaerobic metabolism is known as the Warburg effect, whose reversal inhibits cancer cell proliferation. Studying its regulator pyruvate kinase (PYK) in yeast, we discovered that central metabolism is self-adapting to synchronize redox metabolism when respiration is activated. Low PYK activity activated yeast respiration. However, levels of reactive oxygen species (ROS) did not increase, and cells gained resistance to oxidants. This adaptation was attributable to accumulation of the PYK substrate phosphoenolpyruvate (PEP). PEP acted as feedback inhibitor of the glycolytic enzyme triosephosphate isomerase (TPI). TPI inhibition stimulated the pentose phosphate pathway, increased antioxidative metabolism, and prevented ROS accumulation. Thus, a metabolic feedback loop, initiated by PYK, mediated by its substrate and acting on TPI, stimulates redox metabolism in respiring cells. Originating from a single catalytic step, this autonomous reconfiguration of central carbon metabolism prevents oxidative stress upon shifts between fermentation and respiration.

Oxidative Stress in Aging Human Skin

Oxidative stress in skin plays a major role in the aging process. This is true for intrinsic aging and even more for extrinsic aging. Although the results are quite different in dermis and epidermis, extrinsic aging is driven to a large extent by oxidative stress caused by UV irradiation. In this review the overall effects of oxidative stress are discussed as well as the sources of ROS including the mitochondrial ETC, peroxisomal and ER localized proteins, the Fenton reaction, and such enzymes as cyclooxygenases, lipoxygenases, xanthine oxidases, and NADPH oxidases. Furthermore, the defense mechanisms against oxidative stress ranging from enzymes like superoxide dismutases, catalases, peroxiredoxins, and GSH peroxidases to organic compounds such as L-ascorbate, α-tocopherol, beta-carotene, uric acid, CoQ10, and glutathione are described in more detail. In addition the oxidative stress induced modifications caused to proteins, lipids and DNA are discussed. Finally age-related changes of the skin are also a topic of this review. They include a disruption of the epidermal calcium gradient in old skin with an accompanying change in the composition of the cornified envelope. This modified cornified envelope also leads to an altered anti-oxidative capacity and a reduced barrier function of the epidermis.

Expression profiling of aging in the human skin

During the last years it was shown that the aging process is controlled by specific genes in a large number of organisms (C. elegans, Drosophila, mouse or humans). To investigate genes involved in the natural aging process of the human skin we applied cDNA microarray analysis of naturally aged human foreskin samples. For the array experiments a non-redundant set of 2135 pre-selected EST clones was used. These arrays were used to probe the patterns of gene expression in naturally aged human skin of five young (3-4 years of age) and five old (68-72 years of age) healthy persons. We found that in total 105 genes change their expression over 1.7-fold during the aging process in the human skin. Of these 43 genes were shown to be down-regulated in contrast to 62 up-regulated genes. Expression of regulated genes was confirmed by real-time PCR. These results suggest that the aging process in the human skin is connected with the deregulation of various cellular processes, like cell cycle control, cytoskeletal changes, inflammatory response, signaling and metabolism.

Ribosomal proteins Rpl10 and Rps6 are potent regulators of yeast replicative life span.

The yeast ribosome is composed of two subunits, the large 60S subunit (LSU) and the small 40S subunit (SSU) and harbors 78 ribosomal proteins (RPs), 59 of which are encoded by duplicate genes. Recently, deletions of the LSU paralogs RPL31A and RPL6B were found to increase significantly yeast replicative life span (RLS). RPs Rpl10 and Rps6 are known translational regulators. Here, we report that heterozygosity for rpl10Delta but not for rpl25Delta, both LSU single copy RP genes, increased RLS by 24%. Deletion of the SSU RPS6B paralog, but not of the RPS6A paralog increased replicative life span robustly by 45%, while deletion of both the SSU RPS18A, and RPS18B paralogs increased RLS moderately, but significantly by 15%. Altering the gene dosage of RPL10 reduced the translating ribosome population, whereas deletion of the RPS6A, RPS6B, RPS18A, and RPS18B paralogs produced a large shift in free ribosomal subunit stoichiometry. We observed a reduction in growth rate in all deletion strains and reduced cell size in the SSU RPS6B, RPS6A, and RPS18B deletion strains. Thus, reduction of gene dosage of RP genes belonging to both the 60S and the 40S subunit affect lifespan, possibly altering the aging process by modulation of translation.

Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.

Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Quantitation of (a)symmetric inheritance of functional and of oxidatively damaged mitochondrial aconitase in the cell division of old yeast mother cells.

Asymmetric segregation of oxidatively damaged proteins is discussed in the literature as a mechanism in cell division cycles which at the same time causes rejuvenation of the daughter cell and aging of the mother cell. This process must be viewed as cooperating with the cellular degradation processes like autophagy, proteasomal degradation and others. Together, these two mechanisms guarantee survival of the species and prevent clonal senescence of unicellular organisms, like yeast. It is widely believed that oxidative damage to proteins is primarily caused by oxygen radicals and their follow-up products produced in the mitochondria. As we have shown previously, old yeast mother cells in contrast to young cells contain reactive oxygen species and undergo programmed cell death. Here we show that aconitase of the mitochondrial matrix is readily inactivated by oxidative stress, but even in its inactive form is relatively long-lived and retains fluorescence in the Aco1p-eGFP form. The fluorescent protein is distributed between old mothers and their daughters approximately corresponding to the different sizes of mother and daughter cells. However, the remaining active enzyme is primarily inherited by the daughter cells. This indicates that asymmetric distribution of the still active enzyme takes place and a mechanism for discrimination between active and inactive enzyme must exist. As the aconitase remains mitochondrial during aging and cell division, our findings could indicate discrimination between active and no longer active mitochondria during the process.

Age‐related changes in the composition of the cornified envelope in human skin

The main function of the epidermis is to protect us against a multitude of hostile attacks from the environment. Its main cell type, the keratinocytes have a sophisticated system of different proteins and lipids available to form the cornified envelope, which is responsible for the barrier function of the skin. During ageing, dramatic changes are taking place. Some proteins of the SPRR‐, S100‐ and LCE3‐family are massively up‐regulated, whereas others like loricrin, filaggrin and the LCE1&2 protein families are significantly down‐regulated. The latter ones are known to be under control of calcium and/or ‘calcium response elements’. We were able to show that the calcium peak specific for the stratum granulosum, which is the site where loricrin and the LCE1&2 families are synthesized, is reduced during ageing. The resulting cornified envelope in old skin has an extensively changed composition on the molecular level compared to young skin. This knowledge is of critical importance to understand chronic wound formation and ulcers in old age.

Oxidative Stress in Fungi: Its Function in Signal Transduction, Interaction with Plant Hosts, and Lignocellulose Degradation

In this review article, we want to present an overview of oxidative stress in fungal cells in relation to signal transduction, interaction of fungi with plant hosts, and lignocellulose degradation. We will discuss external oxidative stress which may occur through the interaction with other microorganisms or plant hosts as well as internally generated oxidative stress, which can for instance originate from NADPH oxidases or “leaky” mitochondria and may be modulated by the peroxiredoxin system or by protein disulfide isomerases thus contributing to redox signaling. Analyzing redox signaling in fungi with the tools of molecular genetics is presently only in its beginning. However, it is already clear that redox signaling in fungal cells often is linked to cell differentiation (like the formation of perithecia), virulence (in plant pathogens), hyphal growth and the successful passage through the stationary phase.