The control of translational accuracy is a determinant of healthy ageing in yeast

Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the repiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency

Circulating miRNAs as predictors for morbidity and mortality in coronary artery disease

Micro ribonucleic acids (miRNAs) are small non-coding RNA molecules that control gene expression by translational inhibition. They have been identified to play a role in a multitude of physiological and pathophysiological cellular processes amongst others in the heart. Due to their ability to be released into the blood as well as their stability in body fluids, they appear suitable as biomarkers. This review discusses the role of selected miRNA that currently emerge as biomarkers for coronary artery disease, their potential to discriminate between different diseases, as well as how they might be used as predictive tools for cardiac events or disease outcome. Furthermore, we propose procedural steps of miRNA analysis, to allow better comparison between studies in the future.(VLID)386549

Discovery and Functional Analysis of the Single-Celled Yeast NADPH Oxidase, Yno1

In this chapter, we describe the discovery of the NADPH oxidase gene and protein of the single-celled yeast Saccharomyces cerevisiae, Yno1. This enzyme was characterized with respect to mechanism of action, subcellular location, regulation of gene expression, and physiological function. Yno1 is not involved in defense and is not highly expressed in vegetatively growing cells. However, it is expressed in diverse stress situations. The signaling substance produced by Yno1 in conjunction with the superoxide dismutase Sod1, hydrogen peroxide, consequently leads through a change in the expression of target genes to the modulation of an adaptive cellular response. An example is the formation of pseudohyphae enabling invasive growth of the yeast cells, which is believed to aid in the utilization of new nutrients. The major role of Yno1 is in the switch of the mode of growth from vegetative budding to the formation of pseudohyphae, which are elongated chains of cells. Further examples that are described in this chapter are the response to osmotic stress and mating. All these pathways have in common that they exit the regular cell cycle and are associated with in parts enormous changes in cell morphology. This is accomplished involving a change in the structure of the actin cytoskeleton. Yno1 was shown to directly modulate the actin cytoskeleton

Wiener Medizinische Wochenschrift / The defense and signaling role of NADPH oxidases in eukaryotic cells

This short review article summarizes what is known clinically and biochemically about the seven human NADPH oxidases. Emphasis is put on the connection between mutations in the catalytic and regulatory subunits of Nox2, the phagocyte defense enzyme, with syndromes like chronic granulomatous disease, as well as a number of chronic inflammatory diseases. These arise paradoxically from a lack of reactive oxygen species production needed as second messengers for immune regulation. Both Nox2 and the six other human NADPH oxidases display signaling functions in addition to the functions of these enzymes in specialized biochemical reactions, for instance, synthesis of the hormone thyroxine. NADPH oxidases are also needed by Saccharomyces cerevisiae cells for the regulation of the actin cytoskeleton in times of stress or developmental changes, such as pseudohyphae formation. The article shows that in certain cancer cells Nox4 is also involved in the re-structuring of the actin cytoskeleton, which is required for cell mobility and therefore for metastasis.(VLID)286374

En Route to Targeted Ribosome Editing to Replenish Skin Anchor Protein LAMB3 in Junctional Epidermolysis Bullosa

Severe junctional epidermolysis bullosa is a rare genetic, postpartum lethal skin disease, predominantly caused by nonsense/premature termination codon (PTC) sequence variants in LAMB3 gene. LAMB3 encodes LAMB3, the β subunit of epidermal–dermal skin anchor laminin 332. Most translational reads of a PTC mRNA deliver truncated, nonfunctional proteins, whereas an endogenous PTC readthrough mechanism produces full-length protein at minimal and insufficient levels. Conventional translational readthrough-inducing drugs amplify endogenous PTC readthrough; however, translational readthrough-inducing drugs are either proteotoxic or nonselective. Ribosome editing is a more selective and less toxic strategy. This technique identified ribosomal protein L35/uL29 (ie, RpL35) and RpL35-ligands repurposable drugs artesunate and atazanavir as molecular tools to increase production levels of full-length LAMB3. To evaluate ligand activity in living cells, we monitored artesunate and atazanavir treatment by dual luciferase reporter assays. Production levels of full-length LAMB3 increased up to 200% upon artesunate treatment, up to 150% upon atazanavir treatment, and up to 170% upon combinatorial treatment of RpL35 ligands at reduced drug dosage, with an unrelated PTC reporter being nonresponsive. Proof of bioactivity of RpL35 ligands in selective increase of full-length LAMB3 provides the basis for an alternative, targeted therapeutic route to replenish LAMB3 in severe junctional epidermolysis bullosa

The Human NADPH Oxidase, Nox4, Regulates Cytoskeletal Organization in Two Cancer Cell Lines, HepG2 and SH-SY5Y

NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis

Supplemental Figure 1 from The control of translational accuracy is a determinant of healthy ageing in yeast

Translational accuracy in an rcm1 deletion strain

Raw data files from The control of translational accuracy is a determinant of healthy ageing in yeast

Raw data in excel format, including luciferase assay, ageing and growth rate data