From blood to stress-my life investigating cell differentiation.

This short retrospective covers more than 50 years of research. I spent most of it doing yeast genetics and genetic engineering. It has been my great privilege to be part of the international group of yeast genetics researchers. With many of them named in this retrospective, I am connected in lifelong friendships and the same is true for my students and collaborators. The question which we wanted to ask is "How does the genome of the cell and cell differentiation adapt to changing and stressful environmental conditions?" The two examples we studied were sporulation and pseudohyphal growth. Both forms of differentiation are triggered by the stress of starvation. In the pathway of regulation of pseudohyphal growth, a yeast NADPH oxidase (discovered by our group) plays a major role.

Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa.

INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.

Discordant evolution of mitochondrial and nuclear yeast genomes at population level.

BACKGROUND: Mitochondria are essential organelles partially regulated by their own genomes. The mitochondrial genome maintenance and inheritance differ from the nuclear genome, potentially uncoupling their evolutionary trajectories. Here, we analysed mitochondrial sequences obtained from the 1011 Saccharomyces cerevisiae strain collection and identified pronounced differences with their nuclear genome counterparts. RESULTS: In contrast with pre-whole genome duplication fungal species, S. cerevisiae mitochondrial genomes show higher genetic diversity compared to the nuclear genomes. Strikingly, mitochondrial genomes appear to be highly admixed, resulting in a complex interconnected phylogeny with a weak grouping of isolates, whereas interspecies introgressions are very rare. Complete genome assemblies revealed that structural rearrangements are nearly absent with rare inversions detected. We tracked intron variation in COX1 and COB to infer gain and loss events throughout the species evolutionary history. Mitochondrial genome copy number is connected with the nuclear genome and linearly scale up with ploidy. We observed rare cases of naturally occurring mitochondrial DNA loss, petite, with a subset of them that do not suffer the expected growth defect in fermentable rich media. CONCLUSIONS: Overall, our results illustrate how differences in the biology of two genomes coexisting in the same cells can lead to discordant evolutionary histories.

Behead and live long or the tale of cathepsin L.

In recent decades Saccharomyces cerevisiae has proven to be one of the most valuable model organisms of aging research. Pathways such as autophagy or the effect of substances like resveratrol and spermidine that prolong the replicative as well as chronological lifespan of cells were described for the first time in S. cerevisiae. In this study we describe the establishment of an aging reporter that allows a reliable and relative quick screening of substances and genes that have an impact on the replicative lifespan. A cDNA library of the flatworm Dugesia tigrina that can be immortalized by beheading was screened using this aging reporter. Of all the flatworm genes, only one could be identified that significantly increased the replicative lifespan of S.cerevisiae. This gene is the cysteine protease cathepsin L that was sequenced for the first time in this study. We were able to show that this protease has the capability to degrade such proteins as the yeast Sup35 protein or the human α-synuclein protein in yeast cells that are both capable of forming cytosolic toxic aggregates. The degradation of these proteins by cathepsin L prevents the formation of these unfolded protein aggregates and this seems to be responsible for the increase in replicative lifespan.

The defense and signaling role of NADPH oxidases in eukaryotic cells : Review.

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.

High reactive oxygen species levels are detected at the end of the chronological life span of translocant yeast cells.

Chromosome translocation is a major genomic event for a cell, affecting almost every of its life aspects ranging from metabolism, organelle maintenance and homeostasis to gene maintenance and expression. By using the bridge-induced translocation system, we defined the effects of induced chromosome translocation on the chronological life span (CLS) of yeast with particular interest to the oxidative stress condition. The results demonstrate that every translocant strain has a different CLS, but all have a high increase in reactive oxygen species and in lipid peroxides levels at the end of the life span. This could be due to the very unique and strong deregulation of the oxidative stress network. Furthermore, the loss of the translocated chromosome occurs at the end of the life span and is locus dependent. Additionally, the RDH54 gene may play a role in the correct segregation of the translocant chromosome, since in its absence there is an increase in loss of the bridge-induced translocated chromosome.

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis.

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.

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.

Mitochondria in ageing: there is metabolism beyond the ROS.

Mitochondria are responsible for a series of metabolic functions. Superoxide leakage from the respiratory chain and the resulting cascade of reactive oxygen species-induced damage, as well as mitochondrial metabolism in programmed cell death, have been intensively studied during ageing in single-cellular and higher organisms. Changes in mitochondrial physiology and metabolism resulting in ROS are thus considered to be hallmarks of ageing. In this review, we address 'other' metabolic activities of mitochondria, carbon metabolism (the TCA cycle and related underground metabolism), the synthesis of Fe/S clusters and the metabolic consequences of mitophagy. These important mitochondrial activities are hitherto less well-studied in the context of cellular and organismic ageing. In budding yeast, they strongly influence replicative, chronological and hibernating lifespan, connecting the diverse ageing phenotypes studied in this single-cellular model organism. Moreover, there is evidence that similar processes equally contribute to ageing of higher organisms as well. In this scenario, increasing loss of metabolic integrity would be one driving force that contributes to the ageing process. Understanding mitochondrial metabolism may thus be required for achieving a unifying theory of eukaryotic ageing.

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 ß 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.

Introduction.

Aging in yeast is now a well researched area with hundreds of new research and review papers appearing every year. The chapters following in this book written by some of the leading experts in the field will give an overview of the most relevant areas of yeast aging. The purpose of this chapter is to give the newcomer an introduction to the field including some basic technical questions.

Oxidative stresses and ageing.

Oxidative damage to cellular constituents has frequently been associated with aging in a wide range of organisms. The power of yeast genetics and biochemistry has provided the opportunity to analyse in some detail how reactive oxygen and nitrogen species arise in cells, how cells respond to the damage that these reactive species cause, and to begin to dissect how these species may be involved in the ageing process. This chapter reviews the major sources of reactive oxygen species that occur in yeast cells, the damage they cause and how cells sense and respond to this damage.

Yeast aging and apoptosis.

A concerted balance between proliferation and apoptosis is essential to the survival of multicellular organisms. Thus, apoptosis per se, although it is a destructive process leading to the death of single cells, also serves as a pro-survival mechanism pro-survival mechanism that ensures healthy organismal development and acts as a life-prolonging or anti-aging anti-aging program. The discovery that yeast also possess a functional and, in many cases, highly conserved apoptotic machinery has made it possible to study the relationships between aging and apoptosis in depth using a well-established genetic system and the powerful tools available to yeast researchers for investigating complex physiological and cytological interactions. The aging process of yeast, be it replicative replicative or chronological chronological aging, is closely related to apoptosis, although it remains unclear whether apoptosis is a causal feature of the aging process or vice versa. Nevertheless, experimental results obtained during the past several years clearly demonstrate that yeast serve as a powerful and versatile experimental system for understanding the interconnections between these two fundamentally important cellular and physiological pathways.

The role of mitochondria in the aging processes of yeast.

This chapter reviews the role of mitochondria and of mitochondrial metabolism in the aging processes of yeast and the existing evidence for the "mitochondrial theory of aging mitochondrial theory of aging ". Mitochondria are the major source of ATP in the eukaryotic cell but are also a major source of reactive oxygen species reactive oxygen species (ROS) and play an important role in the process of apoptosis and aging. We are discussing the mitochondrial theory of aging mitochondrial theory of aging (TOA), its origin, similarity with other TOAs, and its ramifications which developed in recent decades. The emphasis is on mother cell-specific aging mother cell-specific aging and the RLS (replicative lifespan) with only a short treatment of CLS (chronological lifespan). Both of these aging processes may be relevant to understand also the aging of higher organisms, but they are biochemically very different, as shown by the fact the replicative aging occurs on rich media and is a defect in the replicative capacity of mother cells, while chronological aging occurs in postmitotic cells that are under starvation conditions in stationary phase leading to loss of viability, as discussed elsewhere in this book. In so doing we also give an overview of the similarities and dissimilarities of the various aging processes of the most often used model organisms for aging research with respect to the mitochondrial theory of aging mitochondrial theory of aging.

Spontaneous Mutation Rates and Spectra of Respiratory-Deficient Yeast.

The yeast petite mutant was first discovered in the yeast Saccharomyces cerevisiae, which shows growth stress due to defects in genes encoding the respiratory chain. In a previous study, we described that deletion of the nuclear-encoded gene MRPL25 leads to mitochondrial genome (mtDNA) loss and the petite phenotype, which can be rescued by acquiring ATP3 mutations. The mrpl25Δ strain showed an elevated SNV (single nucleotide variant) rate, suggesting genome instability occurred during the crisis of mtDNA loss. However, the genome-wide mutation landscape and mutational signatures of mitochondrial dysfunction are unknown. In this study we profiled the mutation spectra in yeast strains with the genotype combination of MRPL25 and ATP3 in their wildtype and mutated status, along with the wildtype and cytoplasmic petite rho0 strains as controls. In addition to the previously described elevated SNV rate, we found the INDEL (insertion/deletion) rate also increased in the mrpl25Δ strain, reinforcing the occurrence of genome instability. Notably, although both are petites, the mrpl25Δ and rho0 strains exhibited different INDEL rates and transition/transversion ratios, suggesting differences in the mutational signatures underlying these two types of petites. Interestingly, the petite-related mutagenesis effect disappeared when ATP3 suppressor mutations were acquired, suggesting a cost-effective mechanism for restoring both fitness and genome stability. Taken together, we present an unbiased genome-wide characterization of the mutation rates and spectra of yeast strains with respiratory deficiency, which provides valuable insights into the impact of respiratory deficiency on genome instability.

Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways.

Reactive oxygen species (ROS) that exceed the antioxidative capacity of the cell can be harmful and are termed oxidative stress. Increasing evidence suggests that ROS are not exclusively detrimental, but can fulfill important signaling functions. Recently, we have been able to demonstrate that a NADPH oxidase-like enzyme (termed Yno1p) exists in the single-celled organism Saccharomyces cerevisiae. This enzyme resides in the peripheral and perinuclear endoplasmic reticulum and functions in close proximity to the plasma membrane. Its product, hydrogen peroxide, which is also produced by the action of the superoxide dismutase, Sod1p, influences signaling of key regulatory proteins Ras2p and Yck1p/2p. In the present work, we demonstrate that Yno1p-derived H2O2 regulates outputs controlled by three MAP kinase pathways that can share components: the filamentous growth (filamentous growth MAPK (fMAPK)), pheromone response, and osmotic stress response (hyperosmolarity glycerol response, HOG) pathways. A key structural component and regulator in this process is the actin cytoskeleton. The nucleation and stabilization of actin are regulated by Yno1p. Cells lacking YNO1 showed reduced invasive growth, which could be reversed by stimulation of actin nucleation. Additionally, under osmotic stress, the vacuoles of a ∆yno1 strain show an enhanced fragmentation. During pheromone response induced by the addition of alpha-factor, Yno1p is responsible for a burst of ROS. Collectively, these results broaden the roles of ROS to encompass microbial differentiation responses and stress responses controlled by MAPK pathways.

The life in a gradient: calcium, the lncRNA SPRR2C and mir542/mir196a meet in the epidermis to regulate the aging process.

The turnover of the epidermis beginning with the progenitor cells in the basal layer to the fully differentiated corneocytes is tightly regulated by calcium. Calcium more than anything else promotes the differentiation of keratinocytes which implies the need for a calcium gradient with low concentrations in the stratum basale and high concentrations in the stratum granulosum. One of the hallmarks of skin aging is a collapse of this gradient that has a direct impact on the epidermal fitness. The rise of calcium in the stratum basale reduces cell proliferation, whereas the drop of calcium in the stratum granulosum leads to a changed composition of the cornified envelope. We showed that keratinocytes respond to the calcium induced block of cell division by a large increase of the expression of several miRNAs (hsa-mir542-5p, hsa-mir125a, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p). The pitfall of this rescue mechanism is a dramatic change in gene expression which causes a further impairment of the epidermal barrier. This effect is attenuated by a pseudogene (SPRR2C) that gives rise to a lncRNA. SPRR2C specifically resides in the stratum granulosum/corneum thus acting as a sponge for miRNAs.

The metabolic growth limitations of petite cells lacking the mitochondrial genome.

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

The actin cytoskeleton, RAS-cAMP signaling and mitochondrial ROS in yeast apoptosis.

The release of reactive oxygen species (ROS) by mitochondria instigates the pathways of programmed cell death in eukaryotic cells. Gourlay and Ayscough present intriguing experimental evidence that mutations in the genes encoding the regulatory proteins End3p and Sla1p, which influence actin dynamics in budding yeast, lead to a loss of mitochondrial membrane potential, resulting in ROS production and apoptosis. This effect can be suppressed by downregulation of the RAS-cAMP signaling pathway, thus establishing the existence of a new and complex regulatory network.

Metabolic changes through hypoxia in humans and in yeast as a comparable cell model.

BACKGROUND: In several investigations on mountaineers under moderate hypoxia, at altitudes between 2,500 m and 4,500 m, weight loss occurs, fat levels in the serum and insulin resistance (in diabetic mountaineers) are reduced. Animal studies with different time dosage regimens of hypoxia in animal cages revealed different and partly confusing results regarding fat metabolism under hypoxia. HYPOTHESES: Hypothesis for the change in glucose and fat metabolism include a HIF promoted higher leptin rate under hypoxia and an increased glucose transport in peripheral organs. DISCUSSION: This short review discusses some of the different investigations in this topic. In a second part it is shown how studies of metabolism in yeast cells with an upregulated glycolysis in the cell itself under hypoxic conditions could help to better understand metabolic changes under hypoxia.