Rena Balzan

Mitoferrin is essential for erythroid iron assimilation

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe–S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe–S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe–S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.

Oxidative Stress and Programmed Cell Death in Yeast

Yeasts, such as Saccharomyces cerevisiae, have long served as useful models for the study of oxidative stress, an event associated with cell death and severe human pathologies. This review will discuss oxidative stress in yeast, in terms of sources of reactive oxygen species (ROS), their molecular targets, and the metabolic responses elicited by cellular ROS accumulation. Responses of yeast to accumulated ROS include upregulation of antioxidants mediated by complex transcriptional changes, activation of pro-survival pathways such as mitophagy, and programmed cell death (PCD) which, apart from apoptosis, includes pathways such as autophagy and necrosis, a form of cell death long considered accidental and uncoordinated. The role of ROS in yeast aging will also be discussed.

Guidelines and recommendations on yeast cell death nomenclature

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

Mitochondrial involvement in aspirin-induced apoptosis in yeast

We have previously reported that aspirin induces apoptosis in manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells when cultivated on the non-fermentable carbon source ethanol. Here, we investigated the role of mitochondria in aspirin-induced apoptosis. We report that aspirin had an inhibitory effect on cellular respiration, and caused the release of most of the mitochondrial cytochrome c and a dramatic drop in the mitochondrial membrane potential (DeltaPsi(m)). Also, aspirin reduced the intracellular cytosolic pH in the MnSOD-deficient cells growing in ethanol medium, but this did not seem to be the initial trigger that committed these cells to aspirin-induced apoptosis. Furthermore, loss of DeltaPsi(m) was not required for aspirin-induced release of cytochrome c, since the initial release of cytochrome c occurred prior to the disruption of the DeltaPsi(m). It is thus possible that cytochrome c release does not involve the early onset of the mitochondrial permeability transition, but only an alteration of the permeability of the outer mitochondrial membrane.

Prokaryotic iron superoxide dismutase replaces cytosolic copper, zinc superoxide dismutase in protecting yeast cells against oxidative stress

The iron superoxide dismutase (FeSOD) gene of Escherichia coli was cloned in Saccharomyces cerevisiae cells deficient in copper, zinc superoxide dismutase (Cu,ZnSOD). FeSOD replaced Cu,ZnSOD in protecting the yeast cells against oxidative stress. In the recombinant strains the FeSOD gene, which was under the transcriptional control of the yeast phosphoglycerate kinase gene promoter, was functionally expressed at two different levels on episomal and centromeric plasmids. Despite suppression of methionine and lysine auxotrophy, the higher level of FeSOD activity was more beneficial to growth of the mutant yeast cells only when these were exposed to higher levels of oxidative stress induced by paraquat or 100% oxygen. In the presence of paraquat, there was a novel stimulation of FeSOD activity. This was associated with a marked increase in catalase activity, and a decrease in glutathione reductase activity.

The Proapoptotic Effect of Traditional and Novel Nonsteroidal Anti-Inflammatory Drugs in Mammalian and Yeast Cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) have long been used to treat pain, fever, and inflammation. However, mounting evidence shows that NSAIDs, such as aspirin, have very promising antineoplastic properties. The chemopreventive, antiproliferative behaviour of NSAIDs has been associated with both their inactivation of cyclooxygenases (COX) and their ability to induce apoptosis via pathways that are largely COX-independent. In this review, the various proapoptotic pathways induced by traditional and novel NSAIDs such as phospho-NSAIDs, hydrogen sulfide-releasing NSAIDs and nitric oxide-releasing NSAIDs in mammalian cell lines are discussed, as well as the proapoptotic effects of NSAIDs on budding yeast which retains the hallmarks of mammalian apoptosis. The significance of these mechanisms in terms of the role of NSAIDs in effective cancer prevention is considered.

Aspirin‐induced apoptosis of yeast cells is associated with mitochondrial superoxide radical accumulation and NAD(P)H oxidation

In previous studies, we observed that aspirin, a promising cancer-preventive agent, induces apoptosis in mitochondrial manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells grown aerobically in ethanol medium. In this study, we show that aspirin-induced apoptosis is associated with a significant increase in mitochondrial and cytosolic O2 ·- and oxidation of mitochondrial NAD(P)H. A concomitant rise in the level of cytosolic CuZnSOD activity failed to compensate for mitochondrial MnSOD deficiency. However, an observed increase in activity of Escherichia coli FeSOD targeted to the mitochondrial matrix of the MnSOD-deficient yeast cells, markedly decreased aspirin-induced accumulation of mitochondrial O2 ·-, significantly increased the mitochondrial NAD(P)H level and rescued the apoptotic phenotype. Indeed, recombinant yeast cells expressing E. coli FeSOD behaved in a similar manner to the parent wild-type yeast cells with native mitochondrial MnSOD activity. Wild-type cells consistently showed a decrease in mitochondrial O2 ·- and an increase in mitochondrial NAD(P)H levels in the presence of aspirin in ethanol medium. In fact, in wild-type cells, our studies supported an antioxidant action of aspirin. Taken together, our results indicate that a pro-oxidant effect of aspirin occurring predominantly in cells with compromised mitochondrial redox balance may be enough to overcome antioxidant defences resulting in apoptosis, as observed in MnSOD-deficient yeast cells.

Cell-cycle involvement in autophagy and apoptosis in yeast.

Regulation of the cell cycle and apoptosis are two eukaryotic processes required to ensure maintenance of genomic integrity, especially in response to DNA damage. The ease with which yeast, amongst other eukaryotes, can switch from cellular proliferation to cell death may be the result of a common set of biochemical factors which play dual roles depending on the cells physiological state. A wide variety of homologues are shared between different yeasts and metazoans and this conservation confirms their importance. This review gives an overview of key molecular players involved in yeast cell-cycle regulation, and those involved in mechanisms which are induced by cell-cycle dysregulation. One such mechanism is autophagy which, depending on the severity and type of DNA damage, may either contribute to the cells survival or death. Cell-cycle dysregulation due to checkpoint deficiency leads to mitotic catastrophe which in turn leads to programmed cell death. Molecular players implicated in the yeast apoptotic pathway were shown to play important roles in the cell cycle. These include the metacaspase Yca1p, the caspase-like protein Esp1p, the cohesin subunit Mcd1p, as well as the inhibitor of apoptosis protein Bir1p. The roles of these molecular players are discussed.