Oxidase enzyme genes are differentially expressed during Acanthamoeba castellanii encystment.

Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A crucial survival strategy of A. castellanii involves the formation of highly resistant cysts during adverse conditions. This study delves into the cellular processes underpinning encystment, focusing on gene expression changes related to reactive oxygen species (ROS) balance, with a particular emphasis on mitochondrial processes. Our findings reveal a dynamic response within the mitochondria during encystment, with the downregulation of key enzymes involved in oxidative phosphorylation (COX, AOX, and NADHalt) during the initial 48 h, followed by their overexpression at 72 h. This orchestrated response likely creates a pro-oxidative environment, facilitating encystment. Analysis of other ROS processing enzymes across the cell reveals differential expression patterns. Notably, antioxidant enzymes, such as catalases, glutaredoxins, glutathione S-transferases, peroxiredoxins, and thioredoxins, mirror the mitochondrial trend of downregulation followed by upregulation. Additionally, glycolysis and gluconeogenesis are downregulated during the early stages in order to potentially balance the metabolic requirement of the cyst. Our study underscores the importance of ROS regulation in Acanthamoeba encystment. Understanding these mechanisms offers insights into infection control and identifies potential therapeutic targets. This work contributes to unraveling the complex biology of A. castellanii and may aid in combatting Acanthamoeba-related infections. Further research into ROS and oxidase enzymes is warranted, given the organism's remarkable respiratory versatility.

Penicillium chrysogenum as a model system for studying cellular effects of methylglyoxal.

BACKGROUND: α-oxoaldehydes are formed as toxic by-products during metabolic activity. The biologically most important compound of this class, methylglyoxal, results from spontaneous phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate which are intermediate glycolysis products. Methylglyoxal-mediated modification of lipids, nucleic acids and proteins is known to lead to the formation of advanced glycation end products. These modifications contribute to the aetiology of severe diseases like diabetes and neurodegenerative disorders. By using simple model organisms it is possible to conveniently study the effects of methylglyoxal on cellular processes. Here, results are presented on the effects of methylglyoxal on mycelium growth, stationary phase entry (monitored by autophagy induction), mitochondrial morphology and protein composition in the filamentous fungus Penicillium chrysogenum. RESULTS: Methylglyoxal leads to growth rate reduction of this fungus so that the entry into the stationary phase is delayed. Mitochondrial morphology is not changed by methylglyoxal. However, rapamycin-mediated fragmentation of mitochondria is prevented by methylglyoxal. Furthermore, three proteins are identified that are present in lower abundance when methylglyoxal is added to the growth medium (aldo-keto reductase [Pc22g04850], 5-methyl-tetrahydropteroyl-triglutamate-homocysteine S-methyltransferase [Pc22g18630] and NAD-dependent formate dehydrogenase [Pc12g04310]). CONCLUSIONS: The presented results contribute to the understanding of cellular pathways and mechanisms that are affected by the ubiquitous α-oxoaldehyde methylglyoxal.

Improving penicillin biosynthesis in Penicillium chrysogenum by glyoxalase overproduction.

Genetic engineering of fungal cell factories mainly focuses on manipulating enzymes of the product pathway or primary metabolism. However, despite the use of strong promoters or strains containing the genes of interest in multiple copies, the desired strongly enhanced enzyme levels are often not obtained. Here we present a novel strategy to improve penicillin biosynthesis by Penicillium chrysogenum by reducing reactive and toxic metabolic by-products, 2-oxoaldehydes. This was achieved by overexpressing the genes encoding glyoxalase I and II, which resulted in a 10% increase in penicillin titers relative to the control strain. The protein levels of two key enzymes of penicillin biosynthesis, isopenicillin N synthase and isopenicillin N acyltransferase, were increased in the glyoxalase transformants, whereas their transcript levels remained unaltered. These results suggest that directed intracellular reduction of 2-oxoaldehydes prolongs the functional lifetime of these enzymes.

Cellular homeostasis in fungi: impact on the aging process.

Cellular quality control pathways are needed for maintaining the biological function of organisms. If these pathways become compromised, the results are usually highly detrimental. Functional impairments of cell components can lead to diseases and in extreme cases to organismal death. Dysfunction of cells can be induced by a number of toxic by-products that are formed during metabolic activity, like reactive oxygen and nitrogen species, for example. A key source of reactive oxygen species (ROS) are the organelles of oxidative phosphorylation, mitochondria. Therefore mitochondrial function is also directly affected by ROS, especially if there is a compromised ROS-scavenging capacity. Biological systems therefore depend on several lines of defence to counteract the toxic effects of ROS and other damaging agents. The first level is active at the molecular level and consists of various proteases that bind and degrade abnormally modified and / or aggregated mitochondrial proteins. The second level is concerned with maintaining the quality of whole mitochondria. Among the pathways of this level are mitochondrial dynamics and autophagy (mitophagy). Mitochondrial dynamics describes the time-dependent fusion and fission of mitochondria. It is argued that this kind of organellar dynamics has the power to restore the function of impaired organelles by content mixing with intact organelles. If the first and second lines of defence against damage fail and mitochondria become damaged too severely, there is the option to remove affected cells before they can elicit more damage to their surrounding environment by apoptosis. This form of programmed cell death is strictly regulated by a complex network of interacting components and can be divided into mitochondria-dependent and mitochondria-independent modes of action. In this review we give an overview on various biological quality control systems in fungi (yeasts and filamentous fungi) with an emphasis on autophagy (mitophagy) and apoptosis and how these pathways allow fungal organisms to maintain a balanced cellular homeostasis.

Characterization of survival and stress resistance in S. cerevisiae mutants affected in peroxisome inheritance and proliferation, Δinp1 and Δpex11.

Baker's yeast is a valuable model system for the study of biological aging as it can be utilized for the measurement of replicative and chronological life spans in response to interventions. Whereas replicative aging in Saccharomyces cerevisiae mirrors dividing mammalian cells, chronological aging is seen in non-dividing cells. Aging is strongly influenced by the cellular organelles, especially by mitochondria which house essential functions like oxidative phosphorylation. Additionally, peroxisomes were shown to modulate the aging process, mainly by their turnover of reactive oxygen species. There is a fundamental interest in understanding how mitochondria and peroxisomes contribute to cellular aging. This work analyzes chronological aging in yeast mutants that are affected in peroxisomal proliferation and inheritance. Deletion of INP1 (retention of peroxisomes in the mother cell) or PEX11 (division of peroxisomes) leads to clearly reduced life spans compared to the wild-type control under conditions which depend on peroxisomal metabolism. Δinp1 cells are long-lived in contrast to the wild type and Δpex11 when assayed under conditions that not necessitate peroxisome function. Neither treatment affects the index of respiratory capacity, indicating fully functional mitochondria. Evaluation of stress resistances reveals that Δinp1 has significantly higher resistance to the apoptosis elicitor acetic acid. Old Δpex11 cells from an oleate culture are more susceptible to hydrogen peroxide treatment compared to Δinp1 and the wild type. Finally, aged cells are hyper-sensitive to heat shock treatment in contrast to young cells.

Carotenoids and carotenogenic genes in Podospora anserina: engineering of the carotenoid composition extends the life span of the mycelium.

Carotenoids have been identified in the fungus Podospora anserina and a parallel pathway to neurosporene and beta-carotene was established. Three genes for the beta-carotene branch have been cloned and their function elucidated. They correspond to the al-1, al-2 and al-3 genes from Neurospora crassa. They were individually and in combinations over-expressed in P. anserina in order to modify the carotenoid composition qualitatively and quantitatively. In the resulting transformants, carotenoid synthesis was up to eightfold increased and several intermediates of the pathway together with special cyclic carotenoids, beta-zeacarotene and 7,8-dihydro-beta-carotene, accumulated. All transformants with an over-expressed al-2 gene (encoding a phytoene synthase and a lycopene cyclase) displayed up to 31% prolonged life span.

Molecular basis of and interference into degenerative processes in fungi: potential relevance for improving biotechnological performance of microorganisms.

Biological systems, from simple microorganisms to humans, are characterized by time-dependent degenerative processes which lead to reduced fitness, disabilities, severe diseases, and, finally, death. These processes are under genetic control but also influenced by environmental conditions and by stochastic processes. Studying the mechanistic basis of degenerative processes in the filamentous ascomycete Podospora anserina and in other systems demonstrated that mitochondria play a key role in the expression of degenerative phenotypes and unraveled a number of underlying molecular pathways. Reactive oxygen species (ROS) which are mainly, but not exclusively, formed at the mitochondrial respiratory chain are crucial players in this network. While being essential for signaling processes and development, ROS are, at the same time, a potential danger because they lead to molecular damage and degeneration. Fortunately, a number of interacting pathways including ROS scavenging, DNA and protein repair, protein degradation, and mitochondrial fission and fusion are involved in keeping cellular damage low. If these pathways are overwhelmed by extensive damage, programmed cell death is induced. The current knowledge of this hierarchical system of mitochondrial quality control, although still incomplete, appears now to be ready for the development of strategies effective in interventions into those pathways leading to degeneration and loss of performance also in microorganisms used in biotechnology. Very promising interdisciplinary interactions and collaborations involving academic and industrial research teams can be envisioned to arise which bear a great potential, in particular, when system biology approaches are used to understand relevant networks of pathways in a holistic way.

Impact of mitochondrial dynamics on organismic aging.

Mitochondria, the cellular powerhouses, are mandatory to keep eukaryotic cells alive. The function of these organelles has to be regulated carefully, as they also have an important impact on the regulation of cell death programs and aging. In the past few years, it became clear that the dynamic morphology changes of mitochondria by division and fusion events play a crucial role in controlling most of these mitochondrial activities. This article examines established and putative mechanisms of how the regulation of mitochondrial dynamics influences various parameters related to aging.

Studying the mechanisms and targets of glycation and advanced glycation end-products in simple eukaryotic model systems.

Biological systems are usually astonishingly complex. This complexity makes it often difficult if not impossible to study their inner workings. In order to address complex research questions more simply structured models (e.g., microorganisms, plants, non-vertebrate animals) are utilized. Findings from these studies can then be translated to more complex systems like mammals. This strategy facilitates the identification of relevant 'leads' that can be specifically addressed in the higher organism. In this review studies to elucidate the relevance, modes of action and molecular targets of reactive carbonyl species using simple model systems are discussed. These dicarbonyls are formed during metabolic activity in all organisms as toxic by-products that lead to the dysfunction of essential cellular components by a process termed glycation, resulting ultimately in the generation of advanced glycation end-products. Understanding how both dicarbonyls and advanced glycation end-products are formed, which environmental conditions influence their levels and what cellular pathways they affect is paramount to develop efficient strategies targeting diseases that are related to reactive carbonyl species, like diabetes, neurodegenerative disorders and cancer. This contribution presents important findings in the field of dicarbonyls and glycation from fungi, plants, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster.

Differential proteomic profiling of mitochondria from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes.

According to the 'free radical theory of ageing', the generation and accumulation of reactive oxygen species are key events during ageing of biological systems. Mitochondria are a major source of ROS and prominent targets for ROS-induced damage. Whereas mitochondrial DNA and membranes were shown to be oxidatively modified with ageing, mitochondrial protein oxidation is not well understood. The purpose of this study was an unbiased investigation of age-related changes in mitochondrial proteins and the molecular pathways by which ROS-induced protein oxidation may disturb cellular homeostasis. In a differential comparison of mitochondrial proteins from young and senescent strains of the fungal ageing model Podospora anserina, from brains of young (5 months) vs. older rats (17 and 31 months), and human cells, with normal and chemically accelerated in vitro ageing, we found certain redundant posttranslationally modified isoforms of subunits of ATP synthase affected across all three species. These appear to represent general susceptible hot spot targets for oxidative chemical changes of proteins accumulating during ageing, and potentially initiating various age-related pathologies and processes. This type of modification is discussed using the example of SAM-dependent O-methyltransferase from P. anserina (PaMTH1), which surprisingly was found to be enriched in mitochondrial preparations of senescent cultures.

OXPHOS Supercomplexes: respiration and life-span control in the aging model Podospora anserina.

Recent biochemical evidence has indicated the existence of respiratory supercomplexes as well as ATP synthase oligomers in the inner mitochondrial membrane of different eukaryotes. We have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This aging model is able to respire by either the standard or the alternative pathway. In the latter, electrons are directly transferred from ubiquinol to the alternative oxidase (AOX) and thus bypass complexes III and IV. We showed that the two pathways are composed of distinct respiratory supercomplexes. These data are of significance for the understanding of both respiratory pathways as well as of life-span control and aging.

Impact of ROS on ageing of two fungal model systems: Saccharomyces cerevisiae and Podospora anserina.

To provide a foundation for the development of effective interventions to counteract various age-related diseases in humans, ageing processes have been extensively studied in various model organisms and systems. However, the mechanisms underlying ageing are still not unravelled in detail in any system including rather simple organisms. In this article, we review some of the molecular mechanisms that were found to affect ageing in two fungal models, the unicellular ascomycete Saccharomyces cerevisiae and the filamentous ascomycete Podospora anserina. A selection of issues like retrograde response, genomic instability, caloric restriction, mtDNA reorganisation and apoptosis is presented and discussed with special emphasis on the role reactive oxygen species (ROS) play in these diverse molecular pathways.

Analysis of autophagy in Penicillium chrysogenum by using starvation pads in combination with fluorescence microscopy.

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular 'self-eating') has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called 'starvation pads' for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Arg354 in the catalytic centre of bovine liver catalase is protected from methylglyoxal-mediated glycation.

BACKGROUND: In addition to controlled post-translational modifications proteins can be modified with highly reactive compounds. Usually this leads to a compromised functionality of the protein. Methylglyoxal is one of the most common agents that attack arginine residues. Methylglyoxal is also regarded as a pro-oxidant that affects cellular redox homeostasis by contributing to the formation of reactive oxygen species. Antioxidant enzymes like catalase are required to protect the cell from oxidative damage. These enzymes are also targets for methylglyoxal-mediated modification which could severely affect their catalytic activity in breaking down reactive oxygen species to less reactive or inert compounds. RESULTS: Here, bovine liver catalase was incubated with high levels of methylglyoxal to induce its glycation. This treatment did not lead to a pronounced reduction of enzymatic activity. Subsequently methylglyoxal-mediated arginine modifications (hydroimidazolone and dihydroxyimidazolidine) were quantitatively analysed by sensitive nano high performance liquid chromatography/electron spray ionisation/tandem mass spectrometry. Whereas several arginine residues displayed low to moderate levels of glycation (e.g., Arg93, Arg365, Arg444) Arg354 in the active centre of catalase was never found to be modified. CONCLUSIONS: Bovine liver catalase is able to tolerate very high levels of the modifying α-oxoaldehyde methylglyoxal so that its essential enzymatic function is not impaired.

Synthetic quantitative array technology identifies the Ubp3-Bre5 deubiquitinase complex as a negative regulator of mitophagy.

Mitophagy is crucial to ensuring mitochondrial quality control. However, the molecular mechanism and regulation of mitophagy are still not fully understood. Here, we developed a quantitative methodology termed synthetic quantitative array (SQA) technology, which allowed us to perform a genome-wide screen for modulators of rapamycin-induced mitophagy in S. cerevisiae. SQA technology can be easily employed for other enzyme-based reporter systems and widely applied in yeast research. We identified 86 positive and 10 negative regulators of mitophagy. Moreover, SQA-based analysis of non-selective autophagy revealed that 63 of these regulators are specific for mitophagy and 33 regulate autophagy in general. The Ubp3-Bre5 deubiquitination complex was found to inhibit mitophagy but, conversely, to promote other types of autophagy, including ribophagy. This complex translocates dynamically to mitochondria upon induction of mitophagy. These findings point to a role of ubiquitination in mitophagy in yeast and suggest a reciprocal regulation of distinct autophagy pathways.

Copper homeostasis and aging in the fungal model system Podospora anserina: differential expression of PaCtr3 encoding a copper transporter.

Lifespan extension of Podospora anserina mutant grisea is caused by a loss-of-function mutation in the nuclear gene Grisea. This gene encodes the copper regulated transcription factor GRISEA recently shown to be involved in the expression of PaSod2 encoding the mitochondrial manganese superoxide dismutase. Here we report the identification and characterization of a second target gene. This gene, PaCtr3, encodes a functional homologue of the Saccharomyces cerevisiae high affinity copper permease yCTR3. PaCtr3 is not expressed in the grisea mutant confirming the assumption that the extension of lifespan is primarily caused by cellular copper limitation and a switch from a cytochrome oxidase (COX)-dependent to and alternative oxidase (AOX)-dependent respiration. Transcript levels of PaCtr3 and PaSod2 respond to copper, iron, manganese and zinc. Transcription of PaCtr3 was found to be down-regulated during senescence of wild-type cultures suggesting that the intracellular copper concentration is raised in old cultures. A two hybrid analysis suggested that GRISEA acts as a homodimer. In accordance, an inverted repeat was identified as a putative binding sequence in the promoter region of PaCtr3 and of PaSod2. Finally, the expression of PaCtr3 in transformants of the grisea mutant led to lifespan shortening. This effect correlates with the activity of the copper-dependent COX demonstrating a strong link between copper-uptake, respiration and lifespan.

Unopposed mitochondrial fission leads to severe lifespan shortening.

Mitochondrial morphology is controlled by the opposing processes of fusion and fission. Previously, in baker's yeast it was shown that reduced mitochondrial fission leads to a network-like morphology, decreased sensitivity for the induction of apoptosis and a remarkable extension of both replicative and chronological lifespan. However, the effects of reduced mitochondrial fusion on aging are so far unknown and complicated by the fact that deletion of genes encoding components of mitochondrial fusion are often lethal to higher organisms. This is also true for the mammalian OPA1 protein, which is a key regulator of mitochondrial inner membrane fusion. Baker's yeast contains an OPA1 ortholog, Mgm1p. Deletion of Mgm1 is possible in yeast due to the fact that mitochondrial function is not essential for growth on glucose-containing media. In this study, we report that absence of mitochondrial fusion in the Δmgm1 mutant leads to a striking reduction of both replicative and chronological lifespan. Concomitantly, sensitivity to apoptosis elicitation via the reactive oxygen species hydrogen peroxide is substantially increased. These results demonstrate that the unopposed mitochondrial fission as displayed by the Δmgm1 mutant strongly affects organismal aging. Moreover, our results bear important clues for translational research to intervene into age-related degenerative processes also in multicellular organisms including humans.

Alternative oxidase dependent respiration leads to an increased mitochondrial content in two long-lived mutants of the aging model Podospora anserina.

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.

Modulation of the glyoxalase system in the aging model Podospora anserina: effects on growth and lifespan.

The eukaryotic glyoxalase system consists of two enzymatic components, glyoxalase I (lactoylglutathione lyase) and glyoxalase II (hydroxyacylglutathione hydrolase). These enzymes are dedicated to the removal of toxic α-oxoaldehydes like methylglyoxal (MG). MG is formed as a by-product of glycolysis and MG toxicity results from its damaging capability leading to modifications of proteins, lipids and nucleic acids. An efficient removal of MG appears to be essential to ensure cellular functionality and viability. Here we study the effects of the genetic modulation of genes encoding the components of the glyoxalase system in the filamentous ascomycete and aging modelPodospora anserina. Overexpression of PaGlo1 leads to a lifespan reduction on glucose rich medium, probably due to depletion of reduced glutathione. Deletion of PaGlo1 leads to hypersensitivity against MG added to the growth medium. A beneficial effect on lifespan is observed when both PaGlo1 and PaGlo2 are overexpressed and the corresponding strains are grown on media containing increased glucose concentrations. Notably, the double mutant has a 'healthy' phenotype without physiological impairments. Moreover, PaGlo1/PaGlo2_OEx strains are not long-lived on media containing standard glucose concentrations suggesting a tight correlation between the efficiency and capacity to remove MG within the cell, the level of available glucose and lifespan. Overall, our results identify the up-regulation of both components of the glyoxalase system as an effective intervention to increase lifespan in P. anserina.

Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina.

Work from more than 50 years of research has unraveled a number of molecular pathways that are involved in controlling aging of the fungal model system Podospora anserina. Early research revealed that wild-type strain aging is linked to gross reorganization of the mitochondrial DNA. Later it was shown that aging of P. anserina does also take place, although at a slower pace, when the wild-type specific mitochondrial DNA rearrangements do not occur. Now it is clear that a network of different pathways is involved in the control of aging. Branches of these pathways appear to be connected and constitute a hierarchical system of responses. Although cross talk between the individual pathways seems to be fundamental in the coordination of the overall system, the precise underlying interactions remain to be unraveled. Such a systematic approach aims at a holistic understanding of the process of biological aging, the ultimate goal of modern systems biology.