Publications by authors named "Mikael Molin"

23 Publications

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Structural determinants of multimerization and dissociation in 2-Cys peroxiredoxin chaperone function.

Structure 2021 Apr 28. Epub 2021 Apr 28.

Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30 Göteborg, Sweden; Department of Biology and Biological Engineering, Chalmers University of Technology, 405 30 Göteborg, Sweden. Electronic address:

Peroxiredoxins (PRDXs) are abundant peroxidases present in all kingdoms of life. Recently, they have been shown to also carry out additional roles as molecular chaperones. To address this emerging supplementary function, this review focuses on structural studies of 2-Cys PRDX systems exhibiting chaperone activity. We provide a detailed understanding of the current knowledge of structural determinants underlying the chaperone function of PRDXs. Specifically, we describe the mechanisms which may modulate their quaternary structure to facilitate interactions with client proteins and how they are coordinated with the functions of other molecular chaperones. Following an overview of PRDX molecular architecture, we outline structural details of the presently best-characterized peroxiredoxins exhibiting chaperone function and highlight common denominators. Finally, we discuss the remarkable structural similarities between 2-Cys PRDXs, small HSPs, and J-domain-independent Hsp40 holdases in terms of their functions and dynamic equilibria between low- and high-molecular-weight oligomers.
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http://dx.doi.org/10.1016/j.str.2021.04.007DOI Listing
April 2021

Protein kinase A controls yeast growth in visible light.

BMC Biol 2020 11 16;18(1):168. Epub 2020 Nov 16.

Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden.

Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400-700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection.

Results: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light.

Conclusion: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination.
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http://dx.doi.org/10.1186/s12915-020-00867-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667738PMC
November 2020

Peroxiredoxin promotes longevity and HO-resistance in yeast through redox-modulation of protein kinase A.

Elife 2020 07 14;9. Epub 2020 Jul 14.

Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden.

Peroxiredoxins are HO scavenging enzymes that also carry out HO signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to HO and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging HO, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by HO and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the HO sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to HO.
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http://dx.doi.org/10.7554/eLife.60346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392609PMC
July 2020

The Third International Symposium on Fungal Stress - ISFUS.

Fungal Biol 2020 05 24;124(5):235-252. Epub 2020 Feb 24.

Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jeruslaem, Rehovot 7610001, Israel.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.
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http://dx.doi.org/10.1016/j.funbio.2020.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438019PMC
May 2020

Intragenic repeat expansion in the cell wall protein gene controls yeast chronological aging.

Genome Res 2020 05 10;30(5):697-710. Epub 2020 Apr 10.

Université Côte d'Azur, CNRS, INSERM, IRCAN, 06107 Nice, France.

Aging varies among individuals due to both genetics and environment, but the underlying molecular mechanisms remain largely unknown. Using a highly recombined population, we found 30 distinct quantitative trait loci (QTLs) that control chronological life span (CLS) in calorie-rich and calorie-restricted environments and under rapamycin exposure. Calorie restriction and rapamycin extended life span in virtually all genotypes but through different genetic variants. We tracked the two major QTLs to the cell wall glycoprotein genes and We found that massive expansion of intragenic tandem repeats within the N-terminal domain of was sufficient to cause pronounced life span shortening. Life span impairment by was buffered by rapamycin but not by calorie restriction. The repeat expansion shifted yeast cells from a sedentary to a buoyant state, thereby increasing their exposure to surrounding oxygen. The higher oxygenation altered methionine, lipid, and purine metabolism, and inhibited quiescence, which explains the life span shortening. We conclude that fast-evolving intragenic repeat expansions can fundamentally change the relationship between cells and their environment with profound effects on cellular lifestyle and longevity.
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http://dx.doi.org/10.1101/gr.253351.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263189PMC
May 2020

A Redox-Sensitive Thiol in Wis1 Modulates the Fission Yeast Mitogen-Activated Protein Kinase Response to HO and Is the Target of a Small Molecule.

Mol Cell Biol 2020 03 16;40(7). Epub 2020 Mar 16.

University of Gothenburg, Department of Chemistry and Molecular Biology, Gothenburg, Sweden

Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast MAPKK Wis1, which belongs to the HO-responsive MAPK Sty1 pathway. Here, we show that HO reversibly inactivates Wis1 through this residue (C458) We found that C458 is oxidized and that serine replacement of this residue significantly enhances Wis1 activation upon addition of HO The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by HO and significantly increased Wis1 activation by low levels of HO We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the MAPKK Wis1.
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http://dx.doi.org/10.1128/MCB.00346-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076255PMC
March 2020

Nonlinear feedback drives homeostatic plasticity in HO stress response.

Elife 2017 04 18;6. Epub 2017 Apr 18.

Developmental Biology and Stem Cells Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France.

Homeostatic systems that rely on genetic regulatory networks are intrinsically limited by the transcriptional response time, which may restrict a cell's ability to adapt to unanticipated environmental challenges. To bypass this limitation, cells have evolved mechanisms whereby exposure to mild stress increases their resistance to subsequent threats. However, the mechanisms responsible for such remain largely unknown. Here, we used live-cell imaging and microfluidics to investigate the adaptive response of budding yeast to temporally controlled HO stress patterns. We demonstrate that acquisition of tolerance is a systems-level property resulting from nonlinearity of HO scavenging by peroxiredoxins and our study reveals that this regulatory scheme induces a striking hormetic effect of extracellular HO stress on replicative longevity. Our study thus provides a novel quantitative framework bridging the molecular architecture of a cellular homeostatic system to the emergence of nonintuitive adaptive properties.
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http://dx.doi.org/10.7554/eLife.23971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438251PMC
April 2017

Light-sensing via hydrogen peroxide and a peroxiredoxin.

Nat Commun 2017 03 24;8:14791. Epub 2017 Mar 24.

Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-413 90 Göteborg, Sweden.

Yeast lacks dedicated photoreceptors; however, blue light still causes pronounced oscillations of the transcription factor Msn2 into and out of the nucleus. Here we show that this poorly understood phenomenon is initiated by a peroxisomal oxidase, which converts light into a hydrogen peroxide (HO) signal that is sensed by the peroxiredoxin Tsa1 and transduced to thioredoxin, to counteract PKA-dependent Msn2 phosphorylation. Upon HO, the nuclear retention of PKA catalytic subunits, which contributes to delayed Msn2 nuclear concentration, is antagonized in a Tsa1-dependent manner. Conversely, peroxiredoxin hyperoxidation interrupts the HO signal and drives Msn2 oscillations by superimposing on PKA feedback regulation. Our data identify a mechanism by which light could be sensed in all cells lacking dedicated photoreceptors. In particular, the use of HO as a second messenger in signalling is common to Msn2 oscillations and to light-induced entrainment of circadian rhythms and suggests conserved roles for peroxiredoxins in endogenous rhythms.
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http://dx.doi.org/10.1038/ncomms14791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376668PMC
March 2017

Lifespan Control by Redox-Dependent Recruitment of Chaperones to Misfolded Proteins.

Cell 2016 Jun 2;166(1):140-51. Epub 2016 Jun 2.

Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Medicinaregatan 7A, 413 90 Göteborg, Sweden. Electronic address:

Caloric restriction (CR) extends the lifespan of flies, worms, and yeast by counteracting age-related oxidation of H2O2-scavenging peroxiredoxins (Prxs). Here, we show that increased dosage of the major cytosolic Prx in yeast, Tsa1, extends lifespan in an Hsp70 chaperone-dependent and CR-independent manner without increasing H2O2 scavenging or genome stability. We found that Tsa1 and Hsp70 physically interact and that hyperoxidation of Tsa1 by H2O2 is required for the recruitment of the Hsp70 chaperones and the Hsp104 disaggregase to misfolded and aggregated proteins during aging, but not heat stress. Tsa1 counteracted the accumulation of ubiquitinated aggregates during aging and the reduction of hyperoxidized Tsa1 by sulfiredoxin facilitated clearance of H2O2-generated aggregates. The data reveal a conceptually new role for H2O2 signaling in proteostasis and lifespan control and shed new light on the selective benefits endowed to eukaryotic peroxiredoxins by their reversible hyperoxidation.
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http://dx.doi.org/10.1016/j.cell.2016.05.006DOI Listing
June 2016

Metagenomics reveals that detoxification systems are underrepresented in marine bacterial communities.

BMC Genomics 2014 Sep 1;15:749. Epub 2014 Sep 1.

Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30 Göteborg, Sweden.

Background: Environmental shotgun sequencing (metagenomics) provides a new way to study communities in microbial ecology. We here use sequence data from the Global Ocean Sampling (GOS) expedition to investigate toxicant selection pressures revealed by the presence of detoxification genes in marine bacteria. To capture a broad range of potential toxicants we selected detoxification protein families representing systems protecting microorganisms from a variety of stressors, such as metals, organic compounds, antibiotics and oxygen radicals.

Results: Using a bioinformatics procedure based on comparative analysis to finished bacterial genomes we found that the amount of detoxification genes present in marine microorganisms seems surprisingly small. The underrepresentation is particularly evident for toxicant transporters and proteins involved in detoxifying metals. Exceptions are enzymes involved in oxidative stress defense where peroxidase enzymes are more abundant in marine bacteria compared to bacteria in general. In contrast, catalases are almost completely absent from the open ocean environment, suggesting that peroxidases and peroxiredoxins constitute a core line of defense against reactive oxygen species (ROS) in the marine milieu.

Conclusions: We found no indication that detoxification systems would be generally more abundant close to the coast compared to the open ocean. On the contrary, for several of the protein families that displayed a significant geographical distribution, like peroxidase, penicillin binding transpeptidase and divalent ion transport protein, the open ocean samples showed the highest abundance. Along the same lines, the abundance of most detoxification proteins did not increase with estimated pollution. The low level of detoxification systems in marine bacteria indicate that the majority of marine bacteria have a low capacity to adapt to increased pollution. Our study exemplifies the use of metagenomics data in ecotoxicology, and in particular how anthropogenic consequences on life in the sea can be examined.
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http://dx.doi.org/10.1186/1471-2164-15-749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161860PMC
September 2014

Linking Peroxiredoxin and Vacuolar-ATPase Functions in Calorie Restriction-Mediated Life Span Extension.

Int J Cell Biol 2014 3;2014:913071. Epub 2014 Feb 3.

Department of Chemistry and Molecular Biology, University of Gothenburg, Medicinaregatan 9C, 413 90 Gothenburg, Sweden ; Department of Molecular Biology and Genetics, Izmir Institute of Technology, 35430 Urla, Izmir, Turkey ; Department of Oncology, Institute of Oncology, Dokuz Eylul University, 35340 Inciralti, Izmir, Turkey.

Calorie restriction (CR) is an intervention extending the life spans of many organisms. The mechanisms underlying CR-dependent retardation of aging are still poorly understood. Despite mechanisms involving conserved nutrient signaling pathways proposed, few target processes that can account for CR-mediated longevity have so far been identified. Recently, both peroxiredoxins and vacuolar-ATPases were reported to control CR-mediated retardation of aging downstream of conserved nutrient signaling pathways. In this review, we focus on peroxiredoxin-mediated stress-defence and vacuolar-ATPase regulated acidification and pinpoint common denominators between the two mechanisms proposed for how CR extends life span. Both the activities of peroxiredoxins and vacuolar-ATPases are stimulated upon CR through reduced activities in conserved nutrient signaling pathways and both seem to stimulate cellular resistance to peroxide-stress. However, whereas vacuolar-ATPases have recently been suggested to control both Ras-cAMP-PKA- and TORC1-mediated nutrient signaling, neither the physiological benefits of a proposed role for peroxiredoxins in H2O2-signaling nor downstream targets regulated are known. Both peroxiredoxins and vacuolar-ATPases do, however, impinge on mitochondrial iron-metabolism and further characterization of their impact on iron homeostasis and peroxide-resistance might therefore increase our understanding of the beneficial effects of CR on aging and age-related diseases.
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http://dx.doi.org/10.1155/2014/913071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930189PMC
June 2014

Enhancing protein disaggregation restores proteasome activity in aged cells.

Aging (Albany NY) 2013 Nov;5(11):802-12

Department of Chemistry and Molecular Biology, University of Gothenburg, Medicinaregatan 9C, SE-413 90 Göteborg, Sweden.

The activity of the ubiquitin-proteasome system, UPS, declines during aging in several multicellular organisms. The reason behind this decline remains elusive. Here, using yeast as a model system, we show that while the level and potential capacity of the 26S proteasome is maintained in replicatively aged cells, the UPS is not functioning properlyin vivo. As a consequence cytosolic UPS substrates, such as ΔssCPY* are stabilized, accumulate, and form inclusions. By integrating a pGPD-HSP104 recombinant gene into the genome, we were able to constitutively elevate protein disaggregase activity, which diminished the accumulation of protein inclusions during aging. Remarkably, this elevated disaggregation restored degradation of a 26S proteasome substrate in aged cells without elevating proteasome levels, demonstrating that age-associated aggregation obstructs UPS function. The data supports the existence of a negative feedback loop that accelerates aging by exacerbating proteostatic decline once misfolded and aggregation-prone proteins reach a critical level.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868723PMC
http://dx.doi.org/10.18632/aging.100613DOI Listing
November 2013

The yeast transcription factor Crz1 is activated by light in a Ca2+/calcineurin-dependent and PKA-independent manner.

PLoS One 2013 15;8(1):e53404. Epub 2013 Jan 15.

Department of Applied Physics, Chalmers University of Technology, Göteborg, Sweden.

Light in the visible range can be stressful to non-photosynthetic organisms. The yeast Saccharomyces cerevisiae has earlier been reported to respond to blue light via activation of the stress-regulated transcription factor Msn2p. Environmental changes also induce activation of calcineurin, a Ca(2+)/calmodulin dependent phosphatase, which in turn controls gene transcription by dephosphorylating the transcription factor Crz1p. We investigated the connection between cellular stress caused by blue light and Ca(2+) signalling in yeast by monitoring the nuclear localization dynamics of Crz1p, Msn2p and Msn4p. The three proteins exhibit distinctly different stress responses in relation to light exposure. Msn2p, and to a lesser degree Msn4p, oscillate rapidly between the nucleus and the cytoplasm in an apparently stochastic fashion. Crz1p, in contrast, displays a rapid and permanent nuclear localization induced by illumination, which triggers Crz1p-dependent transcription of its target gene CMK2. Moreover, increased extracellular Ca(2+) levels stimulates the light-induced responses of all three transcription factors, e.g. Crz1p localizes much quicker to the nucleus and a larger fraction of cells exhibits permanent Msn2p nuclear localization at higher Ca(2+) concentration. Studies in mutants lacking Ca(2+) transporters indicate that influx of extracellular Ca(2+) is crucial for the initial stages of light-induced Crz1p nuclear localization, while mobilization of intracellular Ca(2+) stores appears necessary for a sustained response. Importantly, we found that Crz1p nuclear localization is dependent on calcineurin and the carrier protein Nmd5p, while not being affected by increased protein kinase A activity (PKA), which strongly inhibits light-induced nuclear localization of Msn2/4p. We conclude that the two central signalling pathways, cAMP-PKA-Msn2/4 and Ca(2+)-calcineurin-Crz1, are both activated by blue light illumination.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053404PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546054PMC
June 2013

Peroxiredoxins, gerontogenes linking aging to genome instability and cancer.

Genes Dev 2012 Sep;26(18):2001-8

Department of Cell and Molecular Biology, University of Gothenburg, Göteborg, Sweden.

Age is the highest risk factor known for a large number of maladies, including cancers. However, it is unclear how aging mechanistically predisposes the organism to such diseases and which gene products are the primary targets of the aging process. Recent studies suggest that peroxiredoxins, antioxidant enzymes preventing tumor development, are targets of age-related deterioration and that bolstering their activity (e.g., by caloric restriction) extends cellular life span. This review focuses on how the peroxiredoxin functions (i.e., as peroxidases, signal transducers, and molecular chaperones) fit with contemporary theories of aging and whether peroxiredoxins could be targeted therapeutically in the treatment of age-associated cancers.
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http://dx.doi.org/10.1101/gad.200006.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444726PMC
September 2012

Life span extension and H(2)O(2) resistance elicited by caloric restriction require the peroxiredoxin Tsa1 in Saccharomyces cerevisiae.

Mol Cell 2011 Sep;43(5):823-33

CMB, University of Gothenburg, Medicinaregatan 9C, S-413 90 Göteborg, Sweden.

Caloric restriction (CR) extends the life span of organisms ranging from yeast to primates. Here, we show that the thiol-dependent peroxiredoxin Tsa1 and its partner sulfiredoxin, Srx1, are required for CR to extend the replicative life span of yeast cells. Tsa1 becomes hyperoxidized/inactive during aging, and CR mitigates such oxidation by elevating the levels of Srx1, which is required to reduce/reactivate hyperoxidized Tsa1. CR, by lowering cAMP-PKA activity, enhances Gcn2-dependent SRX1 translation, resulting in increased resistance to H(2)O(2) and life span extension. Moreover, an extra copy of the SRX1 gene is sufficient to extend the life span of cells grown in high glucose concentrations by 20% in a Tsa1-dependent and Sir2-independent manner. The data demonstrate that Tsa1 is required to ensure yeast longevity and that CR extends yeast life span, in part, by counteracting age-induced hyperoxidation of this peroxiredoxin.
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http://dx.doi.org/10.1016/j.molcel.2011.07.027DOI Listing
September 2011

Absence of mitochondrial translation control proteins extends life span by activating sirtuin-dependent silencing.

Mol Cell 2011 May;42(3):390-400

Department of Cell and Molecular Biology, University of Gothenburg, Medicinaregatan 9C, 413 90 Göteborg, Sweden.

Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges with a pathway for life span extension elicited by reducing TOR signaling.
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http://dx.doi.org/10.1016/j.molcel.2011.03.021DOI Listing
May 2011

Revealing the genetic structure of a trait by sequencing a population under selection.

Genome Res 2011 Jul 21;21(7):1131-8. Epub 2011 Mar 21.

The Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

One approach to understanding the genetic basis of traits is to study their pattern of inheritance among offspring of phenotypically different parents. Previously, such analysis has been limited by low mapping resolution, high labor costs, and large sample size requirements for detecting modest effects. Here, we present a novel approach to map trait loci using artificial selection. First, we generated populations of 10-100 million haploid and diploid segregants by crossing two budding yeast strains of different heat tolerance for up to 12 generations. We then subjected these large segregant pools to heat stress for up to 12 d, enriching for beneficial alleles. Finally, we sequenced total DNA from the pools before and during selection to measure the changes in parental allele frequency. We mapped 21 intervals with significant changes in genetic background in response to selection, which is several times more than found with traditional linkage methods. Nine of these regions contained two or fewer genes, yielding much higher resolution than previous genomic linkage studies. Multiple members of the RAS/cAMP signaling pathway were implicated, along with genes previously not annotated with heat stress response function. Surprisingly, at most selected loci, allele frequencies stopped changing before the end of the selection experiment, but alleles did not become fixed. Furthermore, we were able to detect the same set of trait loci in a population of diploid individuals with similar power and resolution, and observed primarily additive effects, similar to what is seen for complex trait genetics in other diploid organisms such as humans.
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http://dx.doi.org/10.1101/gr.116731.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129255PMC
July 2011

H2O2 activates the nuclear localization of Msn2 and Maf1 through thioredoxins in Saccharomyces cerevisiae.

Eukaryot Cell 2009 Sep 6;8(9):1429-38. Epub 2009 Jul 6.

CEA, IBITECS, SBIGEM, LBI, Gif-sur-Yvette Cedex, France.

The cellular response to hydrogen peroxide (H(2)O(2)) is characterized by a repression of growth-related processes and an enhanced expression of genes important for cell defense. In budding yeast, this response requires the activation of a set of transcriptional effectors. Some of them, such as the transcriptional activator Yap1, are specific to oxidative stress, and others, such as the transcriptional activators Msn2/4 and the negative regulator Maf1, are activated by a wide spectrum of stress conditions. How these general effectors are activated in response to oxidative stress remains an open question. In this study, we demonstrate that the two cytoplasmic thioredoxins, Trx1 and Trx2, are essential to trigger the nuclear accumulation of Msn2/4 and Maf1, specifically under H(2)O(2) treatment. Contrary to the case with many stress conditions previously described for yeast, the H(2)O(2)-induced nuclear accumulation of Msn2 and Maf1 does not correlate with the downregulation of PKA kinase activity. Nevertheless, we show that PP2A phosphatase activity is essential for driving Maf1 dephosphorylation and its subsequent nuclear accumulation in response to H(2)O(2) treatment. Interestingly, under this condition, the lack of PP2A activity has no impact on the subcellular localization of Msn2, demonstrating that the H(2)O(2) signaling pathways share a common route through the thioredoxin system and then diverge to activate Msn2 and Maf1, the final integrators of these pathways.
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http://dx.doi.org/10.1128/EC.00106-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747830PMC
September 2009

Dihydroxyacetone-induced death is accompanied by advanced glycation endproduct formation in selected proteins of Saccharomyces cerevisiae and Caenorhabditis elegans.

Proteomics 2007 Oct;7(20):3764-74

Department of Cell and Molecular Biology, Göteborg University, Göteborg, Sweden.

Advanced glycation endproduct (AGE) formation is an important mechanism for protein deterioration during diabetic complications and ageing. The effects on AGE formation following dihydroxyacetone (DHA) stress were studied in two model organisms, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Total protein AGEs, detected using an anti-N(epsilon)-carboxyalkyllysine-specific monoclonal antibody, displayed a strong correlation to DHA-induced yeast cell mortality in the wild-type and hypersensitive as well as resistant mutant strains. During DHA-induced cell death we also detected AGEs as the formation of acidic protein modifications by 2-D PAGE. Furthermore, we confirmed AGE targets immunologically on 2-D gel-separated protein extracted from DHA-treated cells. AGE modification of several metabolic enzymes (Eno2p, Adh1p, Met6 and Pgk1p) and actin (Act1p) displayed a strong correlation to DHA-induced cell death. DHA was toxic to C. elegans even at low concentration and also in this organism AGE formation accompanied death. We propose the use of DHA as a model AGE-generating substance for its apparent lack of a clear oxidative stress connection, and yeast and worm as model organisms to identify genetic determinants of protein AGE formation.
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http://dx.doi.org/10.1002/pmic.200700165DOI Listing
October 2007

Ionizing radiation induces a Yap1-dependent peroxide stress response in yeast.

Free Radic Biol Med 2007 Jul 13;43(1):136-44. Epub 2007 Apr 13.

Laboratoire de PhysioGénomique, SBGM/DBJC/DSV, CEA/Saclay, F-91191, Gif-sur-Yvette Cedex, France.

Repair of DNA damage is fundamental for cellular tolerance to ionizing radiation (IR) and many IR-induced DNA lesions are thought to occur as a result of oxidative stress. We investigated the physiological effects of IR in Saccharomyces cerevisiae by performing protein expression profiles in cells exposed to electron pulse irradiation. Transient induction of several antioxidant enzymes in wild-type cells, but not in cells lacking the oxidative stress regulator Yap1, indicated that IR exposure causes cellular oxidative stress. Yap1 activation involved oxidation to the intramolecular disulfide bond, a signature of activation by peroxide, and was dependent on the Yap1 peroxide sensor Orp1/Gpx3. H(2)O(2) was produced in the culture medium of irradiated cells and was both necessary and sufficient for IR-induced Yap1 activation. When IR was performed in the presence of N(2)O, obviating H(2)O(2) production and increasing hydroxyl radical ((*)OH) production, the Yap1 response was lost, indicating that Yap1 was unable to respond to (*)OH or (*)OH-induced damage. However, the Yap1 response to IR did not seem to be a primary determinant of cellular IR tolerance. Altogether, these data provide a molecular demonstration that cells experience in vivo peroxide stress during IR and indicate that the H(2)O(2) produced cannot account for IR toxicity.
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http://dx.doi.org/10.1016/j.freeradbiomed.2007.04.007DOI Listing
July 2007

Dihydroxyacetone detoxification in Saccharomyces cerevisiae involves formaldehyde dissimilation.

Mol Microbiol 2006 May;60(4):925-38

Department of Cell and Molecular Biology, Microbiology, Göteborg University, Lundberg laboratory, Medicinaregatan 9c, S-413 90 Göteborg, Sweden.

To investigate Saccharomyces cerevisiae physiology during growth on the conditionally toxic triose dihydroxyacetone (DHA), protein expression was studied in strains overexpressing either of the two dihydroxyacetone kinase isogenes, DAK1 or DAK2, that grow well utilizing DHA as a carbon and energy source. DHA metabolism was found mostly similar to ethanol utilization, involving a strong component of glucose derepression, but also involved DHA-specific regulatory changes. A specific and strong (10- to 30-fold induction of formaldehyde dehydrogenase, Fdhlp, indicated activation of the formaldehyde dissimilation pathway in DHA medium. The importance of this pathway was further supported by impaired adaptation to DHA growth and DHA survival in a glutathione-dependent formaldehyde dehydrogenase (SFA1) deletion mutant. Glutathione synthase (GSH1) deletion led to decreased DHA survival in agreement with the glutathione cofactor requirement for the SFA1-encoded activity. DHA toxicity did, however, not solely appear related to formaldehyde accumulation, because SFA1 overexpression only enhanced formaldehyde but not DHA tolerance. In further agreement with a low DHA-to-formaldehyde flux, GSH supplements in the low microM range also fully suppressed the DHA sensitivity of a gsh1Delta strain. Under growth reduction on high (100 mM) DHA medium we report increased levels of advanced glycation end-product (AGE) formation on total protein. Under these high-DHA conditions expression of several stress-related proteins, e.g. a heat-shock protein (Hsp104p) and the oxidative stress indicator, alkyl hydroperoxide reductase (Ahp1p) was also found induced. However, hallmark determinants of oxidative stress tolerance (e.g. YAP1, SKN7, HYR1/GPX3 and SOD2) were redundant for DHA tolerance, thus indicating mechanisms of DHA toxicity largely independent of central oxidative stress defence mechanisms. We conclude that mechanisms for DHA growth and detoxification appear complex and that the evolutionary strive to minimize detrimental effects of this intracellular metabolite links to both formaldehyde and glutathione metabolism.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05154.xDOI Listing
May 2006

Fragmentation of dihydroxyacetone kinase 1 from Saccharomyces cerevisiae indicates a two-domain structure.

Proteomics 2003 May;3(5):752-63

Department of Cell and Molecular Biology--Microbiology, Göteborg University, Göteborg, Sweden.

Global protein expression in Saccharomyces cerevisiae strains either deleted for both yeast dihydroxyacetone kinases (DAK1 and DAK2) or overexpressing DAK1, was characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We found protein expression in the double deletion strain to be highly similar to wild-type. In the strain overexpressing Dak1p, nine spots representing fragments of the Dak1p protein in the size range 40-20 kDa and amounting to approximately 30% of total Dak1p, were discovered (native size Dak1p migrates at roughly 60 kDa). Fragments were characterized by matrix-assisted laser desorption/ionization mass spectrometry and electrospray mass spectrometry analyses to represent either the N- or the C-terminal part of the DAK1 protein. Cleavage points, predicted from mass spectrometry and 2-D PAGE data, mapped almost exclusively in the middle region showing low sequence conservation between Dak1p and its closest homologues. We hypothesize that observed Dak1p fragments represent stable structural domains shielded from access by native endoproteases. Furthermore, overexpressing Dak1p with the non-native N-terminus (M)A-, resulted in native size Dak1p and N-terminal Dak1p fragments appearing in two major 2-D PAGE forms of approximately equal size and abundance, but with slightly different isoelectric points. However, when overexpressing Dak1p with the native N-terminus (M)S-, only the more acidic 2-D PAGE form appeared. In the N-terminal acetyltransferase mutant nat1delta, (M)A-Dak1p species were converted into the basic form, arguing twin spots to represent forms with acetylated and deacetylated N-termini. Data thus indicated that (M)A-N-termini, in the Dak1p context, were NatA substrates recognized with 50% lower efficiency than (M)S-N-termini.
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http://dx.doi.org/10.1002/pmic.200300393DOI Listing
May 2003

Dihydroxyacetone kinases in Saccharomyces cerevisiae are involved in detoxification of dihydroxyacetone.

J Biol Chem 2003 Jan 24;278(3):1415-23. Epub 2002 Oct 24.

Department of Cell and Molecular Biology-Microbiology, Göteborg University, Lundberg Laboratory, Medicinaregatan 9c, 413 90 Göteborg, Sweden..

The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms. The kinetic properties of the two isoforms were similar, exhibiting K(m)((DHA)) of 22 and 5 microm and K(m)((ATP)) of 0.5 and 0.1 mm for Dak1p and Dak2p, respectively. We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK. The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1 Delta dak2 Delta), since this strain was highly sensitive to DHA. In the opposite case, overexpression of either DAK1 or DAK2 made the dak1 Delta dak2 Delta highly resistant to DHA. In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h. The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent. However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source. Salt addition strongly aggravated the DHA toxicity, independent of carbon source. Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde. It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species.
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http://dx.doi.org/10.1074/jbc.M203030200DOI Listing
January 2003