A systematic screen identifies Saf5 as a link between splicing and transcription in fission yeast.

Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several integrated fluorescence-based in vivo splicing reporter constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5' splicing site (5'SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter showing more intron retention with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants -Δcwf12, a member of the NineTeen Complex (NTC) and Δsaf5, a methylosome subunit that acts together with the survival motor neuron (SMN) complex in small nuclear ribonucleoproteins (snRNP) biogenesis. We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5'SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5'SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.

Formation of Transient Protein Aggregate-like Centers Is a General Strategy Postponing Degradation of Misfolded Intermediates.

When misfolded intermediates accumulate during heat shock, the protein quality control system promotes cellular adaptation strategies. In Schizosaccharomyces pombe, thermo-sensitive proteins assemble upon stress into protein aggregate-like centers, PACs, to escape from degradation. The role of this protein deposition strategy has been elusive due to the use of different model systems and reporters, and to the addition of artificial inhibitors, which made interpretation of the results difficult. Here, we compare fission and budding yeast model systems, expressing the same misfolding reporters in experiments lacking proteasome or translation inhibitors. We demonstrate that mild heat shock triggers reversible PAC formation, with the collapse of both reporters and chaperones in a process largely mediated by chaperones. This assembly postpones proteasomal degradation of the misfolding reporters, and their Hsp104-dependent disassembly occurs during stress recovery. Severe heat shock induces formation of cytosolic PACs, but also of nuclear structures resembling nucleolar rings, NuRs, presumably to halt nuclear functions. Our study demonstrates that these distantly related yeasts use very similar strategies to adapt and survive to mild and severe heat shock and that aggregate-like formation is a general cellular scheme to postpone protein degradation and facilitate exit from stress.

Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects.

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.

Spatial sequestration of misfolded proteins as an active chaperone-mediated process during heat stress.

Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another. Hence thermo-sensitive proteins that lose their active conformation might be refolded with the aid of chaperones or removed by the ubiquitin-proteasome system or the process of autophagy. We have recently developed thermo-sensitive reporters to study PQC in fission yeast and shown the relevance of a third adaptation strategy: the sequestration of misfolded proteins into inclusions which will prevent a rapid degradation and allow the refolding once stress ends. These protein inclusions, protein aggregate centers (PACs), contain a broad spectrum of misfolding/aggregation-prone proteins and chaperones involved in their assembly or dissolution. The chaperone couple Mas5/Ssa2 plays a crucial role in PAC formation, whereas the Hsp104 chaperone promotes their disassembly. The absence of aggregates observed in cells lacking Mas5 could be also explained by the activation of the transcription factor Hsf1 and the induction of chaperone genes, we have excluded this possibility here demonstrating that increased Hsf1 activity and the subsequent overexpression of chaperones do not prevent the assembly of protein aggregates. Protein deposition at certain locations also constitutes a tactic to inactivate proteins temporally. This is the case of Pyp1, the main phosphatase of the stress response kinase Sty1. Upon stress imposition, misfolded Pyp1 is sequestered into cytosolic protein foci while active Sty1 at the nucleus switches on the transcriptional response. In conclusion, we propose that the assembly of aggregation-like foci, PACs in fission yeast, is a crucial PQC strategy during heat stress, and that the Hsp40 chaperone Mas5 is required for PAC assembly and connects physiological and heat-shock triggered PQC.

Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase.

The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR). RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 protein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the supply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regulating transcription of S phase genes and constitutive RNR synthesis. Regarding the thioredoxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth.

A genetic approach to study H2O2 scavenging in fission yeast--distinct roles of peroxiredoxin and catalase.

The main peroxiredoxin in Schizosaccharomyces pombe, Tpx1, is important to sustain aerobic growth, and cells lacking this protein are only able to grow on solid plates under anaerobic conditions. We have found that deletion of the gene coding for thioredoxin reductase, trr1, is a suppressor of the sensitivity to aerobic growth of Δtpx1 cells, so that cells lacking both proteins are able to grow on solid plates in the presence of oxygen. We have investigated this suppression effect, and determined that it depends on the presence of catalase, which is constitutively expressed in Δtrr1 cells in a transcription factor Pap1-dependent manner. A complete characterization of the repertoire of hydrogen peroxide scavenging activities in fission yeast suggests that Tpx1 is the only enzyme with sufficient sensitivity for peroxides and cellular abundance as to control the low levels produced during aerobic growth, catalase being the next barrier of detoxification when the steady-state levels of peroxides are increased in Δtpx1 cells. Gpx1, the only glutathione peroxidase encoded by the S. pombe genome, only has a minor secondary role when extracellular peroxides are added. Our study proposes non-overlapping roles for the different hydrogen peroxide scavenging activities of this eukaryotic organism.

Reversible cysteine oxidation in hydrogen peroxide sensing and signal transduction.

Activation of redox cascades through hydrogen peroxide-mediated reversible cysteine oxidation is a major mechanism for intracellular signaling. Understanding why some cysteine residues are specifically oxidized, in competition with other proximal cysteine residues and in the presence of strong redox buffers, is therefore crucial for understanding redox signaling. In this review, we explore the recent advances in thiol-redox chemistry linked to signaling. We describe the last findings in the field of redox sensors, those that are naturally present in different model organisms as well as those that have been engineered to quantify intracellular hydrogen peroxide concentrations. Finally, we provide a summary of the newest approaches developed to study reversible cysteine oxidation at the proteomic level.

Methionine sulphoxide reductases revisited: free methionine as a primary target of H2O2stress in auxotrophic fission yeast.

Amino acid methionine can suffer reversible oxidation to sulphoxide and further irreversible over-oxidation to methionine sulphone. As part of the cellular antioxidant scavenging activities are the methionine sulphoxide reductases (Msrs), with a reported role in methionine sulphoxide reduction, both free and in proteins. Three families of Msrs have been described, but the fission yeast genome only includes one representative for two of these families: MsrA/Mxr1 and MsrB/Mxr2. We have investigated their role in methionine reduction and H2 O2 sensitivity. We show here that MsrA/Mxr1 is able to reduce free oxidized methionine. Cells lacking each one of the genes are not significantly sensitive to different types of oxidative stresses, neither display altered life span. However, only when deletion of msrA/mxr1 is combined with deletion of met6, which confers methionine auxotrophy, the survival upon H2 O2 stress decreases by 100-fold. In fact, cells lacking only Met6, and which therefore require addition of methionine to the growth media, are extremely sensitive to H2 O2 stress. These and other evidences suggest that oxidation of free methionine is a primary target of peroxide toxicity in cells devoid of methionine biosynthetic capacity, and that an important role of Msrs is to recycle this oxidized free amino acid.

Mitotic Cdc6 stabilizes anaphase-promoting complex substrates by a partially Cdc28-independent mechanism, and this stabilization is suppressed by deletion of Cdc55.

Ectopic expression of Cdc6p results in mitotic delay, and this has been attributed to Cdc6p-mediated inhibition of Cdc28 protein kinase and failure to activate the anaphase-promoting complex (APC). Here we show that endogenous Cdc6p delays a specific subset of mitotic events and that Cdc28 inhibition is not sufficient to account for it. The depletion of Cdc6p in G(2)/M cells reveals that Cdc6p is rate limiting for the degradation of the APC/Cdc20 substrates Pds1p and Clb2p. Conversely, the premature expression of Cdc6p delays the degradation of APC/Cdc20 substrates. Abolishing Cdc6p/Cdc28p interaction does not eliminate the Cdc6-dependent delay of these anaphase events. To identify additional Cdc6-mediated, APC-inhibitory mechanisms, we looked for mutants that reversed the mitotic delay. The deletion of SWE1, RAD24, MAD2, or BUB2 had no effect. However, disrupting CDC55, a PP2A regulatory subunit, suppressed the Cdc6p-dependent delay of Pds1 and Clb2 destruction. A specific role for CDC55 was supported by demonstrating that the lethality of Cdc6 ectopic expression in a cdc16-264 mutant is suppressed by the deletion of CDC55, that endogenous Cdc6p coimmunoprecipitates with the Cdc55 and Tpd3 subunits of PP2A, that Cdc6p/Cdc55p/Tpd3 interaction occurs only during mitosis, and that Cdc6 affects PP2A-Cdc55 activity during anaphase. This demonstrates that the levels and timing of accumulation of Cdc6p in mitosis are appropriate for mediating the modulation of APC/Cdc20.

Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress.

Cells have developed protein quality-control strategies to manage the accumulation of misfolded substrates during heat stress. Using a soluble reporter of misfolding in fission yeast, Rho1.C17R-GFP, we demonstrate that upon mild heat shock, the reporter collapses in protein aggregate centers (PACs). They contain and/or require several chaperones, such as Hsp104, Hsp16, and the Hsp40/70 couple Mas5/Ssa2. Stress granules do not assemble at mild temperatures and, therefore, are not required for PAC formation; on the contrary, PACs may serve as nucleation centers for the assembly of stress granules. In contrast to the general belief, the dominant fate of these PACs is not degradation, and the aggregated reporter can be disassembled by chaperones and recovers native structure and activity. Using mass spectrometry, we show that thermo-unstable endogenous proteins form PACs as well. In conclusion, formation of PACs during heat shock is a chaperone-mediated adaptation strategy.

Identification of ubiquitin-proteasome system components affecting the degradation of the transcription factor Pap1.

Signaling cascades respond to specific inputs, but also require active interventions to be maintained in their basal/inactive levels in the absence of the activating signal(s). In a screen to search for protein quality control components required for wild-type tolerance to oxidative stress in fission yeast, we have isolated eight gene deletions conferring resistance not only to H2O2 but also to caffeine. We show that dual resistance acquisition is totally or partially dependent on the transcription factor Pap1. Some gene products, such as the ribosomal-ubiquitin fusion protein Ubi1, the E2 conjugating enzyme Ubc2 or the E3 ligase Ubr1, participate in basal ubiquitin labeling of Pap1, and others, such as Rpt4, are non-essential constituents of the proteasome. We demonstrate here that basal nucleo-cytoplasmic shuttling of Pap1, occurring even in the absence of stress, is sufficient for the interaction of the transcription factor with nuclear Ubr1, and we identify a 30 amino acids peptide in Pap1 as the degron for this important E3 ligase. The isolated gene deletions increase only moderately the concentration of the transcription factor, but it is sufficient to enhance basal tolerance to stress, probably by disturbing the inactive stage of this signaling cascade.

The Hsp40 Mas5 Connects Protein Quality Control and the General Stress Response through the Thermo-sensitive Pyp1.

Upon heat shock, the fission yeast Hsp40 chaperone Mas5 drives temperature-sensitive proteins toward protein aggregate centers (PACs) to avoid their degradation until lower temperatures favor their refolding. We show here that cells lacking Mas5 are resistant to oxidative stress. Components of the general stress pathways, the MAP kinase Sty1 and the transcription factor Atf1, are suppressors of this phenotype. Strain Δmas5 expresses higher levels of Sty1- and Atf1-dependent stress genes than wild-type cells. Pyp1, the main tyrosine phosphatase maintaining Sty1 inactive in the absence of stress, is a temperature-sensitive protein that aggregates upon temperature up-shifts in a Mas5-dependent manner. In strain Δmas5, Pyp1 is sent to proteasomal degradation even in the absence of stress. We propose that Pyp1 is a thermo-sensitive phosphatase, which during heat stress coalescences into PACs in a Mas5-dependent manner, to promote full activation of the anti-stress Sty1-Atf1 cascade.

The Emerging Roles of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 in Skeletal Muscle Redox Signaling and Metabolism.

Significance: Skeletal muscle is a crucial tissue to whole-body locomotion and metabolic health. Reactive oxygen species (ROS) have emerged as intracellular messengers participating in both physiological and pathological adaptations in skeletal muscle. A complex interplay between ROS-producing enzymes and antioxidant networks exists in different subcellular compartments of mature skeletal muscle. Recent evidence suggests that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are a major source of contraction- and insulin-stimulated oxidants production, but they may paradoxically also contribute to muscle insulin resistance and atrophy. Recent Advances: Pharmacological and molecular biological tools, including redox-sensitive probes and transgenic mouse models, have generated novel insights into compartmentalized redox signaling and suggested that NOX2 contributes to redox control of skeletal muscle metabolism. Critical Issues: Major outstanding questions in skeletal muscle include where NOX2 activation occurs under different conditions in health and disease, how NOX2 activation is regulated, how superoxide/hydrogen peroxide generated by NOX2 reaches the cytosol, what the signaling mediators are downstream of NOX2, and the role of NOX2 for different physiological and pathophysiological processes. Future Directions: Future research should utilize and expand the current redox-signaling toolbox to clarify the NOX2-dependent mechanisms in skeletal muscle and determine whether the proposed functions of NOX2 in cells and animal models are conserved into humans.

Monitoring cytosolic H2O2 fluctuations arising from altered plasma membrane gradients or from mitochondrial activity.

Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations.

Modulation of aryl hydrocarbon receptor transactivation by carbaryl, a nonconventional ligand.

Carbaryl (1-naphthyl-N-methylcarbamate), a widely used carbamate insecticide, induces cytochrome P450 1A gene expression in mammalian cells. This activity is usually mediated by the interaction of the compound with the aryl hydrocarbon receptor. However, it has been proposed that this mechanism does not apply to carbaryl because its structure differs from that of typical aryl hydrocarbon receptor ligands. We show here that carbaryl promotes activation of target genes in a yeast-based bioassay expressing both aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator. By contrast, carbaryl acted as a competitive inhibitor, rather than as an agonist, in a simplified yeast system, in which aryl hydrocarbon receptor nuclear translocator function is bypassed by fusing aryl hydrocarbon receptor to a heterologous DNA binding domain. This dual action of carbaryl, agonist and partial antagonist, was also observed by comparing carbaryl response in two vertebrate cell lines. A yeast two-hybrid assay showed that the mammalian coactivator cAMP response element-binding protein readily interacts with aryl hydrocarbon receptor bound to its canonical ligand beta-naphthoflavone, but not with the carbaryl-aryl hydrocarbon receptor complex. We propose that carbaryl interacts with aryl hydrocarbon receptor, but that its peculiar structure imposes a substandard configuration on the aryl hydrocarbon receptor ligand-binding domain that prevents interaction with key coactivators and activates transcription without the need for aryl hydrocarbon receptor nuclear translocator. This effect may be relevant in explaining its physiological effects in exposed animals, and may help to predict its effects, and that of similar compounds, in humans. Our data also identify the aryl hydrocarbon receptor/cAMP response element-binding protein interaction as a molecular target for the identification and development of new aryl hydrocarbon receptor antagonists.

Proteomic Characterization of Reversible Thiol Oxidations in Proteomes and Proteins.

SIGNIFICANCE: Reactive oxygen species are produced during normal metabolism in cells, and their excesses have been implicated in protein damage and toxicity, as well as in the activation of signaling events. In particular, hydrogen peroxide participates in the regulation of different physiological processes as well as in the induction of antioxidant cascades, and often the redox molecular events triggering these pathways are based on reversible cysteine (Cys) oxidation. Recent Advances: Increases in peroxides can cause the accumulation of reversible Cys oxidations in proteomes, which may be either protecting thiols from irreversible oxidations or may just be reporters of future toxicity. It is also becoming clear, however, that only a few proteins, such as the bacterial OxyR or peroxidases, can suffer direct oxidation of their Cys residues by hydrogen peroxide and, therefore, may be the only true sensors initiating signaling events. CRITICAL ISSUES: We will in this study describe some of the methodologies used to characterize at the proteome level reversible thiol oxidations, specifically those combining gel-free approaches with mass spectrometry. In the second part of this review, we will summarize some of the electrophoretic and proteomic techniques used to monitor Cys oxidation at the protein level, needed to confirm that a protein contains redox Cys involved in signaling relays, using as examples some of the best characterized redox sensors such as bacterial OxyR or yeast Tpx1/Pap1. FUTURE DIRECTIONS: While Cys oxidations are often detected in proteomes and in specific proteins, major efforts have to be made to establish that they are physiologically relevant. Antioxid. Redox Signal. 26, 329-344.

A Transcript-Specific eIF3 Complex Mediates Global Translational Control of Energy Metabolism.

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer.

Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry.

Reversible thiol oxidation of cysteine residues occurs in many intracellular catalytic and signaling processes. Here we describe an optimized protocol, which can be completed in ∼5 d, to unambiguously identify specific cysteine residues that are transiently and reversibly oxidized by comparing two complex biological samples obtained from yeast cell cultures at the proteome level. After 'freezing' the in vivo thiol stage of cysteine residues by medium acidification, we first block reduced thiols in extracts with iodoacetamide (IAM), and then we sequentially reduce and label reversible oxidized thiols with the biotin-based heavy or light IAM derivatives, which are known as isotope-coded affinity tag (ICAT) reagents, so that the two samples can be compared at once after combination of the labeled extracts, trypsin digestion, streptavidin-affinity purification of peptides containing oxidized cysteines, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For the same protein extracts, before cysteine-containing peptide enrichment, individual relative protein concentrations are obtained by stable-isotope dimethyl labeling.

Thiol-based H2O2 signalling in microbial systems.

Cysteine residues, and in particular their thiolate groups, react not only with reactive oxygen species but also with electrophiles and with reactive nitrogen species. Thus, cysteine oxidation has often been linked to the toxic effects of some of these reactive molecules. However, thiol-based switches are common in protein sensors of antioxidant cascades, in both prokaryotic and eukaryotic organisms. We will describe here three redox sensors, the transcription factors OxyR, Yap1 and Pap1, which respond by disulfide bond formation to hydrogen peroxide stress, focusing specially on the differences among the three peroxide-sensing mechanisms.

Dissection of a redox relay: H2O2-dependent activation of the transcription factor Pap1 through the peroxidatic Tpx1-thioredoxin cycle.

In fission yeast, the transcription factor Pap1 undergoes H2O2-dependent oxidation that promotes its nuclear accumulation and the activation of an antioxidant gene program. However, the mechanisms that regulate the sensitivity and selectivity of Pap1 activation by peroxides are not fully understood. Here, we demonstrate that the peroxiredoxin Tpx1, the sensor of this signaling cascade, activates the otherwise unresponsive Pap1 protein once the main cytosolic reduced thioredoxin, Trx1, becomes transiently depleted. In other words, Pap1 works as an alternative electron donor for oxidized Tpx1. We have trapped the very transient Tpx1-Pap1 intermediate in cells depleted in Trx1, as we show here using mass spectrometry. Recycling of Tpx1 by Trx1 is required for the efficient signaling to Pap1, suggesting that the complete cycle of H2O2 scavenging by Tpx1 and further recycling of oxidized Tpx1 by Trx1 is required for full downstream activation of the redox cascade.