Search

Summary An old and controversial question in biology is whether information perceived by the nervous system of an animal can "cross the Weismann barrier" to alter the phenotypes and fitness of their progeny. Here, we show that such intergenerational transmission of sensory information occurs in the model organism, C. elegans, with a major effect on fitness. Specifically, that perception of social pheromones by chemosensory neurons controls the post-embryonic timing of the development of one tissue, the germline, relative to others in the progeny of an animal. Neuronal perception of the social environment thus intergenerationally controls the generation time of this animal.

Funder: Ubbo Emmius fonds, Funder: Boehringer Ingelheim Fonds (BIF); Id: http://dx.doi.org/10.13039/501100001645, Funder: Cornelis de Cock, Funder: FP7 People Marie‐Curie Actions (PEOPLE), Funder: BCN Brain RUG, Funder: KU Leuven, Post‐doctoral Mandate PDM/20/150 and the Industrial Research Fund; Id: http://dx.doi.org/10.13039/501100004040, While aggregation‐prone proteins are known to accelerate aging and cause age‐related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG‐4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid‐promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG‐4 to neutralize charge. Our data indicate that MOAG‐4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation‐prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age‐related protein toxicity.

Background After embryonic development, Caenorhabditis elegans progress through for larval stages, each of them finishing with molting. The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homologue. Nevertheless, each larval stage has a defined duration and entails specific events. Since the overall duration of development is controlled by numerous factors, we have asked whether different rate-limiting interventions impact all stages equally. Results We have measured the duration of each stage of development for over 2500 larvae, under varied environmental conditions known to alter overall developmental rate. We applied changes in temperature and in the quantity and quality of nutrition and analysed the effect of genetically reduced insulin signalling. Our results show that the distinct developmental stages respond differently to these perturbations. The changes in the duration of specific larval stages seem to depend on stage-specific events. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. Conclusions Altogether, our results show that multiple factors fine tune developmental timing, impacting larval stages independently. Further understanding of the regulation of this process will allow modelling the mechanisms that control developmental timing.

SUMMARYThe repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homolog. Nevertheless, each larval stage has a defined duration and entails specific events. We have measured the duration of each stage of development for over 2,500 larvae, under varied environmental conditions known to alter overall developmental rate. Our results show that distinct developmental stages respond differently to environmental perturbations, including changes in temperature, in food quantity and quality, and amount of insulin signaling. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. Altogether, our results support a model of linear progression of C. elegans development, with the duration of each stage determined by a unique program.

Developmental programs are under strict genetic control that favors robustness of the process. In order to guarantee the same outcome in different environmental situations, development is modulated by input pathways, which inform about external conditions. In the nematode Caenorhabditis elegans, the process of postembryonic development involves a series of stereotypic cell divisions, the progression of which is controlled by the nutritional status of the animal. C. elegans can arrest development at different larval stages, leading to cell arrest of the relevant divisions of the stage. This means that studying the nutritional control of development in C. elegans we can learn about the mechanisms controlling cell division in an in vivo model. In this work, we reviewed the current knowledge about the nutrient sensing pathways that control the progression or arrest of development in response to nutrient availability, with a special focus on the arrest at the L1 stage.

Developmental programs are under strict genetic control that favors robustness of the process. In order to guarantee the same outcome in different environmental situations, development is modulated by input pathways, which inform about external conditions. In the nematode Caenorhabditis elegans, the process of postembryonic development involves a series of stereotypic cell divisions, the progression of which is controlled by the nutritional status of the animal. C. elegans can arrest development at different larval stages, leading to cell arrest of the relevant divisions of the stage. This means that studying the nutritional control of development in C. elegans we can learn about the mechanisms controlling cell division in an in vivo model. In this work, we reviewed the current knowledge about the nutrient sensing pathways that control the progression or arrest of development in response to nutrient availability, with a special focus on the arrest at the L1 stage.

An old and controversial question in biology is whether information perceived by the nervous system of an animal can ‘cross the Weismann barrier’ to alter the phenotypes and fitness of their progeny. Here we show that such intergenerational transmission of sensory information occurs in the model organism, C. elegans, with a major effect on fitness. Specifically, that perception of social pheromones by chemosensory neurons controls the post-embryonic timing of development of one tissue – the germline – relative to others in an animal’s progeny. Neuronal perception of the social environment thus intergenerationally controls the generation time of this animal.

While aggregation-prone proteins are known to accelerate ageing and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A and SERF2 have recently been identified as cellular modifiers of such cytotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with specific patterns of negatively charged and hydrophobic, aromatic amino acids. The absence of such patterns, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In a protein aggregation model in the nematodeC. elegans, protein aggregation was suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that charge interactions are required for MOAG-4 and SERF2 to promote aggregation. Such charged interactions might accelerate the primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our finding that negatively charged segments are overrepresented in amyloid-forming proteins suggests that inhibition of charge interactions deserves exploration as a strategy to target age-related protein toxicity.Significance StatementHow aging causes relatively common diseases such as Alzheimer’s and Parkinson’s is still a mystery. Since toxic structural changes in proteins are likely to be responsible, we investigated biological mechanisms that could drive such changes. We made use of a modifying factor called SERF2, which accelerates structural changes and aggregation of several disease-related proteins. Through a peptide-binding screen, we found that SERF2 acts on negatively charged protein regions. The abundance of such regions in the disease-related proteins explains why SERF has its effect. Removing positive charge in SERF was sufficient to suppress protein aggregation in models for disease. We propose that blocking charge-interactions with SERF or other modifiers could serve as a general approach to treat age-related protein toxicity.

An old and controversial question in biology is whether information perceived by the nervous system of an animal can ‘cross the Weismann barrier’ to alter the phenotypes and fitness of their progeny. Here we show that such intergenerational transmission of sensory information occurs in the model organism,C. elegans, with a major effect on fitness. Specifically, that perception of social pheromones by chemosensory neurons controls the post-embryonic timing of development of one tissue – the germline – relative to others in an animal’s progeny. Neuronal perception of the social environment thus intergenerationally controls the generation time of this animal.

© 2020 Mata-Cabana, Gómez-Delgado, Romero-Expósito, Rodríguez-Palero, Artal-Sanz and Olmedo., In a population, chemical communication determines the response of animals to changing environmental conditions, what leads to an enhanced resistance against stressors. In response to starvation, the nematode Caenorhabditis elegans arrest post-embryonic development at the first larval stage (L1) right after hatching. As arrested L1 larvae, C. elegans become more resistant to diverse stresses, allowing them to survive for several weeks expecting to encounter more favorable conditions. L1 arrested at high densities display an enhanced resistance to starvation, dependent on soluble compounds released beyond hatching and the first day of arrest. Here, we show that this chemical communication also influences recovery after prolonged periods in L1 arrest. Animals at high density recovered faster than animals at low density. We found that the density effect on survival depends on the final effector of the insulin signaling pathway, the transcription factor DAF-16. Moreover, DAF-16 activation was higher at high density, consistent with a lower expression of the insulin-like peptide DAF-28 in the neurons. The improved recovery of animals after arrest at high density depended on soluble compounds present in the media of arrested L1s. In an effort to find the nature of these compounds, we investigated the disaccharide trehalose as putative signaling molecule, since its production is enhanced during L1 arrest and it is able to activate DAF-16. We detected the presence of trehalose in the medium of arrested L1 larvae at a low concentration. The addition of this concentration of trehalose to animals arrested at low density was enough to rescue DAF-28 production and DAF-16 activation to the levels of animals arrested at high density. However, despite activating DAF-16, trehalose was not capable of reversing survival and recovery phenotypes, suggesting the participation of additional signaling molecules. With all, here we describe a molecular mechanism underlying social communication that allows C. elegans to maintain arrested L1 larvae ready to quickly recover as soon as they encounter nutrient sources., MO was supported by the Ramón y Cajal program of the Spanish Ministerio de Economía y Competitividad, (RYC-2014-15551). Our work was supported by the Agencia Estatal de Investigación (AEI) y al Fondo Europeo de Desarrollo Regional (FEDER) (BFU2016-74949-P, AEI/FEDER, UE), the European Research Council (ERC-2011-StG-281691) and a Marie-Curie Intra-European Fellowship (FP7-PEOPLE-2013-627263).

Summary Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer’s and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases., Graphical Abstract, Highlights • Inactivation of MOAG-2/LIR-3 reduces polyglutamine aggregation • MOAG-2/LIR-3 regulates Pol III-mediated transcription of small non-coding RNAs • Polyglutamine mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol • Polyglutamine converts MOAG-2/LIR-3 into an aggregation-promoting factor, The cellular mechanisms that drive polyglutamine aggregation are poorly understood. Sin et al. show that polyglutamine relocates MOAG-2/LIR-3 from the nucleus to the cytosol, thereby converting this protein into an aggregation-promoting factor to drive protein aggregation and toxicity.

SummaryCells can enter quiescence in adverse conditions and resume proliferation when the environment becomes favourable. Prolonged quiescence comes with a cost, reducing proliferation potential and survival. Interestingly, cellular quiescence also occurs in normal development, with many cells spending most of their lifetime at this state. Elucidating the mechanisms involved in surviving long-term quiescence and in maintenance of cellular proliferation potential will contribute to a better understanding of the process of tissue regeneration. Developmental arrest ofC. elegansat the L1 stage is an emerging model for the study of cellular quiescence and reactivation. During arrest, L1 larvae undergo a process that shares phenotypic hallmarks with the ageing of the adult. Interestingly, insulin signalling, a prominent pathway in the regulation of ageing, also balances cell proliferation and activation of stress resistance pathways during quiescence, becoming a candidate regulator of proliferation potential. Here we report that prolonged L1 quiescence delays reactivation of blast cell divisions inC. elegans, leading to a delay in the initiation of postembryonic development. This delay is accompanied by increased inter-individual variability. We propose that the delay in cell division results from the decline that animals suffer during L1 arrest. To that end, we show that insulin signalling modulates the rate of L1 ageing, affecting proliferative potential after quiescence. These findings support that the insulin signalling pathway has a comparable role in L1 arrest to that in ageing adults. Furthermore, we show that variable yolk provisioning to the embryos as a consequence of maternal age is one of the sources of inter-individual variability in recovery after quiescence of genetically identical animals. Taken together, these results support the relevance of L1 arrest as a model to studyin vivoproliferation after quiescence and to understand the mechanisms for maintenance of proliferation potential.

C. elegans is widely used to investigate biological processes related to health and disease. To study protein localization, fluorescently-tagged proteins can be used in vivo or immunohistochemistry can be performed in whole worms. Here, we describe a technique to localize a protein of interest at a subcellular level in C. elegans lysates, which can give insight into the location, function and/or toxicity of proteins National Institutes of Health National Centre for Research Resources (NIH) European Research Council (ERC) USANIH National Center for Research Resources (NCRR) Japan National BioResource Project

As the population is aging, the incidence of age-related neurodegenerative diseases, such as Alzheimer and Parkinson disease, is growing. The pathology of neurodegenerative diseases is characterized by the presence of protein aggregates of disease specific proteins in the brain of patients. Under certain conditions these disease proteins can undergo structural rearrangements resulting in misfolded proteins that can lead to the formation of aggregates with a fibrillar amyloid-like structure. Cells have different mechanisms to deal with this protein aggregation, where the molecular chaperone machinery constitutes the first line of defense against misfolded proteins. Proteins that cannot be refolded are subjected to degradation and compartmentalization processes. Amyloid formation has traditionally been described as responsible for the proteotoxicity associated with different neurodegenerative disorders. Several mechanisms have been suggested to explain such toxicity, including the sequestration of key proteins and the overload of the protein quality control system. Here, we review different aspects of the involvement of amyloid-forming proteins in disease, mechanisms of toxicity, structural features, and biological functions of amyloids, as well as the cellular mechanisms that modulate and regulate protein aggregation, including the presence of enhancers and suppressors of aggregation, and how aging impacts the functioning of these mechanisms, with special attention to the molecular chaperones.

Aberrant protein aggregation underlies a variety of age-related neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Little is known, however, about the molecular mechanisms that modulate the aggregation process in the cellular environment. Recently, MOAG-4/SERF has been identified as a class of evolutionarily conserved proteins that positively regulates aggregate formation. Here, by using nuclear magnetic resonance (NMR) spectroscopy, we examine the mechanism of action of MOAG-4 by characterizing its interaction with α-synuclein (α-Syn). NMR chemical shift perturbations demonstrate that a positively charged segment of MOAG-4 forms a transiently populated α-helix that interacts with the negatively charged C terminus of α-Syn. This process interferes with the intramolecular interactions between the N- and C-terminal regions of α-Syn, resulting in the protein populating less compact forms and aggregating more readily. These results provide a compelling example of the complex competition between molecular and cellular factors that protect against protein aggregation and those that promote it.

Redox regulation based on disulfide-dithiol conversion catalyzed by thioredoxins is an important component of chloroplast function. The reducing power is provided by ferredoxin reduced by the photosynthetic electron transport chain. In addition, chloroplasts are equipped with a peculiar NADPH-dependent thioredoxin reductase, termed NTRC, with a joint thioredoxin domain at the carboxyl terminus. Because NADPH can be produced by the oxidative pentose phosphate pathway during the night, NTRC is important to maintain the chloroplast redox homeostasis under light limitation. NTRC is exclusive for photosynthetic organisms such as plants, algae, and some, but not all, cyanobacteria. Phylogenetic analysis suggests that chloroplast NTRC originated from an ancestral cyanobacterial enzyme. While the biochemical properties of plant NTRC are well documented, little is known about the cyanobacterial enzyme. With the aim of comparing cyanobacterial and plant NTRCs, we have expressed the full-length enzyme from the cyanobacterium Anabaena species PCC 7120 as well as site-directed mutant variants and truncated polypeptides containing the NTR or the thioredoxin domains of the protein. Immunological and kinetic analysis showed a high similarity between NTRCs from plants and cyanobacteria. Both enzymes efficiently reduced 2-Cys peroxiredoxins from plants and from Anabaena but not from the cyanobacterium Synechocystis. Arabidopsis (Arabidopsis thaliana) NTRC knockout plants were transformed with the Anabaena NTRC gene. Despite a lower content of NTRC than in wild-type plants, the transgenic plants showed significant recovery of growth and pigmentation. Therefore, the Anabaena enzyme fulfills functions of the plant enzyme in vivo, further emphasizing the similarity between cyanobacterial and plant NTRCs. © 2011 American Society of Plant Biologists., This work was supported by the Ministerio de Ciencia e Innovacio´n, Spain (grant nos. BIO2010–15430 and BFU2007–60300) and the Junta de Andalucı´a, Spain (grant nos. P06–CVI–01578, BIO– 182, and BIO–284).

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.

Cyanobacteria perform oxygenic photosynthesis, which gives rise to the continuous production of reactive oxygen species, such as superoxide anion radicals and hydrogen peroxide, particularly under unfavorable growth conditions. Peroxiredoxins, which are present in both chloroplasts and cyanobacteria, constitute a class of thiol-dependent peroxidases capable of reducing hydrogen peroxide as well as alkyl hydroperoxides. Chloroplast peroxiredoxins have been studied extensively and have been found to use a variety of endogenous electron donors, such as thioredoxins, glutaredoxins, or cyclophilin, to sustain their activities. To date, however, the endogenous reduction systems for cyanobacterial peroxiredoxins have not been systematically studied. We have expressed and purified all five Synechocystis sp. strain PCC 6803 peroxiredoxins, which belong to the classes 1-Cys Prx, 2-Cys Prx, type II Prx (PrxII), and Prx Q, and we have examined their capacities to interact with and receive electrons from the m -, x -, and y -type thioredoxins from the same organism, which are called TrxA, TrxB, and TrxQ, respectively. Assays for peroxidase activity demonstrated that all five enzymes could use thioredoxins as electron donors, whereas glutathione and Synechocystis sp. strain PCC 6803 glutaredoxins were inefficient. The highest catalytic efficiency was obtained for the couple consisting of PrxII and TrxQ thioredoxin. Studies of transcript levels for the peroxiredoxins and thioredoxins under different stress conditions highlighted the similarity between the PrxII and TrxQ thioredoxin expression patterns.

Cyanobacteria perform oxygenic photosynthesis, which makes them unique among the prokaryotes, and this feature together with their abundance and worldwide distribution renders them a central ecological role. Cyanobacteria and chloroplasts of plants and algae are believed to share a common ancestor and the modern chloroplast would thus be the remnant of an endosymbiosis between a eukaryotic cell and an ancestral oxygenic photosynthetic prokaryote. Chloroplast metabolic processes are coordinated with those of the other cellular compartments and are strictly controlled by means of regulatory systems that commonly involve redox reactions. Disulphide/dithiol exchange catalysed by thioredoxin is a fundamental example of such regulation and represents the molecular mechanism for light-dependent redox control of an ever-increasing number of chloroplast enzymatic activities. In contrast to chloroplast thioredoxins, the functions of the cyanobacterial thioredoxins have long remained elusive, despite their common origin. The sequenced genomes of several cyanobacterial species together with novel experimental approaches involving proteomics have provided new tools for re-examining the roles of the thioredoxin systems in these organisms. Thus, each cyanobacterial genome encodes between one and eight thioredoxins and all components necessary for the reduction of thioredoxins. Screening for thioredoxin target proteins in cyanobacteria indicates that assimilation and storage of nutrients, as well as some central metabolic pathways, are regulated by mechanisms involving disulphide/dithiol exchange, which could be catalysed by thioredoxins or related thiol-containing proteins.

Aims: Protein phosphorylation is a principal signaling mechanism that mediates regulation of enzymatic activities, modulation of gene expression, and adaptation to environmental changes. Recent studies have shown a ubiquitous distribution of eukaryotic-type Serine/Threonine protein kinases in prokaryotic genomes, though the functions, substrates, and possible regulation of these enzymes remain largely unknown. In this study, we investigated whether cyanobacterial protein phosphorylation may be subject to redox regulation through modulation of the cysteine redox state, as has previously been reported for animals and plants. We also explored the role of a cyanobacterial Serine/Threonine kinase in oxidative stress tolerance. Results: The Synechocystis sp. PCC 6803 Serine/Threonine kinase SpkB was found to be inhibited by oxidation and reactivated by thioredoxin-catalyzed reduction. A Synechocystis mutant devoid of the SpkB kinase was unable to phosphorylate the glycyl-tRNA synthetase β-subunit (GlyS), one of the most prominent phosphoproteins in the wild type, and recombinant purified SpkB could phosphorylate purified GlyS. In vivo characterization of the SpkB mutant showed a pronounced hypersensitivity to oxidative stress and displayed severe growth retardation or death in response to menadione, methyl viologen, and elevated light intensities. Innovation: This study points out a previously unrecognised complexity of prokaryotic regulatory pathways in adaptation to the environment and extends the roles of bacterial eukaryotic-like Serine/Threonine kinases to oxidative stress response. Conclusion: The SpkB kinase is required for survival of the cyanobacterium Synechocystis sp. PCC 6803 under conditions implying increased concentrations of reactive oxygen species, and the activity of SpkB depends on the redox state of its cysteines. © Copyright 2012, Mary Ann Liebert, Inc., This work was financed by the Spanish Ministry of Science and Innovation (MICINN) through grants no. BFU2007-6300 and BFU2010-15708, co-funded to 70% by FEDER. A.M.-C. was the recipient of an FPU fellowship from the Spanish Ministry of Education and Science (MEC)

Ten years ago, proteomics techniques designed for large-scale investigations of redox-sensitive proteins started to emerge. The proteomes, defined as sets of proteins containing reactive cysteines that undergo oxidative post-translational modifications, have had a particular impact on research concerning the redox regulation of cellular processes. These proteomes, which are hereafter termed "disulfide proteomes," have been studied in nearly all kingdoms of life, including animals, plants, fungi, and bacteria. Disulfide proteomics has been applied to the identification of proteins modified by reactive oxygen and nitrogen species under stress conditions. Other studies involving disulfide proteomics have addressed the functions of thioredoxins and glutaredoxins. Hence, there is a steadily growing number of proteins containing reactive cysteines, which are probable targets for redox regulation. The disulfide proteomes have provided evidence that entire pathways, such as glycolysis, the tricarboxylic acid cycle, and the Calvin-Benson cycle, are controlled by mechanisms involving changes in the cysteine redox state of each enzyme implicated. Synthesis and degradation of proteins are processes highly represented in disulfide proteomes and additional biochemical data have established some mechanisms for their redox regulation. Thus, combined with biochemistry and genetics, disulfide proteomics has a significant potential to contribute to new discoveries on redox regulation and signaling.

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.

The light-dependent regulation of stromal enzymes by thioredoxin-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that thioredoxin targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Thioredoxin-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed thioredoxin-linked redox control in the chloroplast lumen of Arabidopsis thaliana.Using complementary proteomics approaches, we identified 19 thioredoxin target proteins, thus covering more than 40 percent of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox-controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.

Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. In the present work, we have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under nonreducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the 38 newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases.