A redox signalling globin is essential for reproduction in Caenorhabditis elegans.

Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers.

A simplified hydroethidine method for fast and accurate detection of superoxide production in isolated mitochondria.

Because superoxide is involved in various physiological processes, many efforts have been made to improve its accurate quantification. We optimized and validated a superoxide-specific and -sensitive detection method. The protocol is based on fluorescence detection of the superoxide-specific hydroethidine (HE) oxidation product, 2-hydroxyethidium. We established a method for the quantification of superoxide production in isolated mitochondria without the need for acetone extraction and purification chromatography as described in previous studies.

Exploring real-time in vivo redox biology of developing and aging Caenorhabditis elegans.

Reactive oxygen species (ROS) are no longer considered merely toxic by-products of the oxidative metabolism. Tightly controlled concentrations of ROS and fluctuations in redox potential may be important mediators of signaling processes. Understanding the role of ROS and redox status in physiology, stress response, development, and aging requires their nondisruptive, spatiotemporal, real-time quantification in a living organism. We established Caenorhabditis elegans strains bearing the genetically encoded fluorescent biosensors HyPer and Grx1-roGFP2 for the detection of hydrogen peroxide (H(2)O(2)) and the glutathione redox potential, respectively. Although, given its transparency and genetic tractability, C. elegans is perfectly suitable as a model organism for such approaches, they have never been tried before in this nematode. We found that H(2)O(2) treatment clearly induces a dose-dependent, reversible response of both biosensors in the living worms. The ratio of oxidized to reduced glutathione decreases during postembryonic development. H(2)O(2) levels increase with age and this effect is delayed when life span is extended by dietary restriction. In young adults, we detected several regions with distinct redox properties that may be linked to their biological function. Our findings demonstrate that genetically encoded biosensors can reveal previously unknown details of in vivo redox biology in multicellular organisms.

Globins in Caenorhabditis elegans.

Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for globins that are all transcribed. These globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four globins (GLB-1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two globins (GLB-5 and GLB-26), and overlays of the globins were constructed to highlight the structural diversity among them. It is clear that although they all share the globin fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the globin family of C. elegans and suggest potential functions.

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

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

Protein metabolism and lifespan in Caenorhabditis elegans.

Lifespan of the versatile model system Caenorhabditis elegans can be extended by a decrease of insulin/IGF-1 signaling, TOR signaling, mitochondrial function, protein synthesis and dietary intake. The exact molecular mechanisms by which these modulations confer increased life expectancy are yet to be determined but increased stress resistance and improved protein homeostasis seem to be of major importance. In this chapter, we explore the interactions among several genetic pathways and cellular functions involved in lifespan extension and their relation to protein homeostasis in C. elegans. Several of these processes have been associated, however some relevant data are conflicting and further studies are needed to clarify these interactions. In mammals, protein homeostasis is also implicated in several neurodegenerative diseases, many of which can be modeled in C. elegans.

DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO.

Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e(-11)). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16::DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF 'parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.

Disruption of insulin signalling preserves bioenergetic competence of mitochondria in ageing Caenorhabditis elegans.

BACKGROUND: The gene daf-2 encodes the single insulin/insulin growth factor-1-like receptor of Caenorhabditis elegans. The reduction-of-function allele e1370 induces several metabolic alterations and doubles lifespan. RESULTS: We found that the e1370 mutation alters aerobic energy production substantially. In wild-type worms the abundance of key mitochondrial proteins declines with age, accompanied by a dramatic decrease in energy production, although the mitochondrial mass, inferred from the mitochondrial DNA copy number, remains unaltered. In contrast, the age-dependent decrease of both key mitochondrial proteins and bioenergetic competence is considerably attenuated in daf-2(e1370) adult animals. The increase in daf-2(e1370) mitochondrial competence is associated with a higher membrane potential and increased reactive oxygen species production, but with little damage to mitochondrial protein or DNA. Together these results point to a higher energetic efficiency of daf-2(e1370) animals. CONCLUSIONS: We conclude that low daf-2 function alters the overall rate of ageing by a yet unidentified mechanism with an indirect protective effect on mitochondrial function.

Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties.

BACKGROUND: The genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression. RESULTS: We expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 --> Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three alpha-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding. CONCLUSION: The presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.

Effects of sod gene overexpression and deletion mutation on the expression profiles of reporter genes of major detoxification pathways in Caenorhabditis elegans.

Reactive oxygen species have long been considered a major cause of aging. However, previous work showed that loss of superoxide dismutase (SOD) only weakly affects lifespan of Caenorhabditis elegans. Here, we examined the impact of sod gene deletion and overexpression on the mRNA levels of the remaining sod genes and other detoxification genes. We detected no compensatory upregulation of other sod genes in any of the sod deletion mutants in both wild-type and daf-2(m577) genetic backgrounds when L4 larvae were shifted from 17 to 24 degrees C, and harvested as young adults. Elimination of MnSOD increased transcription of SKN-1 regulated genes and reduced transcription of multiple DAF-16 targets. Loss of the major Cu/ZnSOD isoform SOD-1 caused enhanced expression of subsets of both SKN-1 and DAF-16 targets when the animals were grown continuously at 24 degrees C, and strong overexpression of sod-1 induced a compensatory decrease in all tested SKN-1 regulated gst genes. When combined, these results suggest that low cytosolic SOD may activate SKN-1 signaling, whereas high levels may be repressive. Overall, our results suggest that sod gene manipulation causes complex, combinatorial regulation of expression of individual targets of stress sensitive transcription factors.

Intermediary metabolism.

Caenorhabditis elegans has orthologs for most of the key enzymes involved in eukaryotic intermediary metabolism, suggesting that the major metabolic pathways are probably present in this species. We discuss how metabolic patterns and activity change as the worm traverses development and ages, or responds to unfavorable external factors, such as temperature extremes or shortages in food or oxygen. Dauer diapause is marked by an enhanced resistance to oxidative stress and a shift toward microaerobic and anaplerotic metabolic pathways and hypometabolism, as indicated by the increased importance of the malate dismutation and glyoxylate pathways and the repression of citric acid cycle activity. These alterations promote prolonged survival of the dauer larva; some of these changes also accompany the extended lifespan of insulin/IGF-1 and several mitochondrial mutants. We also present a brief overview of the nutritional requirements, energy storage and waste products generated by C. elegans.

Against the oxidative damage theory of aging: superoxide dismutases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans.

The superoxide radical (O(2)(-)) has long been considered a major cause of aging. O(2)(-) in cytosolic, extracellular, and mitochondrial pools is detoxified by dedicated superoxide dismutase (SOD) isoforms. We tested the impact of each SOD isoform in Caenorhabditis elegans by manipulating its five sod genes and saw no major effects on life span. sod genes are not required for daf-2 insulin/IGF-1 receptor mutant longevity. However, loss of the extracellular Cu/ZnSOD sod-4 enhances daf-2 longevity and constitutive diapause, suggesting a signaling role for sod-4. Overall, these findings imply that O(2)(-) is not a major determinant of aging in C. elegans.

The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification.

BACKGROUND: Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. RESULTS: In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. CONCLUSION: Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently dampened further evolution and facilitated fixation of the duplicated genes in the genome.

Prooxidant activity of the superoxide dismutase (SOD)-mimetic EUK-8 in proliferating and growth-arrested Escherichia coli cells.

Numerous studies have aimed to alleviate oxidative stress in a wide range of organisms by increasing superoxide dismutase (SOD) activity. However, experimental approaches have yielded contradictory evidence, and kinetics models have shown that increases in SOD activity may increase, decrease, or not change hydrogen peroxide (H2O2) production, depending on the balance of the various processes that produce and consume superoxide (O2-). In this study we tested whether administration of EUK-8, a synthetic mimetic of the SOD enzyme, can protect starving Escherichia coli cells against stasis-induced oxidative stress. Surprisingly, administration of EUK-8 to starving E. coli cells enhances the production of reactive oxygen species (ROS), resulting in a massive increase of oxidative damage and replicative death of the bacteria. Our results confirm that manipulation of ROS levels by increasing SOD activity does not necessarily result in a consequent decline of oxidative stress and can yield opposite results in a relatively simple model system such as starving E. coli cells.

Molecular phylogeny of the Tylenchina and evolution of the female gonoduct (Nematoda: Rhabditida).

Tylenchina are a morphologically and functionally diverse group of nematode species that range from free-living bacteriovores, over transitory grazing root-hair feeders to highly specialized plant-parasites with complex host associations. We performed phylogenetic analyses of small subunit rDNA sequences from 97 species including an analysis that account for the RNA secondary structure in the models of evolution. The present study confirms the sister relationship of the bacteriovore Cephalobidae with the predominantly plant-parasitic Tylenchomorpha. All analyses appoint the fungal-feeding Aphelenchidae and Aphelenchoididae as being polyphyletic but the morphology based hypothesis of their monophyly could not be significantly rejected. Within the Tylenchomorpha, the families that exclusively parasitize higher plants are joined in a single clade. However, only the monophyletic position of the (super)families Hoplolaimidae and Criconematoidea were supported; Anguinidae, Tylenchidae, Belonolaimidae and Pratylenchidae appeared to be paraphyletic or polyphyletic. Parsimony and likelihood ancestral state reconstruction revealed that burrowing endoparasitism and sedentary endoparasitism each evolved, respectively, at least six and at least three times independently, mostly from migratory ectoparasitic ancestors. Only root-knot nematodes have evolved from burrowing endoparasitic nematodes. Traditional classifications are partially misled by this convergent evolution of feeding type and associated morphology. Contrastingly, mapping attributes of the gonoduct cellular architecture, including newly obtained data of 18 species belonging to the Aphelenchoidea, Criconematoidea, Anguinidae and Panagrolaimidae, revealed a broad congruence of the gonoduct characters and the molecular phylogenetic hypothesis. Yet, the presence of an offset spermatheca and proliferation of uterus cells has evolved multiple times, the latter associated with derived endoparasitic feeding specialization and resulting reproduction mode. Ancestral state reconstruction further revealed that the gonoduct of the morphologically and ecologically dissimilar tylenchid and cephalobid nematodes evolved from a common ancestor.

Dietary restriction of Caenorhabditis elegans by axenic culture reflects nutritional requirement for constituents provided by metabolically active microbes.

In Caenorhabditis elegans, several manipulations that affect nutrition slow development, reduce fecundity, and increase life span. These are viewed as dietary restriction (DR) and include culture in semidefined, nutrient-rich liquid medium that is axenic (i.e., there is no microbial food source). Here we describe convenient ways to exert DR by culture on agar plates containing axenic medium. We used these to explore whether effects of axenic culture really reflect DR. Our results imply that major nutrient components of axenic medium, and overall caloric content, are not limiting for life span. However, adding growth-arrested Escherichia coli as an additional food source rescued the effects of axenic culture. We then sought to identify the component of E. coli that is critical for normal C. elegans nutrition using add-back experiments. Our results suggest that C. elegans has a nutritional requirement for live, metabolically active microbes or, possibly, an unidentified, heat-labile, nonsoluble component present in live microbes.

Dietary restriction by growth in axenic medium induces discrete changes in the transcriptional output of genes involved in energy metabolism in Caenorhabditis elegans.

Dietary restriction increases life span in a wide range of species, including the nematode worm Caenorhabditis elegans. The mechanism by which it does so remains largely unknown, although it is commonly thought that a reduction of reactive oxygen species (ROS) plays a pivotal role. More specifically, for C. elegans, it has been proposed that food restriction reduces energy expenditure, possibly in conjunction with an anaerobic shift in energy production, with consequent reduction in the formation of ROS. We have measured differential transcript abundance of 49 genes known to play roles in energy metabolism in axenic culture medium, which causes a nutritional deficit and leads to a substantial increase of life span. We found no evidence for a reduction in metabolic rate or a shift to anaerobic metabolism in axenic culture. Major changes induced by growth in axenic medium include down-regulation of lipid degradation and up-regulation of glyoxylate cycle activity glyceroneogenesis and, possibly, gluconeogenesis. The activities determined in worm extracts for pyruvate kinase, phosphoenolpyruvate carboxykinase and isocitrate lyase followed a similar trend. We conclude that growth in axenic culture is marked by a general up-regulation of replenishing pathways.

Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans.

BACKGROUND: In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism by which the Ins/IGF-1 signaling pathway regulates these processes. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression levels. The aim of this study was to establish a reliable set of reference genes for gene expression analysis in C. elegans. RESULTS: Real-time quantitative PCR was used to evaluate the expression stability of 12 candidate reference genes (act-1, ama-1, cdc-42, csq-1, eif-3.C, mdh-1, gpd-2, pmp-3, tba-1, Y45F10D.4, rgs-6 and unc-16) in wild-type, three Ins/IGF-1 pathway mutants, dauers and L3 stage larvae. After geNorm analysis, cdc-42, pmp-3 and Y45F10D.4 showed the most stable expression pattern and were used to normalize 5 sod expression levels. Significant differences in mRNA levels were observed for sod-1 and sod-3 in daf-2 relative to wild-type animals, whereas in dauers sod-1, sod-3, sod-4 and sod-5 are differentially expressed relative to third stage larvae. CONCLUSION: Our findings emphasize the importance of accurate normalization using stably expressed reference genes. The methodology used in this study is generally applicable to reliably quantify gene expression levels in the nematode C. elegans using quantitative PCR.

Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family.

BACKGROUND: The emergence of high throughput genome sequencing facilities and powerful high performance bioinformatic tools has highlighted hitherto unexpected wide occurrence of globins in the three kingdoms of life. In silico analysis of the genome of C. elegans identified 33 putative globin genes. It remains a mystery why this tiny animal might need so many globins. As an inroad to understanding this complexity we initiated a structural and functional analysis of the globin family in C. elegans. RESULTS: All 33 C. elegans putative globin genes are transcribed. The translated sequences have the essential signatures of single domain bona fide globins, or they contain a distinct globin domain that is part of a larger protein. All globin domains can be aligned so as to fit the globin fold, but internal interhelical and N- and C-terminal extensions and a variety of amino acid substitutions generate much structural diversity among the globins of C. elegans. Likewise, the encoding genes lack a conserved pattern of intron insertion positioning. We analyze the expression profiles of the globins during the progression of the life cycle, and we find that distinct subsets of globins are induced, or repressed, in wild-type dauers and in daf-2(e1370)/insulin-receptor mutant adults, although these animals share several physiological features including resistance to elevated temperature, oxidative stress and hypoxic death. Several globin genes are upregulated following oxygen deprivation and we find that HIF-1 and DAF-2 each are required for this response. Our data indicate that the DAF-2 regulated transcription factor DAF-16/FOXO positively modulates hif-1 transcription under anoxia but opposes expression of the HIF-1 responsive globin genes itself. In contrast, the canonical globin of C. elegans, ZK637.13, is not responsive to anoxia. Reduced DAF-2 signaling leads to enhanced transcription of this globin and DAF-16 is required for this effect. CONCLUSION: We found that all 33 putative globins are expressed, albeit at low or very low levels, perhaps indicating cell-specific expression. They show wide diversity in gene structure and amino acid sequence, suggesting a long evolutionary history. Ten globins are responsive to oxygen deprivation in an interacting HIF-1 and DAF-16 dependent manner. Globin ZK637.13 is not responsive to oxygen deprivation and regulated by the Ins/IGF pathway only suggesting that this globin may contribute to the life maintenance program.

A model of globin evolution.

Putative globins have been identified in 426 bacterial, 32 Archaeal and 67 eukaryote genomes. Among these sequences are the hitherto unsuspected presence of single domain sensor globins within Bacteria, Fungi, and a Euryarchaeote. Bayesian phylogenetic trees suggest that their occurrence in the latter two groups could be the result of lateral gene transfer from Bacteria. Iterated psiblast searches based on groups of globin sequences indicate that bacterial flavohemoglobins are closer to metazoan globins than to the other two lineages, the 2-over-2 globins and the globin-coupled sensors. Since Bacteria is the only kingdom to have all the subgroups of the three globin lineages, we propose a working model of globin evolution based on the assumption that all three lineages originated and evolved only in Bacteria. Although the 2-over-2 globins and the globin-coupled sensors recognize flavohemoglobins, there is little recognition between them. Thus, in the first stage of globin evolution, we favor a flavohemoglobin-like single domain protein as the ancestral globin. The next stage comprised the splitting off to single domain 2-over-2 and sensor-like globins, followed by the covalent addition of C-terminal domains resulting in the chimeric flavohemoglobins and globin-coupled sensors. The last stage encompassed the lateral gene transfers of some members of the three globin lineages to specific groups of Archaea and Eukaryotes.