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Reactive Oxygen Species (ROS) in the form of H2O2can act both as physiological signaling molecules as well as damaging agents, depending on its concentration and localization. The downstream biological effects of H2O2were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2production by for instance mitochondrial respiration. The enzyme D-Amino Acid Oxidase (DAAO) catalyzes H2O2formation using D-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H2O2. However, a method to directly quantify the amount of H2O2produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2production. The oxygen consumption rate of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, allowing to estimate whether the ensuing level of H2O2production is within the range of physiological mitochondrial ROS production. We show that the assay can also be used to select clones that express differently localized DAAO with the same absolute level of H2O2production to be able to discriminate the effects of H2O2production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.

Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of genomic DNA damage and subsequent mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondrial can directly damage nuclear DNA under (patho)physiological conditions has been largely lacking. In this study we modeled the effects of mitochondrial H2O2release and compared this to H2O2production at the nucleosomes in an untransformed human cell line. We used a chemogenetic approach to produce localized H2O2and combined it with a new method we developed to directly quantify the amount of H2O2produced. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2generation. Nuclear H2O2production gives rise to DNA strand breaks, subsequent activation of the DNA damage response, cell cycle arrest and eventually senescence. Release of H2O2from mitochondria on the other hand shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. Artificially high levels of mitochondrial H2O2release do result in DNA strand breaks, but in parallel invariably cause ferroptosis-mediated cell death, preventing propagation of DNA damage-induced mutations. This study shows that H2O2released from mitochondria is unlikely to directly damage genomic DNA, limiting its contribution to oncogenic transformation and aging.

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N-and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 +/- 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3′UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans. Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3′UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

Actomyosin-based contractility in smooth muscle and nonmuscle cells is regulated by signaling through the small GTPase Rho and by calcium-activated pathways. We use the myoepithelial cells of the Caenorhabditis elegans spermatheca to study the mechanisms of coordinated myosin activation in vivo. Here, we show that redox signaling modulates RHO-1/Rho activity in this contractile tissue. Exogenously added as well as endogenously generated hydrogen peroxide decreases spermathecal contractility by inhibition of RHO-1, which depends on a conserved cysteine in its nucleotide binding site (C20). Further, we identify an endogenous gradient of H2O2 across the spermathecal tissue, which depends on the activity of cytosolic superoxide dismutase, SOD-1. Collectively, we show that SOD-1-mediated H2O2 production regulates the redox environment and fine tunes Rho activity across the spermatheca through oxidation of RHO-1 C20.

Summary The relative positioning of organelles underlies fundamental cellular processes, including signaling, polarization, and cellular growth. Here, we describe the usage of a light-dependent heterodimerization system, LOVpep-ePDZ, to alter organelle positioning locally and reversibly in order to study the functional consequences of organelle positioning. The protocol gives details on how to accomplish expression of fusion proteins encoding this system, describes the imaging parameters to achieve subcellular activation in C. elegans, and may be adapted for use in other model systems. For complete details on the use and execution of this protocol, please refer to De Henau et al. (2020) .

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stableCaenorhabditis eleganstransgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3’UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system forC. elegans(Schwartz and Jorgensen 2016 Genetics, 202:1277-1288). Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3’UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in theC. elegansgermline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

Summary Symmetry breaking is an essential step in cell differentiation and early embryonic development. However, the molecular cues that trigger symmetry breaking remain largely unknown. Here, we show that mitochondrial H2O2 acts as a symmetry-breaking cue in the C. elegans zygote. We find that symmetry breaking is marked by a local H2O2 increase and coincides with a relocation of mitochondria to the cell cortex. Lowering endogenous H2O2 levels delays the onset of symmetry breaking, while artificially targeting mitochondria to the cellular cortex using a light-induced heterodimerization technique is sufficient to initiate symmetry breaking in a H2O2-dependent manner. In wild-type development, both sperm and maternal mitochondria contribute to symmetry breaking. Our findings reveal that mitochondrial H2O2-signaling promotes the onset of polarization, a fundamental process in development and cell differentiation, and this is achieved by both mitochondrial redistribution and differential H2O2-production.

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.

Globins constitute a superfamily of heme-binding proteins that is widely present in many species. There are 33 putative globins in the genome of Caenorhabditis elegans, where glb-13 is a homolog of neuroglobin (Ngb) based on sequence analysis and specific expression in neurons. Here we examined whether glb-13 as well as Ngb is also associated with resistance to reactive oxygen species (ROS) induced by paraquat. Our results showed that the mRNA level of glb-13 was significantly upregulated after paraquat exposure. Expression of a green fluorescent protein (GFP) reporter gene directed by the glb-13 promoter was increased by paraquat exposure. The mutant C. elegans strain glb-13(tm2825) was sensitive to paraquat-induced oxidative stress. Overexpression of human Ngb (hNgb) in C. elegans neuronal cells can rescue the paraquat sensitive phenotype of the mutant strain. glb-13 mutation or hNgb overexpression did not affect the expression of antioxidant enzymes such as superoxide dismutase (SOD). To examine the ROS-scavenging capabilities of hNgb and glb-13, we further examined the level of ROS in glb-13 mutant and hNgb transgenic (hNgb-Tg) worms. There was no statistical difference in ROS levels in the untreated controls; however in paraquat-treated worms, the ROS level was statistically repressed in the hNgb-Tg relative to enhanced green fluorescent protein (EGFP)-Tg worms or wildtype animals. Additionally, the ROS level of glb-13 mutant was statistically higher than the wildtype animals. Furthermore, hNgb overexpression diminished the ROS level of glb-13 mutant. In conclusion, hNgb can rescue the ROS sensitive phenotype of the glb-13 mutant strain. The protein GLB-13 seems to have an hNgb-like function, suggesting the importance of the globin protein family in maintaining the homeostasis of ROS signals. Our data provided evidence for the first time that glb-13 is associated with the resistance against oxidative stress-induced toxicity.

The balance between protein synthesis and protein breakdown is a major determinant of protein homeostasis, and loss of protein homeostasis is one of the hallmarks of aging. Here we describe pulsed SILAC-based experiments to estimate proteome-wide turnover rates of individual proteins. We applied this method to determine protein turnover rates in Caenorhabditis elegans models of longevity and Parkinson's disease, using both developing and adult animals. Whereas protein turnover in developing, long-lived daf-2(e1370) worms is about 30% slower than in controls, the opposite was observed in day 5 adult worms, in which protein turnover in the daf-2(e1370) mutant is twice as fast as in controls. In the Parkinson's model, protein turnover is reduced proportionally over the entire proteome, suggesting that the protein homeostasis network has a strong ability to adapt. The findings shed light on the relationship between protein turnover and healthy aging.

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., Globins are best known for their role in respiration, but recent studies suggest they might contribute to redox signalling as well. Here, the authors present biochemical, structural and in vivo evidence that the roundworm globin Glb-12 acts as a superoxide generator necessary for germline development.

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.

Summary 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 (GLB1, 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. 2011 IUBMB IUBMB Life, 63(3): 166–174, 2011

SummaryFrataxin is a nuclear-encoded mitochondrial protein involved in the biogenesis of Fe-S-cluster-containing proteins and consequently in the functionality of the mitochondrial respiratory chain. Similar to other proteins that regulate mitochondrial respiration, severe frataxin deficiency leads to pathology in humans—Friedreich’s ataxia, a life-threatening neurodegenerative disorder—and to developmental arrest in the nematode C. elegans. Interestingly, partial frataxin depletion extends C. elegans lifespan, and a similar anti-aging effect is prompted by reduced expression of other mitochondrial regulatory proteins from yeast to mammals. The beneficial adaptive responses to mild mitochondrial stress are still largely unknown and, if characterized, may suggest novel potential targets for the treatment of human mitochondria-associated, age-related disorders. Here we identify mitochondrial autophagy as an evolutionarily conserved response to frataxin silencing, and show for the first time that, similar to mammals, mitophagy is activated in C. elegans in response to mitochondrial stress in a pdr-1/Parkin-, pink-1/Pink-, and dct-1/Bnip3-dependent manner. The induction of mitophagy is part of a hypoxia-like, iron starvation response triggered upon frataxin depletion and causally involved in animal lifespan extension. We also identify non-overlapping hif-1 upstream (HIF-1-prolyl-hydroxylase) and downstream (globins) regulatory genes mediating lifespan extension upon frataxin and iron depletion. Our findings indicate that mitophagy induction is part of an adaptive iron starvation response induced as a protective mechanism against mitochondrial stress, thus suggesting novel potential therapeutic strategies for the treatment of mitochondrial-associated, age-related disorders.

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.

Globins occur in all kingdoms of life where they fulfill a wide variety of functions. In the past they used to be primarily characterized as oxygen transport/storage proteins, but since the discovery of new members of the globin family like neuroglobin and cytoglobin, more diverse and complex functions have been assigned to this heterogeneous family. Here we propose a function for a membrane-bound globin of C. elegans, GLB-26. This globin was predicted to be myristoylated at its N-terminus, a post-translational modification only recently described in the globin family. In vivo, this globin is found in the membrane of the head mesodermal cell and in the tail stomato-intestinal and anal depressor muscle cells. Since GLB-26 is almost directly oxidized when exposed to oxygen, we postulate a possible function as electron transfer protein. Phenotypical studies show that GLB-26 takes part in regulating the length of the defecation cycle in C. elegans under oxidative stress conditions.

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 α-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.

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.