Mitophagy-promoting agents and their ability to promote healthy-aging.

The removal of damaged mitochondrial components through a process called mitochondrial autophagy (mitophagy) is essential for the proper function of the mitochondrial network. Hence, mitophagy is vital for the health of all aerobic animals, including humans. Unfortunately, mitophagy declines with age. Many age-associated diseases, including Alzheimer's and Parkinson's, are characterized by the accumulation of damaged mitochondria and oxidative damage. Therefore, activating the mitophagy process with small molecules is an emerging strategy for treating multiple aging diseases. Recent studies have identified natural and synthetic compounds that promote mitophagy and lifespan. This article aims to summarize the existing knowledge about these substances. For readers' convenience, the knowledge is presented in a table that indicates the chemical data of each substance and its effect on lifespan. The impact on healthspan and the molecular mechanism is reported if known. The article explores the potential of utilizing a combination of mitophagy-inducing drugs within a therapeutic framework and addresses the associated challenges of this strategy. Finally, we discuss the process that balances mitophagy, i.e. mitochondrial biogenesis. In this process, new mitochondrial components are generated to replace the ones cleared by mitophagy. Furthermore, some mitophagy-inducing substances activate biogenesis (e.g. resveratrol and metformin). Finally, we discuss the possibility of combining mitophagy and biogenesis enhancers for future treatment. In conclusion, this article provides an up-to-date source of information about natural and synthetic substances that activate mitophagy and, hopefully, stimulates new hypotheses and studies that promote healthy human aging worldwide.

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Distinct designer diamines promote mitophagy, and thereby enhance healthspan in C. elegans and protect human cells against oxidative damage.

Impaired mitophagy is a primary pathogenic event underlying diverse aging-associated diseases such as Alzheimer and Parkinson diseases and sarcopenia. Therefore, augmentation of mitophagy, the process by which defective mitochondria are removed, then replaced by new ones, is an emerging strategy for preventing the evolvement of multiple morbidities in the elderly population. Based on the scaffold of spermidine (Spd), a known mitophagy-promoting agent, we designed and tested a family of structurally related compounds. A prototypic member, 1,8-diaminooctane (VL-004), exceeds Spd in its ability to induce mitophagy and protect against oxidative stress. VL-004 activity is mediated by canonical aging genes and promotes lifespan and healthspan in C. elegans. Moreover, it enhances mitophagy and protects against oxidative injury in rodent and human cells. Initial structural characterization suggests simple rules for the design of compounds with improved bioactivity, opening the way for a new generation of agents with a potential to promote healthy aging.

The phosphatidylinositol transfer protein PITP-1 facilitates fast recovery of eating behavior after hypoxia in the nematode Caenorhabditis elegans.

Among the fascinating adaptations to limiting oxygen conditions (hypoxia) is the suppression of food intake and weight loss. In humans, this phenomenon is called high-altitude anorexia and is observed in people suffering from acute mountain syndrome. The high-altitude anorexia appears to be conserved in evolution and has been seen in species across the animal kingdom. However, the mechanism underlying the recovery of eating behavior after hypoxia is still not known. Here, we show that the phosphatidylinositol transfer protein PITP-1 is essential for the fast recovery of eating behavior after hypoxia in the nematode Caenorhabditis elegans. Unlike the neuroglobin GLB-5 that accelerates the recovery of eating behavior through its function in the oxygen (O2 )-sensing neurons, PITP-1 appears to act downstream, in neurons that express the mod-1 serotonin receptor. Indeed, pitp-1 mutants display wild-type-like O2 -evoked-calcium responses in the URX O2 -sensing neuron. Intriguingly, loss-of-function of protein kinase C 1 (PKC-1) rescues pitp-1 mutants' recovery after hypoxia. Increased diacylglycerol (DAG), which activates PKC-1, attenuates the recovery of wild-type worms. Together, these data suggest that PITP-1 enables rapid recovery of eating behavior after hypoxia by limiting DAG's availability, thereby limiting PKC activity in mod-1-expressing neurons.

Signal Decoding for Glutamate Modulating Egg Laying Oppositely in Caenorhabditis elegans under Varied Environmental Conditions.

Animals' ability to sense environmental cues and to integrate this information to control fecundity is vital for continuing the species lineage. In this study, we observed that the sensory neurons Amphid neuron (ASHs and ADLs) differentially regulate egg-laying behavior in Caenorhabditis elegans under varied environmental conditions via distinct neuronal circuits. Under standard culture conditions, ASHs tonically release a small amount of glutamate and inhibit Hermaphrodite specific motor neuron (HSN) activities and egg laying via a highly sensitive Glutamate receptor (GLR)-5 receptor. In contrast, under Cu2+ stimulation, ASHs and ADLs may release a large amount of glutamate and inhibit Amphid interneuron (AIA) interneurons via low-sensitivity Glutamate-gated chloride channel (GLC)-3 receptor, thus removing the inhibitory roles of AIAs on HSN activity and egg laying. However, directly measuring the amount of glutamate released by sensory neurons under different conditions and assaying the binding kinetics of receptors with the neurotransmitter are still required to support this study directly.

Aripiprazole Cytotoxicity Coincides with Activation of the Unfolded Protein Response in Human Hepatic Cells.

Schizophrenia is a mental disease that results in decreased life expectancy and well-being by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. Although the second-generation antipsychotics (SGA), Olanzapine and Aripiprazole, are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain, and dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We therefore investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma, and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of inositol-requiring enzyme 1 (IRE1)and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), two major transducers of the UPR. Cells underwent apoptosis with Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2, a human liver cancer cell line, protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress. SIGNIFICANCE STATEMENT: The antischizophrenic drug Aripiprazole exerts cytotoxic properties at high concentrations. This study shows that this cytotoxicity is associated with the induction of endoplasmic reticulum (ER) stress and IRE1 activation, mechanisms involved in diet-induced obesity. Aripiprazole induced ER stress and calcium mobilization from the ER in human and mouse hepatocytes. Our study highlights a new mechanism of Aripiprazole that is not related to its effect on dopamine signaling.

Ferritin is regulated by a neuro-intestinal axis in the nematode Caenorhabditis elegans.

Iron is vital for the life of most organisms. However, when dysregulated, iron can catalyze the formation of oxygen (O2) radicals that can destroy any biological molecule and thus lead to oxidative injury and death. Therefore, iron metabolism must be tightly regulated at all times, as well as coordinated with the metabolism of O2. However, how is this achieved at the whole animal level is not well understood. Here, we explore this question using the nematode Caenorhabditis elegans. Exposure of worms to O2 starvation conditions (i.e. hypoxia) induces a major upregulation in levels of the conserved iron-cage protein ferritin 1 (ftn-1) in the intestine, while exposure to 21% O2 decreases ftn-1 level. This O2-dependent inhibition is mediated by O2-sensing neurons that communicate with the intestine through neurotransmitter and neuropeptide signalling, and requires the activity of hydroxylated HIF-1. By contrast, the induction of ftn-1 in hypoxia appears to be HIF-1-independent. This upregulation provides protection against Pseudomonas aeruginosa bacteria and oxidative injury. Taken together, our studies uncover a neuro-intestine axis that coordinates O2 and iron responses at the whole animal level.

A method for measuring sulfide toxicity in the nematode Caenorhabditis elegans.

Cysteine catabolism by gut microbiota produces high levels of sulfide. Excessive sulfide can interfere with colon function, and therefore may be involved in the etiology and risk of relapse of ulcerative colitis, an inflammatory bowel disease affecting millions of people worldwide. Therefore, it is crucial to understand how cells/animals regulate the detoxification of sulfide generated by bacterial cysteine catabolism in the gut. Here we describe a simple and cost-effective way to explore the mechanism of sulfide toxicity in the nematode Caenorhabditis elegans (C. elegans). •A rapid cost-effective method to quantify and study sulfide tolerance in C. elegans and other free-living nematodes.•A cost effective method to measure the concentration of sulfide in the inverted plate assay.

Characterization of gene expression associated with the adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity.

Oxygen (O2) is a double-edged sword to cells, for while it is vital for energy production in all aerobic animals and insufficient O2 (hypoxia) can lead to cell death, the reoxygenation of hypoxic tissues may trigger the generation of reactive oxygen species (ROS) that can destroy any biological molecule. Indeed, both hypoxia and hypoxia-reoxygenation (H/R) stress are harmful, and may play a critical role in the pathophysiology of many human diseases, such as myocardial ischemia and stroke. Therefore, understanding how animals adapt to hypoxia and H/R stress is critical for developing better treatments for these diseases. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from H/R stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to Pseudomonas aeruginosa pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Mechanisms of defense against products of cysteine catabolism in the nematode Caenorhabditis elegans.

Cysteine catabolism presents cells with a double-edged sword. On the one hand, cysteine degradation provides cells with essential molecules such as taurine and sulfide. The formation of sulfide in cells is thought to regulate important and diverse physiological processes including blood circulation, synaptic activity and inflammation. On the other hand, the catabolism of cysteine by gut microbiota can release high levels of sulfide that may underlie the development or relapse of ulcerative colitis, an inflammatory bowel disease affecting millions of people worldwide. Here, we have used the nematode C. elegans to explore how cells tolerate high levels of sulfide produced by cysteine degradation in bacteria. We have identified mutations in genes coding for thioredoxin family proteins, mitochondrial proteins, and collagens that confer tolerance to sulfide toxicity. Exposure to sulfide induces the unfolded protein response in the endoplasmic reticulum and mitochondria. Moreover, our results suggest that sulfide toxicity is mediated by reactive oxygen species (ROS). Indeed, pre-treatment of worms with antioxidants increases their tolerance to sulfide toxicity. Intriguingly, sub-toxic levels of the superoxide generator paraquat can also increase the tolerance of worms to sulfide. Therefore, it appears that activation of ROS detoxification pathway prior to the exposure to sulfide, can increase the tolerance to sulfide toxicity. Our results suggest that these detoxification pathways are mediated by the hypoxia inducible factor HIF-1. Finally, we show that sulfide resistance varies among wild C. elegans and other nematode species, suggesting that tolerance to sulfide was naturally selected in certain habitats.

Synergism between soluble guanylate cyclase signaling and neuropeptides extends lifespan in the nematode Caenorhabditis elegans.

Oxygen (O2 ) homeostasis is important for all aerobic animals. However, the manner by which O2 sensing and homeostasis contribute to lifespan regulation is poorly understood. Here, we use the nematode Caenorhabditis elegans to address this question. We demonstrate that a loss-of-function mutation in the neuropeptide receptor gene npr-1 and a deletion mutation in the atypical soluble guanylate cyclase gcy-35 O2 sensor interact synergistically to extend worm lifespan. The function of npr-1 and gcy-35 in the O2 -sensing neurons AQR, PQR, and URX shortens the lifespan of the worm. By contrast, the activity of the atypical soluble guanylate cyclase O2 sensor gcy-33 in these neurons is crucial for lifespan extension. In addition to AQR, PQR, and URX, we show that the O2 -sensing neuron BAG and the interneuron RIA are also important for the lifespan lengthening. Neuropeptide processing by the proprotein convertase EGL-3 is essential for lifespan extension, suggesting that the synergistic effect of joint loss of function of gcy-35 and npr-1 is mediated through neuropeptide signal transduction. The extended lifespan is regulated by hypoxia and insulin signaling pathways, mediated by the transcription factors HIF-1 and DAF-16. Moreover, reactive oxygen species (ROS) appear to play an important function in lifespan lengthening. As HIF-1 and DAF-16 activities are modulated by ROS, we speculate that joint loss of function of gcy-35 and npr-1 extends lifespan through ROS signaling.

Regulation of Neuronal Oxygen Responses in C. elegans Is Mediated through Interactions between Globin 5 and the H-NOX Domains of Soluble Guanylate Cyclases.

Soluble guanylate cyclases (sGCs) are gas-binding proteins that control diverse physiological processes such as vasodilation, platelet aggregation, and synaptic plasticity. In the nematode Caenorhabditis elegans, a complex of sGCs, GCY-35 and GCY-36, functions in oxygen (O2) sensing. Previous studies suggested that the neuroglobin GLB-5 genetically interacts with GCY-35, and that the inhibitory effect of GLB-5 on GCY-35 function is necessary for fast recovery from prolonged hypoxia. In this study, we identified mutations in gcy-35 and gcy-36 that impact fast recovery and other phenotypes associated with GLB-5, without undermining sGC activity. These mutations, heb1 and heb3, change conserved amino acid residues in the regulatory H-NOX domains of GCY-35 and GCY-36, respectively, and appear to suppress GLB-5 activity by different mechanisms. Moreover, we observed that short exposure to 35% O2 desensitized the neurons responsible for ambient O2 sensing and that this phenomenon does not occur in heb1 animals. These observations may implicate sGCs in neuronal desensitization mechanisms far beyond the specific case of O2 sensing in nematodes. The conservation of functionally important regions of sGCs is supported by examining site-directed mutants of GCY-35, which suggested that similar regions in the H-NOX domains of O2 and NO-sensing sGCs are important for heme/gas interactions. Overall, our studies provide novel insights into sGC activity and regulation, and implicate similar structural determinants in the control of both O2 and NO sensors. Significance statement: Soluble guanylate cyclases (sGCs) control essential and diverse physiological processes, including memory processing. We used Caenorhabditis elegans to explore how a neuroglobin inhibits a complex of oxygen-sensing sGCs, identifying sGC mutants that resist inhibition. Resistance appears to arise by two different mechanisms: increased basal sGC activity or disruption of an interaction with neuroglobin. Our findings demonstrate that the inhibition of sGCs by neuroglobin is essential for rapid adaptation to either low or high oxygen levels, and that similar structural regions are key for regulating both oxygen and nitric oxide sensors. Based on our structural and functional analyses, we present the hypothesis that neuroglobin-sGC interactions may be generally important for adaptation processes, including those in organisms with more complex neurological functions.

GLOBIN-5-dependent O2 responses are regulated by PDL-1/PrBP that targets prenylated soluble guanylate cyclases to dendritic endings.

Aerobic animals constantly monitor and adapt to changes in O2 levels. The molecular mechanisms involved in sensing O2 are, however, incompletely understood. Previous studies showed that a hexacoordinated globin called GLB-5 tunes the dynamic range of O2-sensing neurons in natural C. elegans isolates, but is defective in the N2 lab reference strain (McGrath et al., 2009; Persson et al., 2009). GLB-5 enables a sharp behavioral switch when O2 changes between 21 and 17%. Here, we show that GLB-5 also confers rapid behavioral and cellular recovery from exposure to hypoxia. Hypoxia reconfigures O2-evoked Ca(2+) responses in the URX O2 sensors, and GLB-5 enables rapid recovery of these responses upon re-oxygenation. Forward genetic screens indicate that GLB-5's effects on O2 sensing require PDL-1, the C. elegans ortholog of mammalian PrBP/PDE6δ protein. In mammals, PDE6δ regulates the traffic and activity of prenylated proteins (Zhang et al., 2004; Norton et al., 2005). PDL-1 promotes localization of GCY-33 and GCY-35, atypical soluble guanylate cyclases that act as O2 sensors, to the dendritic endings of URX and BAG neurons, where they colocalize with GLB-5. Both GCY-33 and GCY-35 are predicted to be prenylated. Dendritic localization is not essential for GCY-35 to function as an O2 sensor, but disrupting pdl-1 alters the URX neuron's O2 response properties. Functional GLB-5 can restore dendritic localization of GCY-33 in pdl-1 mutants, suggesting GCY-33 and GLB-5 are in a complex. Our data suggest GLB-5 and the soluble guanylate cyclases operate in close proximity to sculpt O2 responses.

Cross-modulation of homeostatic responses to temperature, oxygen and carbon dioxide in C. elegans.

Different interoceptive systems must be integrated to ensure that multiple homeostatic insults evoke appropriate behavioral and physiological responses. Little is known about how this is achieved. Using C. elegans, we dissect cross-modulation between systems that monitor temperature, O2 and CO2. CO2 is less aversive to animals acclimated to 15°C than those grown at 22°C. This difference requires the AFD neurons, which respond to both temperature and CO2 changes. CO2 evokes distinct AFD Ca²âº responses in animals acclimated at 15°C or 22°C. Mutants defective in synaptic transmission can reprogram AFD CO2 responses according to temperature experience, suggesting reprogramming occurs cell autonomously. AFD is exquisitely sensitive to CO2. Surprisingly, gradients of 0.01% CO2/second evoke very different Ca²âº responses from gradients of 0.04% CO2/second. Ambient O2 provides further contextual modulation of CO2 avoidance. At 21% O2 tonic signalling from the O2-sensing neuron URX inhibits CO2 avoidance. This inhibition can be graded according to O2 levels. In a natural wild isolate, a switch from 21% to 19% O2 is sufficient to convert CO2 from a neutral to an aversive cue. This sharp tuning is conferred partly by the neuroglobin GLB-5. The modulatory effects of O2 on CO2 avoidance involve the RIA interneurons, which are post-synaptic to URX and exhibit CO2-evoked Ca²âº responses. Ambient O2 and acclimation temperature act combinatorially to modulate CO2 responsiveness. Our work highlights the integrated architecture of homeostatic responses in C. elegans.

Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans.

Behaviours evolve by iterations of natural selection, but we have few insights into the molecular and neural mechanisms involved. Here we show that some Caenorhabditis elegans wild strains switch between two foraging behaviours in response to subtle changes in ambient oxygen. This finely tuned switch is conferred by a naturally variable hexacoordinated globin, GLB-5. GLB-5 acts with the atypical soluble guanylate cyclases, which are a different type of oxygen binding protein, to tune the dynamic range of oxygen-sensing neurons close to atmospheric (21%) concentrations. Calcium imaging indicates that one group of these neurons is activated when oxygen rises towards 21%, and is inhibited as oxygen drops below 21%. The soluble guanylate cyclase GCY-35 is required for high oxygen to activate the neurons; GLB-5 provides inhibitory input when oxygen decreases below 21%. Together, these oxygen binding proteins tune neuronal and behavioural responses to a narrow oxygen concentration range close to atmospheric levels. The effect of the glb-5 gene on oxygen sensing and foraging is modified by the naturally variable neuropeptide receptor npr-1 (refs 4, 5), providing insights into how polygenic variation reshapes neural circuit function.

Yeast Mpd1p reveals the structural diversity of the protein disulfide isomerase family.

Oxidoreductases belonging to the protein disulfide isomerase (PDI) family promote proper disulfide bond formation in substrate proteins in the endoplasmic reticulum. In plants and metazoans, new family members continue to be identified and assigned to various functional niches. PDI-like proteins typically contain tandem thioredoxin-fold domains. The limited information available suggested that the relative orientations of these domains may be quite uniform across the family, and structural models based on this assumption are appearing. However, the X-ray crystal structure of the yeast PDI family protein Mpd1p, described here, demonstrates the radically different domain orientations and surface properties achievable with multiple copies of the thioredoxin fold. A comparison of Mpd1p with yeast Pdi1p expands our perspective on the contexts in which redox-active motifs are presented in the PDI family.

Modulation of cellular disulfide-bond formation and the ER redox environment by feedback regulation of Ero1.

Introduction of disulfide bonds into proteins entering the secretory pathway is catalyzed by Ero1p, which generates disulfide bonds de novo, and Pdi1p, which transfers disulfides to substrate proteins. A sufficiently oxidizing environment must be maintained in the endoplasmic reticulum (ER) to allow for disulfide formation, but a pool of reduced thiols is needed for isomerization of incorrectly paired disulfides. We have found that hyperoxidation of the ER is prevented by attenuation of Ero1p activity through noncatalytic cysteine pairs. Deregulated Ero1p mutants lacking certain cysteines show increased enzyme activity, a decreased lag phase in kinetic assays, and growth defects in vivo. We hypothesize that noncatalytic cysteine pairs in Ero1p sense the level of potential substrates in the ER and correspondingly modulate Ero1p activity as part of a homeostatic regulatory system governing the thiol-disulfide balance in the ER.

Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p.

Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.

Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell.

The flavoenzyme Ero1p produces disulfide bonds for oxidative protein folding in the endoplasmic reticulum. Disulfides generated de novo within Ero1p are transferred to protein disulfide isomerase and then to substrate proteins by dithiol-disulfide exchange reactions. Despite this key role of Ero1p, little is known about the mechanism by which this enzyme catalyzes thiol oxidation. Here, we present the X-ray crystallographic structure of Ero1p, which reveals the molecular details of the catalytic center, the role of a CXXCXXC motif, and the spatial relationship between functionally significant cysteines and the bound cofactor. Remarkably, the Ero1p active site closely resembles that of the versatile thiol oxidase module of Erv2p, a protein with no sequence homology to Ero1p. Furthermore, both Ero1p and Erv2p display essential dicysteine motifs on mobile polypeptide segments, suggesting that shuttling electrons to a rigid active site using a flexible strand is a fundamental feature of disulfide-generating flavoenzymes.