Calcium and magnesium ions modulate the oligomeric state and function of mitochondrial 2-Cys peroxiredoxins in Leishmania parasites.
ABSTRACT: Leishmania parasites have evolved a number of strategies to cope with the harsh environmental changes during mammalian infection. One of these mechanisms involves the functional gain that allows mitochondrial 2-Cys peroxiredoxins to act as molecular chaperones when forming decamers. This function is critical for parasite infectivity in mammals, and its activation has been considered to be controlled exclusively by the enzyme redox state under physiological conditions. Herein, we have revealed that magnesium and calcium ions play a major role in modulating the ability of these enzymes to act as molecular chaperones, surpassing the redox effect. These ions are directly involved in mitochondrial metabolism and participate in a novel mechanism to stabilize the decameric form of 2-Cys peroxiredoxins in Leishmania mitochondria. Moreover, we have demonstrated that a constitutively dimeric Prx1m mutant impairs the survival of Leishmania under heat stress, supporting the central role of the chaperone function of Prx1m for Leishmania parasites during the transition from insect to mammalian hosts.
Project description:Many 2-Cys-peroxiredoxins (2-Cys-Prxs) are dual-function proteins, either acting as peroxidases under non-stress conditions or as chaperones during stress. The mechanism by which 2-Cys-Prxs switch functions remains to be defined. Our work focuses on Leishmania infantum mitochondrial 2-Cys-Prx, whose reduced, decameric subpopulation adopts chaperone function during heat shock, an activity that facilitates the transition from insects to warm-blooded host environments. Here, we have solved the cryo-EM structure of mTXNPx in complex with a thermally unfolded client protein, and revealed that the flexible N-termini of mTXNPx form a well-resolved central belt that contacts and encapsulates the unstructured client protein in the center of the decamer ring. In vivo and in vitro cross-linking studies provide further support for these interactions, and demonstrate that mTXNPx decamers undergo temperature-dependent structural rearrangements specifically at the dimer-dimer interfaces. These structural changes appear crucial for exposing chaperone-client binding sites that are buried in the peroxidase-active protein.
Project description:2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.
Project description:The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 A resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone.
Project description:Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.
Project description:Sperm peroxiredoxins (PRDXs) are moonlighting proteins which, in addition to their antioxidant activity, also act as redox signal transducers through PRDX-induced oxidative post-translational modifications of proteins (oxPTMs). Despite extensive knowledge on the antioxidant activity of PRDXs, the mechanisms related to PRDX-mediated oxPTMs are poorly understood. The present study aimed to investigate the effect of bull sperm 2-Cys PRDX inhibition by Conoidin A on changes in oxPTM levels under control and oxidative stress conditions. The results showed that a group of sperm mitochondrial (LDHAL6B, CS, ACO2, SDHA, ACAPM) and actin cytoskeleton proteins (CAPZB, ALDOA, CCIN) is oxidized due to the action of 2-Cys PRDXs under control conditions. In turn, under oxidative stress conditions, 2-Cys PRDX activity seems to be focused on antioxidant function protecting glycolytic, TCA pathway, and respiratory chain enzymes; chaperones; and sperm axonemal tubulins from oxidative damage. Interestingly, the inhibition of PRDX resulted in oxidation of a group of rate-limiting glycolytic proteins, which is known to trigger the switching of glucose metabolism from glycolysis to pentose phosphate pathway (PPP). The obtained results are expected to broaden the knowledge of the potential role of bull sperm 2-Cys in both redox signal transmission and antioxidant activity.
Project description:Many 2-Cys-peroxiredoxins (2-Cys-Prxs) serve as dual-function proteins. Active as peroxidases under non-stress conditions, they convert into effective chaperones under stress conditions. While their peroxidase activity has been extensively studied and shown to involve cycles of redox-mediated oligomeric changes, the mechanisms by which 2-Cys-Prxs sense stress and convert into general chaperones remain to be defined. Here we focus on the Leishmania infantum mitochondrial 2-Cys-Prx (mTXNPx, Prx1m), which, in its reduced, decameric form, readily adopts chaperone function upon exposure to heat shock temperatures. This activity is crucial for parasite survival in mammalian hosts. Here we have determined the cryo-EM structure of mTXNPx in complex with a thermally unfolded client protein that identifies the flexible N-termini of mTXNPx to form a well-resolved central belt that contacts and encapsulates the unstructured client protein in the center of the decamer ring. In vivo cross-linking studies combined with quantitative in vitro cross-linking experiments further support these interactions, and demonstrate that mTXNPx decamers undergo substantial temperature-dependent structural rearrangements specifically at the dimer-dimer interfaces. These structural changes appear crucial for exposing chaperone client binding sites that are otherwise buried in the peroxidase-active protein. Based on this mechanism, we propose that mTXNPx is the founding member of heat-stress activated chaperones in parasitic mitochondria that facilitate the transition to warm-blooded host environments.
Project description:Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H<sub>2</sub>O<sub>2</sub> acting as a second messenger. However, H<sub>2</sub>O<sub>2</sub> actually reacts poorly with most cysteine thiols and it is not clear how H<sub>2</sub>O<sub>2</sub> discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H<sub>2</sub>O<sub>2</sub> scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H<sub>2</sub>O<sub>2</sub>-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.
Project description:Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 220.127.116.11) of cysteine-dependent peroxidases with roles in oxidant protection and signal transduction. An intriguing biophysical property of typical 2-Cys Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers at physiological concentrations. The functional consequences of this linkage are unknown, but on the basis of structural considerations, we hypothesized that decamer-building (dimer-dimer) interactions serve to stabilize a loop that forms the peroxidatic active site. Here, we address this important issue by studying mutations of Thr77 at the decamer-building interface of AhpC from Salmonella typhimurium. Ultracentrifugation studies revealed that two of the substitutions (T77I and T77D) successfully disrupted the decamer, while the third (T77V) actually enhanced decamer stability. Crystal structures of the decameric forms of all three mutant proteins provide a rationale for their properties. A new assay allowed the first ever measurement of the true k(cat) and K(m) values of wild-type AhpC with H(2)O(2), placing the catalytic efficiency at 4 x 10(7) M(-)(1) s(-)(1). T77V had slightly higher activity than wild-type enzyme, and both T77I and T77D exhibited ca. 100-fold lower catalytic efficiency, indicating that the decameric structure is quite important for, but not essential to, activity. The interplay between decamer formation and active site loop dynamics is emphasized by a decreased susceptibility of T77I and T77D to peroxide-mediated inactivation, and by an increase in the crystallographic B-factors in the active site loop, rather than at the site of the mutation, in the T77D variant.
Project description:Summary Protein oligomerization is central to biological function and regulation, yet its experimental quantification and measurement of dynamic transitions in solution remain challenging. Here, we show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution. We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs). Comparison of the polydispersity of plant and human 2CPs as a function of concentration and redox state revealed features conserved among all 2CPs. In addition, we also find species-specific differences in oligomeric transitions, the occurrence of intermediates and the formation of high molecular weight complexes, which are associated with chaperone activity or act as a storage pool for more efficient dimers outlining the functional differentiation of human 2CPs. Our results point to a diversified functionality of oligomerization for 2CPs and illustrate the power of mass photometry for characterizing heterogeneous oligomeric protein distributions in near native conditions. Graphical abstract Highlights • Mass photometry resolves oligomerization dynamics of 2-Cys peroxiredoxins• The thiol redox transition can be tuned by mutagenesis of amino acid residues• Plant and human 2-Cys peroxiredoxins have common and unique features Biophysical chemistry; Protein; Structural biology
Project description:Hydrogen peroxide (H2O2) acts as a signaling messenger by triggering the reversible oxidation of redox-regulated proteins. It remains unclear how proteins can be oxidized by signaling levels of H2O2 in the presence of peroxiredoxins, which are highly efficient peroxide scavengers. Here we show that the rapid formation of disulfide bonds in cytosolic proteins is enabled, rather than competed, by cytosolic 2-Cys peroxiredoxins. Under the conditions tested, the combined deletion or depletion of cytosolic peroxiredoxins broadly frustrated H2O2-dependent protein thiol oxidation, which is the exact opposite of what would be predicted based on the assumption that H2O2 oxidizes proteins directly. We find that peroxiredoxins enable rapid and sensitive protein thiol oxidation by relaying H2O2-derived oxidizing equivalents to other proteins. Although these findings do not rule out the existence of Prx-independent H2O2 signaling mechanisms, they suggest a broader role for peroxiredoxins as sensors and transmitters of H2O2 signals than hitherto recognized.