Mitochondrial fusion occurs in many eukaryotes, including animals, plants, and fungi. It is essential for cellular homeostasis, and yet the underlying mechanisms remain elusive. Comparative analyses ...and phylogenetic reconstructions revealed that fungal Fzo1 and animal Mitofusin proteins are highly diverged from one another and lack strong sequence similarity. Bioinformatic analysis showed that fungal Fzo1 proteins exhibit two predicted transmembrane domains, whereas metazoan Mitofusins contain only a single transmembrane domain. This prediction contradicts the current models, suggesting that both animal and fungal proteins share one topology. This newly predicted topology of Mfn1 and Mfn2 was demonstrated biochemically, confirming that the C-terminal, redox-sensitive cysteine residues reside within the intermembrane space (IMS). Functional experiments established that redox-mediated disulfide modifications within the IMS domain are key modulators of reversible Mfn oligomerization that drives fusion. Together, these results lead to a revised understanding of Mfns as single-spanning outer membrane proteins with an N
-C
orientation, providing functional insight into the IMS contribution to redox-regulated fusion events.
Oxidative protein folding is confined to few compartments, including the endoplasmic reticulum, the mitochondrial intermembrane space and the bacterial periplasm. Conversely, in compartments in which ...proteins are translated such as the cytosol, the mitochondrial matrix and the chloroplast stroma proteins are kept reduced by the thioredoxin and glutaredoxin systems that functionally overlap. The highly reducing NADPH pool thereby serves as electron donor that enables glutathione reductase and thioredoxin reductase to keep glutathione pools and thioredoxins in their reduced redox state, respectively. Notably, also compartments containing oxidizing machineries are linked to these reducing pathways. Reducing pathways aid in proofreading of disulfide bond formation by isomerization or they provide reducing equivalents for the reduction of disulfides prior to degradation. In addition, they contribute to the thiol-dependent regulation of protein activities, and they help to counteract oxidative stress. The existence of oxidizing and reducing pathways in the same compartment poses a potential problem as the cell has to avoid futile cycles of oxidation and subsequent reduction reactions. Thus, compartments that contain oxidizing machineries have developed sophisticated ways to spatiotemporally balance and regulate oxidation and reduction. In this review, we discuss oxidizing and reducing pathways in the endoplasmic reticulum, the periplasm and the mitochondrial intermembrane space and highlight the role of glutathione especially in the endoplasmic reticulum and the intermembrane space. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.
•Oxidative folding is confined to few compartments like ER, periplasm and IMS.•Other compartments are kept reducing by dedicated machineries.•Compartments harboring oxidizing machineries are also linked to reducing pathways.•Balancing reducing and oxidizing pathways poses a challenge in these compartments.•Mechanisms of balancing have been described for ER and periplasm but not the IMS.
The essential oxidoreductase Mia40/CHCHD4 mediates disulfide bond formation and protein folding in the mitochondrial intermembrane space. Here, we investigated the interactome of Mia40 thereby ...revealing links between thiol-oxidation and apoptosis, energy metabolism, and Ca2+ signaling. Among the interaction partners of Mia40 is MICU1—the regulator of the mitochondrial Ca2+ uniporter (MCU), which transfers Ca2+ across the inner membrane. We examined the biogenesis of MICU1 and find that Mia40 introduces an intermolecular disulfide bond that links MICU1 and its inhibitory paralog MICU2 in a heterodimer. Absence of this disulfide bond results in increased receptor-induced mitochondrial Ca2+ uptake. In the presence of the disulfide bond, MICU1-MICU2 heterodimer binding to MCU is controlled by Ca2+ levels: the dimer associates with MCU at low levels of Ca2+ and dissociates upon high Ca2+ concentrations. Our findings support a model in which mitochondrial Ca2+ uptake is regulated by a Ca2+-dependent remodeling of the uniporter complex.
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•Mia40 interactome links thiol redox to apoptosis, energy metabolism, Ca2+ signaling•MCU serves as platform for disulfide-dependent MICU1-MICU2 dimerization by Mia40•Absence of the disulfide that links MICU1 and MICU2 leads to increased Ca2+ uptake•Ca2+ uptake is controlled by Ca2+-dependent dissociation of the MICU dimer from MCU
Petrungaro et al. characterize the interactome of the human mitochondrial oxidoreductase Mia40, among which is MICU1, the regulator of the mitochondrial Ca2+ uniporter (MCU). Mia40 primes MICU1 for heterodimerization with MICU2, and the dimer associates with MCU in a Ca2+-dependent manner to control mitochondrial Ca2+ uptake.
The endoplasmic reticulum (ER) was long considered to be the only compartment of the eukaryotic cell in which protein folding is accompanied by enzyme-catalyzed disulfide bond formation. However, it ...has recently become evident that cells harbor a second oxidizing compartment, the mitochondrial intermembrane space, where disulfide formation facilitates protein translocation from the cytosol. Moreover, protein oxidation has been implicated in many mitochondria-associated processes central for human health such as apoptosis, aging, and regulation of the respiratory chain. Whereas the machineries of ER and mitochondria both form disulfides between cysteine residues, they do not share evolutionary origins and exhibit distinct mechanistic properties. Here, we summarize the current knowledge of these oxidation systems and discuss their functional similarities and differences.
Oxidative protein folding Meyer, Andreas J.; Riemer, Jan; Rouhier, Nicolas
The New phytologist,
February 2019, Letnik:
221, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Disulfide bonds are post-translational modifications crucial for the structure and function of thousands of proteins. Their formation and isomerization, referred to as oxidative folding, require ...specific protein machineries found in oxidizing subcellular compartments, namely the endoplasmic reticulum and the associated endomembrane system, the intermembrane space of mitochondria and the thylakoid lumen of chloroplasts. At least one protein component is required for transferring electrons from substrate proteins to an acceptor that is usually molecular oxygen. For oxidation reactions, incoming reduced substrates are oxidized by thiol-oxidoreductase proteins (or domains in case of chimeric proteins), which are usually themselves oxidized by a single thiol oxidase, the enzyme generating disulfide bonds de novo. By contrast, the description of the molecular actors and pathways involved in proofreading and isomerization of misfolded proteins, which require a tightly controlled redox balance, lags behind. Herein we provide a general overview of the knowledge acquired on the systems responsible for oxidative protein folding in photosynthetic organisms, highlighting their particularities compared to other eukaryotes. Current research challenges are discussed including the importance and specificity of these oxidation systems in the context of the existence of reducing systems in the same compartments.
Genetically encoded hydrogen peroxide (H
O
) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available ..."toolbox" for monitoring cellular H
O
changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔC
offer considerably improved H
O
sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H
O
levels.
The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain.
The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H
O
levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H
O
probes, roGFP2-based H
O
sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.
Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (EGSH) dynamics is critical to understand the network of redox ...processes and their influence on cellular function. Using dynamic oxidant recovery assays together with EGSH‐specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of EGSH in the IMS, thus explaining a steady‐state EGSH in the IMS which is similar to the cytosol. Moreover, we show that the local EGSH contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells.
Glutathione pools in the cytosol and mitochondrial intermembrane space (IMS) are connected. Interestingly, the redox state of Glutathione in the IMS is mainly regulated by the cytosolic Glutathione reductase system, which directly impinges on the activity of IMS enzymes such as Mia40.
Loss of TP53 and RB1 in treatment-naïve small cell lung cancer (SCLC) suggests selective pressure to inactivate cell death pathways prior to therapy. Yet, which of these pathways remain available in ...treatment-naïve SCLC is unknown. Here, through systemic analysis of cell death pathway availability in treatment-naïve SCLC, we identify non-neuroendocrine (NE) SCLC to be vulnerable to ferroptosis through subtype-specific lipidome remodeling. While NE SCLC is ferroptosis resistant, it acquires selective addiction to the TRX anti-oxidant pathway. In experimental settings of non-NE/NE intratumoral heterogeneity, non-NE or NE populations are selectively depleted by ferroptosis or TRX pathway inhibition, respectively. Preventing subtype plasticity observed under single pathway targeting, combined treatment kills established non-NE and NE tumors in xenografts, genetically engineered mouse models of SCLC and patient-derived cells, and identifies a patient subset with drastically improved overall survival. These findings reveal cell death pathway mining as a means to identify rational combination therapies for SCLC.
Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we ...report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.
The disulfide relay system in the intermembrane space of mitochondria is of crucial importance for mitochondrial biogenesis. Major players in this pathway are the oxidoreductase Mia40 that oxidizes ...substrates and the sulfhydryl oxidase Erv1 that reoxidizes Mia40. To analyze in detail the mechanism of this oxidative pathway and the interplay of its components, we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40, and Cox19. Here, we demonstrate that Erv1 dimerizes noncovalently and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proofreading during oxidative protein folding by the mitochondrial disulfide relay.
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► Mia40, Erv1, and oxidized cytochrome c form the minimal mitochondrial disulfide relay ► The oxidoreductase Mia40 can form both disulfide bonds in the substrate Cox19 ► Mia40 is oxidized by intersubunit electron transfer in the sulfhydryl oxidase Erv1 ► Reduced glutathione prevents accumulation of unproductive oxidation intermediates
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