Calcium signaling in mitochondrial intermembrane space.

The mitochondrial intermembrane space (IMS) is a highly protected compartment, second only to the matrix. It is a crucial bridge, coordinating mitochondrial activities with cellular processes such as metabolites, protein, lipid, and ion exchange. This regulation influences signaling pathways for metabolic activities and cellular homeostasis. The IMS harbors various proteins critical for initiating apoptotic cascades and regulating reactive oxygen species production by controlling the respiratory chain. Calcium (Ca2+), a key intracellular secondary messenger, enter the mitochondrial matrix via the IMS, regulating mitochondrial bioenergetics, ATP production, modulating cell death pathways. IMS acts as a regulatory site for Ca2+ entry due to the presence of different Ca2+ sensors such as MICUs, solute carriers (SLCs); ion exchangers (LETM1/SCaMCs); S100A1, mitochondrial glycerol-3-phosphate dehydrogenase, and EFHD1, each with unique Ca2+ binding motifs and spatial localizations. This review primarily emphasizes the role of these IMS-localized Ca2+ sensors concerning their spatial localization, mechanism, and molecular functions. Additionally, we discuss how these sensors contribute to the progression and pathogenesis of various human health conditions and diseases.

Oxidation of Arabidopsis thaliana COX19 Using the Combined Action of ERV1 and Glutathione.

Protein import and oxidative folding within the intermembrane space (IMS) of mitochondria relies on the MIA40-ERV1 couple. The MIA40 oxidoreductase usually performs substrate recognition and oxidation and is then regenerated by the FAD-dependent oxidase ERV1. In most eukaryotes, both proteins are essential; however, MIA40 is dispensable in Arabidopsis thaliana. Previous complementation experiments have studied yeast mia40 mutants expressing a redox inactive, but import-competent versions of yeast Mia40 using A. thaliana ERV1 (AtERV1) suggest that AtERV1 catalyzes the oxidation of MIA40 substrates. We assessed the ability of both yeast and Arabidopsis MIA40 and ERV1 recombinant proteins to oxidize the apo-cytochrome reductase CCMH and the cytochrome c oxidase assembly protein COX19, a typical MIA40 substrate, in the presence or absence of glutathione, using in vitro cysteine alkylation and cytochrome c reduction assays. The presence of glutathione used at a physiological concentration and redox potential was sufficient to support the oxidation of COX19 by AtERV1, providing a likely explanation for why MIA40 is not essential for the import and oxidative folding of IMS-located proteins in Arabidopsis. The results point to fundamental biochemical differences between Arabidopsis and yeast ERV1 in catalyzing protein oxidation.

H2O2 transit through the mitochondrial intermembrane space promotes tumor cell growth in vitro and in vivo.

Cancer cells experience increased levels of oxidant stress as a consequence of oncogene activation, nucleotide biosynthesis, and growth factor receptor signaling. Mitochondria contribute to this redox stress by generating reactive oxygen species (ROS) along the electron transport chain, which are released to the matrix and the intermembrane space (IMS). Assessing the contribution of mitochondrial ROS in cancer cells is technically difficult, as electron transport chain inhibitors can increase or decrease ROS generation, while they also block oxidative phosphorylation and ATP synthesis. Mitochondria-targeted antioxidant compounds can scavenge ROS in the matrix compartment but do not act on ROS released to the IMS. We assessed the importance of mitochondrial ROS for tumor cell proliferation, survival, and for tumor xenograft growth by stably expressing a hydrogen peroxide (H2O2) scavenger, peroxiredoxin-5, in the mitochondrial IMS (IMS-Prdx5) in 143B osteosarcoma and HCT116 colorectal cancer cell lines. IMS-Prdx5 attenuates hypoxia-induced ROS signaling as assessed independently in cytosol and IMS, HIF-1α stabilization and activity, and cellular proliferation under normoxic and hypoxic culture conditions. It also suppressed tumor growth in vivo. Stable expression of nondegradable HIF-1α only partially rescued proliferation in IMS-Prdx5-expressing cells, indicating that mitochondrial H2O2 signaling contributes to tumor cell proliferation and survival through HIF-dependent and HIF-independent mechanisms.

Resistance to Obesity in SOD1 Deficient Mice with a High-Fat/High-Sucrose Diet.

Metabolic syndrome (Mets) is an important condition because it may cause stroke and heart disease in the future. Reactive oxygen species (ROSs) influence the pathogenesis of Mets; however, the types of ROSs and their localization remain largely unknown. In this study, we investigated the effects of SOD1, which localize to the cytoplasm and mitochondrial intermembrane space and metabolize superoxide anion, on Mets using SOD1 deficient mice (SOD1-/-). SOD1-/- fed on a high-fat/high-sucrose diet (HFHSD) for 24 weeks showed reduced body weight gain and adipose tissue size compared to wild-type mice (WT). Insulin secretion was dramatically decreased in SOD1-/- fed on HFHSD even though blood glucose levels were similar to WT. Ambulatory oxygen consumption was accelerated in SOD1-/- with HFHSD; however, ATP levels of skeletal muscle were somewhat reduced compared to WT. Reflecting the reduced ATP, the expression of phosphorylated AMPK (Thr 172) was more robust in SOD1-/-. SOD1 is involved in the ATP production mechanism in mitochondria and may contribute to visceral fat accumulation by causing insulin secretion and insulin resistance.

Human mitochondrial AAA+ ATPase SKD3/CLPB assembles into nucleotide-stabilized dodecamers.

SKD3, also known as human CLPB, belongs to the AAA+ family of ATPases associated with various activities. Mutations in the SKD3/CLPB gene cause 3-methylglutaconic aciduria type VII and congenital neutropenia. SKD3 is upregulated in acute myeloid leukemia, where it contributes to anti-cancer drug resistance. SKD3 resides in the mitochondrial intermembrane space, where it forms ATP-dependent high-molecular weight complexes, but its biological function and mechanistic links to the clinical phenotypes are currently unknown. Using sedimentation equilibrium and dynamic light scattering, we show that SKD3 is monomeric at low protein concentration in the absence of nucleotides, but it forms oligomers at higher protein concentration or in the presence of adenine nucleotides. The apparent molecular weight of the nucleotide-bound SKD3 is consistent with self-association of 12 monomers. Image-class analysis and averaging from negative-stain electron microscopy (EM) of SKD3 in the ATP-bound state visualized cylinder-shaped particles with an open central channel along the cylinder axis. The dimensions of the EM-visualized particle suggest that the SKD3 dodecamer is formed by association of two hexameric rings. While hexameric structure has been often observed among AAA+ ATPases, a double-hexamer sandwich found for SKD3 appears uncommon within this protein family. A functional significance of the non-canonical structure of SKD3 remains to be determined.

DeepPred-SubMito: A Novel Submitochondrial Localization Predictor Based on Multi-Channel Convolutional Neural Network and Dataset Balancing Treatment.

Mitochondrial proteins are physiologically active in different compartments, and their abnormal location will trigger the pathogenesis of human mitochondrial pathologies. Correctly identifying submitochondrial locations can provide information for disease pathogenesis and drug design. A mitochondrion has four submitochondrial compartments, the matrix, the outer membrane, the inner membrane, and the intermembrane space, but various existing studies ignored the intermembrane space. The majority of researchers used traditional machine learning methods for predicting mitochondrial protein localization. Those predictors required expert-level knowledge of biology to be encoded as features rather than allowing the underlying predictor to extract features through a data-driven procedure. Besides, few researchers have considered the imbalance in datasets. In this paper, we propose a novel end-to-end predictor employing deep neural networks, DeepPred-SubMito, for protein submitochondrial location prediction. First, we utilize random over-sampling to decrease the influence caused by unbalanced datasets. Next, we train a multi-channel bilayer convolutional neural network for multiple subsequences to learn high-level features. Third, the prediction result is outputted through the fully connected layer. The performance of the predictor is measured by 10-fold cross-validation and 5-fold cross-validation on the SM424-18 dataset and the SubMitoPred dataset, respectively. Experimental results show that the predictor outperforms state-of-the-art predictors. In addition, the prediction of results in the M983 dataset also confirmed its effectiveness in predicting submitochondrial locations.

A homozygous variant in NDUFA8 is associated with developmental delay, microcephaly, and epilepsy due to mitochondrial complex I deficiency.

Mitochondrial complex I deficiency is caused by pathogenic variants in mitochondrial and nuclear genes associated with complex I structure and assembly. We report the case of a patient with　NDUFA8-related mitochondrial disease. The patient presented with developmental delay, microcephaly, and epilepsy. His fibroblasts showed apparent biochemical defects in mitochondrial complex I. Whole-exome sequencing revealed that the patient carried a homozygous variant in NDUFA8. His fibroblasts showed a reduction in the protein expression level of not only NDUFA8, but also the other complex I subunits, consistent with assembly defects. The enzyme activity of complex I and oxygen consumption rate were restored by reintroducing wild-typeNDUFA8 cDNA into patient fibroblasts. The functional properties of the variant in NDUFA8 were also investigated using NDUFA8 knockout cells expressing wild-type or mutated NDUFA8 cDNA. These experiments further supported the pathogenicity of the variant in complex I assembly. This is the first report describing that the loss of NDUFA8, which has not previously been associated with mitochondrial disease, causes severe defect in the assembly of mitochondrial complex I, leading to progressive neurological and developmental abnormalities.

A High Resolution Mass Spectrometry Study Reveals the Potential of Disulfide Formation in Human Mitochondrial Voltage-Dependent Anion Selective Channel Isoforms (hVDACs).

The voltage-dependent anion-selective channels (VDACs), which are also known as eukaryotic porins, are pore-forming proteins, which allow for the passage of ions and small molecules across the outer mitochondrial membrane (OMM). They are involved in complex interactions that regulate organelle and cellular metabolism. We have recently reported the post-translational modifications (PTMs) of the three VDAC isoforms purified from rat liver mitochondria (rVDACs), showing, for the first time, the over-oxidation of the cysteine residues as an exclusive feature of VDACs. Noteworthy, this peculiar PTM is not detectable in other integral membrane mitochondrial proteins, as defined by their elution at low salt concentration by a hydroxyapatite column. In this study, the association of tryptic and chymotryptic proteolysis with UHPLC/High Resolution nESI-MS/MS, allowed for us to extend the investigation to the human VDACs. The over-oxidation of the cysteine residues, essentially irreversible in cell conditions, was as also certained in VDAC isoforms from human cells. In human VDAC2 and 3 isoforms the permanently reduced state of a cluster of close cysteines indicates the possibility that disulfide bridges are formed in the proteins. Importantly, the detailed oxidative PTMs that are found in human VDACs confirm and sustain our previous findings in rat tissues, claiming for a predictable characterization that has to be conveyed in the functional role of VDAC proteins within the cell. Data are available via ProteomeXchange with identifier PXD017482.

High resolution mass spectrometry characterization of the oxidation pattern of methionine and cysteine residues in rat liver mitochondria voltage-dependent anion selective channel 3 (VDAC3).

Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys229 is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys122 could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein.

Oxidative folding in the mitochondrial intermembrane space: A regulated process important for cell physiology and disease.

Mitochondria are fundamental organelles with a complex internal architecture that fulfill important diverse functions including iron-sulfur cluster assembly and cell respiration. Intense work for more than 30 years has identified the key protein import components and the pathways involved in protein targeting and assembly. More recently, oxidative folding has been discovered as one important mechanism for mitochondrial proteostasis whilst several human disorders have been linked to this pathway. We describe the molecular components of this pathway in view of their putative redox regulation and we summarize available evidence on the connections of these pathways to human disorders.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

SOD1 misplacing and mitochondrial dysfunction in amyotrophic lateral sclerosis pathogenesis.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease presenting as sporadic (sALS) or familial (fALS) forms. Even if the list of the genes underlining ALS greatly expanded, defects in superoxide dismutase 1 (SOD1), encoding the copper/zinc SOD1, still remain a major cause of fALS and are likely involved also in apparently sporadic presentations. The pathogenesis of ALS is still unknown, but several lines of evidence indicate that the mitochondrial accumulation of mutant SOD1 is an important mechanism of mitochondrial dysfunction, leading to motor neuron pathology and death. The intramitochondrial localization of mutant SOD1 is debated. Mutant SOD1 might accumulate inside the intermembrane space (IMS), overriding the physiological retention regulated by the copper chaperone for superoxide dismutase (CCS). On the other hand, misfolded SOD1 might deposit onto the outer mitochondrial membrane (OMM), clumping the transport across mitochondrial membranes and engaging mitochondrial-dependent cell apoptosis. The elucidation of the mechanisms ruling SOD1 localization and misplacing might shed light on peculiar ALS features such as cell selectivity and late onset. More importantly, these studies might disclose novel targets for therapeutic intervention in familial ALS as well as non-genetic forms. Finally, pharmacological or genetic manipulation aimed to prevent or counteract the intracellular shifting of mutant SOD1 could be effective for other neurodegenerative disorders featuring the toxic accumulation of misfolded proteins.

Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity.

Mia40 participates in oxidative protein folding within the mitochondrial intermembrane space (IMS) by mediating the transfer of reducing equivalents from client proteins to FAD-linked oxidoreductases of the Erv1 family (lfALR in mammals). Here we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Reduced pancreatic ribonuclease A (rRNase), avian lysozyme, and riboflavin binding protein are all competent substrates of the Mia40/lfALR system, although they lack those sequence features previously thought to direct disulfide bond formation in cognate IMS substrates. The oxidation of rRNase by Mia40 does not limit overall turnover of unfolded substrate by the Mia40/lfALR system. Mia40 is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40's ability to bind amphipathic peptides is evident by avid binding to the isolated B-chain during the insulin reductase assay. In aggregate these data suggest that the Mia40/lfALR system has a broad sequence specificity and that potential substrates may be protected from adventitious oxidation by kinetic sequestration within the mitochondrial IMS.

Mia40 combines thiol oxidase and disulfide isomerase activity to efficiently catalyze oxidative folding in mitochondria.

Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein.

Balancing oxidative protein folding: the influences of reducing pathways on disulfide bond formation.

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.

The ins and outs of the intermembrane space: diverse mechanisms and evolutionary rewiring of mitochondrial protein import routes.

BACKGROUND: Mitochondrial biogenesis is an essential process in all eukaryotes. Import of proteins from the cytosol into mitochondria is a key step in organelle biogenesis. Recent evidence suggests that a given mitochondrial protein does not take the same import route in all organisms, suggesting that pathways of mitochondrial protein import can be rewired through evolution. Examples of this process so far involve proteins destined to the mitochondrial intermembrane space (IMS). SCOPE OF REVIEW: Here we review the components, substrates and energy sources of the known mechanisms of protein import into the IMS. We discuss evolutionary rewiring of the IMS import routes, focusing on the example of the lactate utilisation enzyme cytochrome b2 (Cyb2) in the model yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. MAJOR CONCLUSIONS: There are multiple import pathways used for protein entry into the IMS and they form a network capable of importing a diverse range of substrates. These pathways have been rewired, possibly in response to environmental pressures, such as those found in the niches in the human body inhabited by C. albicans. GENERAL SIGNIFICANCE: We propose that evolutionary rewiring of mitochondrial import pathways can adjust the metabolic fitness of a given species to their environmental niche. This article is part of a Special Issue entitled Frontiers of Mitochondrial.

C1orf163/RESA1 is a novel mitochondrial intermembrane space protein connected to respiratory chain assembly.

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60kDa and 150kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).

Extramitochondrial OPA1 and adrenocortical function.

We have previously described that silencing of the mitochondrial protein OPA1 enhances mitochondrial Ca(2+) signaling and aldosterone production in H295R adrenocortical cells. Since extramitochondrial OPA1 (emOPA1) was reported to facilitate cAMP-induced lipolysis, we hypothesized that emOPA1, via the enhanced hydrolysis of cholesterol esters, augments aldosterone production in H295R cells. A few OPA1 immunopositive spots were detected in ∼40% of the cells. In cell fractionation studies OPA1/COX IV (mitochondrial marker) ratio in the post-mitochondrial fractions was an order of magnitude higher than that in the mitochondrial fraction. The ratio of long to short OPA1 isoforms was lower in post-mitochondrial than in mitochondrial fractions. Knockdown of OPA1 failed to reduce db-cAMP-induced phosphorylation of hormone-sensitive lipase (HSL), Ca(2+) signaling and aldosterone secretion. In conclusion, OPA1 could be detected in the post-mitochondrial fractions, nevertheless, OPA1 did not interfere with the cAMP - PKA - HSL mediated activation of aldosterone secretion.

Mia40 and MINOS act in parallel with Ccs1 in the biogenesis of mitochondrial Sod1.

Superoxide dismutase 1 (Sod1) is a major superoxide-scavenging enzyme in the eukaryotic cell, and is localized in the cytosol and intermembrane space of mitochondria. Sod1 requires its specific chaperone Ccs1 and disulfide bond formation in order to be retained in the intermembrane space. Our study identified a pool of Sod1 that is present in the reduced state in mitochondria that lack Ccs1. We created yeast mutants with mutations in highly conserved amino acid residues corresponding to human mutations that cause amyotrophic lateral sclerosis, and found that some of the mutant proteins were present in the reduced state. These mutant variants of Sod1 were efficiently localized in mitochondria. Localization of the reduced, Ccs1-independent forms of Sod1 relied on Mia40, an essential component of the mitochondrial intermembrane space import and assembly pathway that is responsible for the biogenesis of intermembrane space proteins. Furthermore, the mitochondrial inner membrane organizing system (MINOS), which is responsible for mitochondrial membrane architecture, differentially modulated the presence of reduced Sod1 in mitochondria. Thus, we identified novel mitochondrial players that are possibly involved in pathological conditions caused by changes in the biogenesis of Sod1.