Intermembrane Space Proteins Research Articles

Systematic mapping of mitochondrial calcium uniporter channel (MCUC)-mediated calcium signaling networks

The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases.

Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

Endogenous BAX and BAK form mosaic rings of variable size and composition on apoptotic mitochondria

One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same spatiotemporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wild type cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK- single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings in both wild type as well as single-knockout cells corroborates the toroidal apoptotic pore model.

CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.

Analysis of targeting signals for mitochondrial intermembrane space import.

The mitochondrial intermembrane space (IMS) is the smallest sub-mitochondrial compartment, containing only 5%-10%of mitochondrial proteins. Despite its size, it exhibits the most diverse array of protein import mechanisms. These are underpinned by several different types of targeting signals that are quite distinct from targeting signals for other mitochondrial sub-compartments. In this chapter we outlined our current understanding of some of the main IMS import pathways, the primary oxidative protein folding targeting signal, and explore the remarkable variety of alternative import methods. Unlike proteins destined for the matrix or inner membrane (IM), IMS proteins need only traverse the outer mitochondrial membrane. This process doesn't require energy from ATP hydrolysis in the matrix or the IM electrochemical potential. We also examine unconventional IMS import pathways that remain poorly understood, often guided by ill-defined or unknown targeting peptides. Many IMS proteins are implicated in human diseases, making it crucial to comprehend how they reach their functional location within the IMS. The chapter concludes by discussing current insights into how understanding IMS targeting pathways can contribute to improved understanding of a wide range of human disorders.

CHCHD10 mutations induce tissue-specific mitochondrial DNA deletions with a distinct signature.

Mutations affecting the mitochondrial intermembrane space protein CHCHD10 cause human disease, but it is not known why different amino acid substitutions cause markedly different clinical phenotypes, including amyotrophic lateral sclerosis-frontotemporal dementia, spinal muscular atrophy Jokela-type, isolated autosomal dominant mitochondrial myopathy and cardiomyopathy. CHCHD10 mutations have been associated with deletions of mitochondrial DNA (mtDNA deletions), raising the possibility that these explain the clinical variability. Here, we sequenced mtDNA obtained from hearts, skeletal muscle, livers and spinal cords of WT and Chchd10 G58R or S59L knockin mice to characterise the mtDNA deletion signatures of the two mutant lines. We found that the deletion levels were higher in G58R and S59L mice than in WT mice in some tissues depending on the Chchd10 genotype, and the deletion burden increased with age. Furthermore, we observed that the spinal cord was less prone to the development of mtDNA deletions than the other tissues examined. Finally, in addition to accelerating the rate of naturally occurring deletions, Chchd10 mutations also led to the accumulation of a novel set of deletions characterised by shorter direct repeats flanking the deletion breakpoints. Our results indicate that Chchd10 mutations in mice induce tissue-specific deletions which may also contribute to the clinical phenotype associated with these mutations in humans.

Cytoplasmic Endonuclease G promotes nonalcoholic fatty liver disease via mTORC2-AKT-ACLY and endoplasmic reticulum stress

Endonuclease G (ENDOG), a nuclear-encoded mitochondrial intermembrane space protein, is well known to be translocated into the nucleus during apoptosis. Recent studies have shown that ENDOG might enter the mitochondrial matrix to regulate mitochondrial genome cleavage and replication. However, little is known about the role of ENDOG in the cytosol. Our previous work showed that cytoplasmic ENDOG competitively binds with 14-3-3γ, which released TSC2 to repress mTORC1 signaling and induce autophagy. Here, we demonstrate that cytoplasmic ENDOG could also release Rictor from 14-3-3γ to activate the mTORC2-AKT-ACLY axis, resulting in acetyl-CoA production. Importantly, we observe that ENDOG could translocate to the ER, bind with Bip, and release IRE1a/PERK to activate the endoplasmic reticulum stress response, promoting lipid synthesis. Taken together, we demonstrate that loss of ENDOG suppresses acetyl-CoA production and lipid synthesis, along with reducing endoplasmic reticulum stress, which eventually alleviates high-fat diet-induced nonalcoholic fatty liver disease in female mice.

Mitochondrial double membrane fission: A mystery solved?

It has long been an unresolved question whether the division machineries that assemble on the mitochondrial surface cooperate with factors inside the organelle. Now, two studies by Connor et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202303147) and Fukuda et al. (2023. Mol. Cell.https://doi.org/10.1016/j.molcel.2023.04.022) have identified an intermembrane space protein that is crucial for mitochondrial double membrane division.

Completion of mitochondrial division requires the intermembrane space protein Mdi1/Atg44.

Mitochondria are highly dynamic double membrane-bound organelles that maintain their shape in part through fission and fusion. Mitochondrial fission is performed by a dynamin-related protein, Dnm1 (Drp1 in humans), that constricts and divides the mitochondria in a GTP hydrolysis-dependent manner. However, it is unclear whether factors inside mitochondria help coordinate the process and if Dnm1/Drp1 activity is sufficient to complete the fission of both mitochondrial membranes. Here, we identify an intermembrane space protein required for mitochondrial fission in yeast, which we propose to name Mdi1 (also named Atg44). Loss of Mdi1 causes mitochondrial hyperfusion due to defects in fission, but not the lack of Dnm1 recruitment to mitochondria. Mdi1 is conserved in fungal species, and its homologs contain an amphipathic α-helix, mutations of which disrupt mitochondrial morphology. One model is that Mdi1 distorts mitochondrial membranes to enable Dnm1 to robustly complete fission. Our work reveals that Dnm1 cannot efficiently divide mitochondria without the coordinated function of Mdi1 inside mitochondria.

Hypoosmosis alters hepatocyte mitochondrial morphology and induces selective release of carbamoyl phosphate synthetase 1.

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.

Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Yeast UPS1 deficiency leads to UVC radiation sensitivity and shortened lifespan.

UPS1/YLR193C of Saccharomyces cerevisiae (S. cerevisiae) encodes a mitochondrial intermembrane space protein. A previous study found that Ups1p is needed for normal mitochondrial morphology and that UPS1 deficiency disrupts the intramitochondrial transport of phosphatidic acid in yeast cells and leads to an altered unfolded protein response and mTORC1 signaling activation. In this paper, we first provide evidence showing that the UPS1 gene is involved in the UVC-induced DNA damage response and aging. We show that UPS1 deficiency leads to sensitivity to ultraviolet C (UVC) radiation and that this effect is accompanied by elevated DNA damage, increased intracellular ROS levels, abnormal mitochondrial respiratoryfunction, an increased early apoptosis rate, and shortened replicative lifespan and chronological lifespan. Moreover, we show that overexpression of the DNA damage-induced checkpoint gene RAD9 effectivelyeliminates the senescence-related defects observed in the UPS1-deficient strain. Collectively, these results suggest a novel role for UPS1 in the UVC-induced DNA damage response and aging.

Abstract 1472: An inside job: Identification of a mitochondrial intermembrane space protein that works from the inside to help divide the organelle

Abstract 1472: An inside job: Identification of a mitochondrial intermembrane space protein that works from the inside to help divide the organelle

Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle

Effective protein import from cytosol is critical for mitochondrial functions and metabolic regulation. We describe here the mammalian muscle-specific and systemic consequences to disrupted mitochondrial matrix protein import by targeted deletion of the mitochondrial HSP70 co-chaperone GRPEL1. Muscle-specific loss of GRPEL1 caused rapid muscle atrophy, accompanied by shut down of oxidative phosphorylation and mitochondrial fatty acid oxidation, and excessive triggering of proteotoxic stress responses. Transcriptome analysis identified new responders to mitochondrial protein import toxicity, such as the neurological disease-linked intermembrane space protein CHCHD10. Besides communication with ER and nucleus, we identified crosstalk of distressed mitochondria with peroxisomes, in particular the induction of peroxisomal Acyl-CoA oxidase 2 (ACOX2), which we propose as an ATF4-regulated peroxisomal marker of integrated stress response. Metabolic profiling indicated fatty acid enrichment in muscle, a shift in TCA cycle intermediates in serum and muscle, and dysregulated bile acids. Our results demonstrate the fundamental importance of GRPEL1 and provide a robust model for detecting mammalian inter-organellar and systemic responses to impaired mitochondrial matrix protein import and folding.

AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long‐lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

Modulation of synaptic plasticity, motor unit physiology, and TDP-43 pathology by CHCHD10

Mutations in CHCHD10, a gene coding for a mitochondrial intermembrane space protein, are associated with Frontotemporal dementia (FTD)-Amyotrophic lateral sclerosis (ALS) spectrum disorders, which are pathologically characterized by cytoplasmic inclusions containing TDP-43. FTD/ALS-linked CHCHD10 mutations and TDP-43 inclusions similarly induce mitochondrial defects in respiration, fusion/fission, mtDNA stability, and cristae structure, while sizeable amounts of cytoplasmic TDP-43 aggregates are found in mitochondria. However, the mechanistic link between CHCHD10 and TDP-43 pathogenesis remains unclear. In this study, we present immunohistochemical and biochemical evidence demonstrating that insoluble CHCHD10 aggregates accumulate and colocalize with phospho-TDP-43 inclusions in brains of FTLD-TDP and AD patients, and that insoluble CHCHD10 levels tightly correlate with insoluble TDP-43 levels in control and FTLD-TDP brains. In an experimental exploration of this pathological phenotype, transgenic mice neuronally expressing FTD/ALS-linked CHCHD10R15L or CHCHDS59L mutations but not CHCHD10WT transgenic mice exhibit significantly increased CHCHD10 aggregation and phospho-TDP-43 pathology, which often colocalize within the same inclusions. Such pathologies are reflected in poor functional outcomes in long-term synaptic plasticity, motor unit physiology, and behavior in CHCHD10R15L and CHCHDS59L transgenic mice. In contrast, expression of CHCHD10WT in hTDP-43 transgenic mice (TAR4;CHCHD10WT) significantly mitigates phospho-TDP-43 pathology and rescues TDP-43-induced impairments in synaptic integrity and long-term synaptic plasticity. In isolated mitochondria, the S59L mutation induces the aggregation of resident CHCHD10S59L protein as well as the aggregation and slower turnover of recombinant TDP-43 imported into mitochondria. Likewise, in an in vitro cell-free system, the S59L mutation induces the aggregation of CHCHD10S59L protein while simultaneously enhancing the aggregation of recombinant TDP-43, as evidenced by filter trap assays and atomic force microscopy. In contrast, recombinant CHCHD10WT inhibits the growth of TDP-43 aggregates. These results in human brains, transgenic mice, and in vitro systems substantiate the role of wild type and mutant CHCHD10 in modulating mitochondrial CHCHD10 and TDP-43 pathogenesis together with associated phenotypes in long-term synaptic plasticity and motor unit physiology in mice and humans.

Abstract 2133: In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355

Abstract Appropriate control of cell death is a fundamental biological process which is frequently dysregulated during tumor development and therapeutic resistance. Apoptosis is a form of regulated cell death initiated by either the extracellular environment (extrinsic) or following internal cellular damage (intrinsic). It is controlled by the BCL-2 family which includes anti-apoptotic regulators like BCL-2, BCL-XL and MCL-1 that bind and sequester various pro-apoptotic BH3-only proteins (BIM, BAD, BID, NOXA, PUMA, etc.), and the pro-apoptotic effectors (BAK, BAX, etc.) responsible for mitochondrial pore formation and MOMP (mitochondrial outer membrane permeabilization). MOMP results in intermembrane space protein release, leading to caspase activation in an irreversible path to programmed cell death. Of the anti-apoptotic regulators, MCL-1 is one of the most frequently and highly amplified genes in human cancers such as myeloid leukemia making it a compelling therapeutic target. Since BCL-2 proteins interact through protein-protein interactions, they have long been elusive targets. The success of selective BCL-2 protein inhibitor Venetoclax in the treatment of various hematological cancers, however spurred interest in MCL-1 as an oncology target. Using structure-based drug design, major breakthroughs were made in the development of MCL-1 inhibitors, with several candidates entering clinical studies in the past five years. JNJ-4355, a highly potent 1,4-indolyl macrocycle (MCL-1 Ki = 18 pM, Cell (MOLP8) AC50 = 8.7 nM) was optimized to address shortcomings from first generation MCL-1 inhibitors: it has improved physicochemical properties (CHI LogD7.4 = 2.35, EPSA = 151 Å2), resulting in greatly improved equilibrium solubility (3.14 mM in buffer pH 7) and reduced protein binding (99.93%). JNJ-4355 showed promising in vitro potency data in cancer cell lines and AML patient-derived samples (cell killing AC50 0.29-75 nM in 25/27 evaluable samples). In vivo MCL-1:BAK complex disruption was confirmed in a mouse MOLM13 (AML) xenograft. Efficacy was demonstrated in a mouse MOLP8 (multiple myeloma) xenograft resulting in complete tumor regression after a single IV dose of JNJ-4355. Citation Format: Frederik J. Rombouts, Lento William, Ingrid Velter, Ann Vos, Aldo Peschiulli, Reuillon Tristan, Maria Dominguez Blanco, Matthieu Jouffroy, Lisa McQueen, Helena Steyvers, Mariette Bekkers, Cristina Altrocchi, Beth Pietrak, Seong Joo Koo, Lawrence Szewczuk, David Walker, Kathryn Packman, Ruud Bueters, Petra Vinken, Amy Johnson, Ricardo Attar, Ulrike Philippar. In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2133.

Chloroplast import of an intermembrane space protein is facilitated by translocon components Toc75 and Tic236.

Chloroplasts are divided into six subcompartments: the outer membrane, intermembrane space, and inner membrane of the envelope, the stroma, the thylakoid membrane, and the thylakoid lumen. Compared with our knowledge of protein import into other subcompartments, extremely little is known about how proteins are imported into the intermembrane space of the envelope. Tic22 was one of the first proteins identified as localizing to the intermembrane space and the only one for which import has been analyzed in some detail. However, conflicting results have been obtained concerning whether the general translocon is used to import Tic22 into the intermembrane space. Taking advantage of available translocon component mutants, we reanalyzed import of Tic22. We reveal reduced in vitro import of Tic22 preprotein (prTic22) into chloroplasts isolated from the Arabidopsis mar1 and tic236 mutants, which are functional knockdown mutants of the outer‐membrane channel Toc75 and the intermembrane space linker Tic236, respectively. Import competition experiments also showed that prTic22 import was reduced by excess amounts of a stroma‐targeted preprotein. Our results indicate that prTic22 uses at least part of the general translocon for import into the intermembrane space.

Short-form OPA1 is a molecular chaperone in mitochondrial intermembrane space.

Mitochondria, double-membrane organelles, are known to participate in a variety of metabolic and signal transduction pathways. The intermembrane space (IMS) of mitochondria is proposed to subject to multiple damages emanating from the respiratory chain. The optic atrophy 1 (OPA1), an important protein for mitochondrial fusion, is cleaved into soluble short-form (S-OPA1) under stresses. Here we report that S-OPA1 could function as a molecular chaperone in IMS. We purified the S-OPA1 (amino acid sequence after OPA1 isoform 5 S1 site) protein and showed it protected substrate proteins from thermally and chemically induced aggregation and strengthened the thermotolerance of Escherichia coli (E. coli). We also showed that S-OPA1 conferred thermotolerance on IMS proteins, e.g., neurolysin. The chaperone activity of S-OPA1 may be required for maintaining IMS homeostasis in mitochondria.

Moschamindole induces glioma cell apoptosis by blocking Mia40-dependent mitochondrial intermembrane space assembly and oxidative respiration.

Glioblastoma multiforme (GBM) is the most frequent, lethal, and aggressive tumor of the central nervous system in adults. In this study, we found for the first time that moschamindole (MCD), a rare phenolic amide with 8/6/6/5/5 rings, is a major bioactive constituent derived from Phragmites communis Trin (Poaceae) that exhibits a potential cytotoxic effect on both TMZ-resistant GBM cell lines and xenograft models. MCD-induced intrinsic apoptosis signals and mitochondrial dysfunction were confirmed by cell cycle arrest, caspase-3/7 activation, and membrane potential depolarization. Furthermore, investigations exploring the mechanism showed that MCD specifically inhibits Mia40-mediated oxidative folding of mitochondrial intermembrane space (IMS) proteins via PCR assay and immunoblot analysis. MCD relies on its positive charge to associate with mitochondrial oxidative respiration, thus blocking energy metabolism and inducing apoptosis. Overexpression and upregulation of Mia40 were proven to reverse MCD-induced apoptosis and were correlated with the chemoresistance of GBM in vitro and in vivo, respectively. Taken together, our study demonstrates that Mia40 is a potential target of the chemoresistance of glioblastoma and suggests that MCD might be a potential agent for the individualized treatment of chemoresistant GBM based on mitochondrial metabolic characteristics and Mia40 expression.