Mitochondrial Proteome Research Articles

Multiomics-based energy metabolism heterogeneity and its regulation by antiparasite drug ivermectin.

e18080 Background: Energy metabolism heterogeneity is a hallmark in ovarian cancer, namely the Warburg effect and the reverse Warburg effects coexist in ovarian cancer. Exploration of energy metabolism heterogeneity benefits for discovery of the effective biomarkers for ovarian cancers. Methods: Comprehensive analysis of mitochondrial proteomics data (1198 mitochondrial differentially expressed proteins), mitochondrial phosphorpoteomics data (67 mitochondrial phosphorproteins), proteomics data (205 differentially expressed proteins), and transcriptomics data (20115 genes in 419 ovarian cancer samples) was useful. Results: It revealed (i) the upregulations of rate-limiting enzymes PKM2 in glycolysis, IDH2 in Kreb’s cycle, and UQCRH in oxidative phosphorylation (OXPHOS) pathways, (ii) the upregulation of PDHB that converts pyruvate from glycolysis into acetyl-CoA in Kreb’s cycle. Anti-parasite drug ivermectin demonstrated its strong abilities to inhibit proliferation and cell cycle progression and promote apoptosis in EOC cells, through molecular networks to target PFKP in glycolysis, IDH2 and IDH3B in Kreb’s cycle, ND2, ND5, CYTB, and UQCRH in OXPHOS, and MCT1 and MCT4 in lactate shuttle to inhibit EOC growth. Those results were further confirmed in the ovarian cancer cell models and tissues. Conclusions: It clearly concluded that ivermectin might have new potential for ovarian cancer treatment through regulating energy metabolism pathways. These findings provide more accurate understanding of molecular mechanisms of ovarian cancers and discovery of effective energy-metabolism-heterogeneity-based therapeutic drugs for ovarian cancers.

Mitochondrial Protein Quality Control Mechanisms.

Mitochondria serve as a hub for many cellular processes, including bioenergetics, metabolism, cellular signaling, redox balance, calcium homeostasis, and cell death. The mitochondrial proteome includes over a thousand proteins, encoded by both the mitochondrial and nuclear genomes. The majority (~99%) of proteins are nuclear encoded that are synthesized in the cytosol and subsequently imported into the mitochondria. Within the mitochondria, polypeptides fold and assemble into their native functional form. Mitochondria health and integrity depend on correct protein import, folding, and regulated turnover termed as mitochondrial protein quality control (MPQC). Failure to maintain these processes can cause mitochondrial dysfunction that leads to various pathophysiological outcomes and the commencement of diseases. Here, we summarize the current knowledge about the role of different MPQC regulatory systems such as mitochondrial chaperones, proteases, the ubiquitin-proteasome system, mitochondrial unfolded protein response, mitophagy, and mitochondria-derived vesicles in the maintenance of mitochondrial proteome and health. The proper understanding of mitochondrial protein quality control mechanisms will provide relevant insights to treat multiple human diseases.

Mitochondrial proteomics profile points oxidative phosphorylation as main target for beauvericin and enniatin B mixture

Beauvericin (BEA) and enniatin B (EN B) are non-legislated Fusarium mycotoxins usually found in cereal and cereal-based products all around the world. By the proteomic analysis of mitochondria enriched extracts from Jurkat cells exposed for 24 h to three concentrations of BEA:EN B (0.01–0.1–0.5 μM), a number of 1821 proteins (202 mitochondrial) were identified and relatively quantified. 340 proteins (59 mitochondrial) were statistically significant altered in our samples (Anova p-value ≤ 0.05 and fold change (FC) ≥1.5). The protein mitochondrial translational release factor 1 like (MTRF1L) was the most abundant protein in the three mycotoxin exposures studied. The mycotoxins mixture exposure induced concentration dependent changes at mitochondrial proteins levels that mainly involve inner and outer membrane complexes, Electron Transport Chain (ETC) and ribosomes. These results showed alteration of proteins levels related to oxidative phosphorylation, metabolic and neurodegenerative diseases related pathways.

De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment.

The translocase of outer mitochondrial membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyper-reflexia, ataxia, dystonia and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1 was found to have a p.Thr607Ile variant while Individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4-driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knockdown of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals.

Mitochondrial Quality Control Governed by Ubiquitin.

Mitochondria are essential organelles important for energy production, proliferation, and cell death. Biogenesis, homeostasis, and degradation of this organelle are tightly controlled to match cellular needs and counteract chronic stress conditions. Despite providing their own DNA, the vast majority of mitochondrial proteins are encoded in the nucleus, synthesized by cytosolic ribosomes, and subsequently imported into different mitochondrial compartments. The integrity of the mitochondrial proteome is permanently challenged by defects in folding, transport, and turnover of mitochondrial proteins. Therefore, damaged proteins are constantly sequestered from the outer mitochondrial membrane and targeted for proteasomal degradation in the cytosol via mitochondrial-associated degradation (MAD). Recent studies identified specialized quality control mechanisms important to decrease mislocalized proteins, which affect the mitochondrial import machinery. Interestingly, central factors of these ubiquitin-dependent pathways are shared with the ER-associated degradation (ERAD) machinery, indicating close collaboration between both tubular organelles. Here, we summarize recently described cellular stress response mechanisms, which are triggered by defects in mitochondrial protein import and quality control. Moreover, we discuss how ubiquitin-dependent degradation is integrated with cytosolic stress responses, particularly focused on the crosstalk between MAD and ERAD.

A Uniquely Complex Mitochondrial Proteome from Euglena gracilis.

Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

The Regulatory Role of Phosphorylation of Mitochondrial Transcription Factor B2 (TFB2M)

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome is encoded by the nuclear genome; which includes additional oxidative phosphorylation subunits, as well as the proteins necessary for mtDNA replication, expression and stability. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible protein post‐translational modifications of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. To address this gap in knowledge, this research focuses on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow for the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, threonine 313 and serine 197. To mimic the behavior of phosphorylation on these three sites, individual amino acids were mutated to glutamate, an amino acid that mimics the size and charge of the phosphoryl group. WT and phosphomimetic TFB2M were expressed in E. coli and purified. The purified proteins were analyzed for their ability to bind mtDNA through a pull‐down assay and fluorescence polarization was used to determine the binding dissociation constants for the light strand promoter. Interactions between TFB2M phosphomimics and POLRMT were also assessed. The data support the hypothesis that the phosphorylation at these sites may decrease the ability of TFB2M to bind to mtDNA. In vitro transcription assays were performed to determine the impact of these modifications on TFB2M’s role in transcription initiation. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.

Wiring the powerhouse: Systems‐to‐structure approaches for defining mitochondrial protein function

Mitochondria—epicenters of cellular metabolism and bioenergetics—are much less well understood than their iconic “powerhouse” moniker implies. Hundreds of mitochondrial proteins remain uncharacterized, classic pathways include many steps not yet assigned to specific enzyme functions, a large percentage of mitochondrial diseases remain unresolved, and there are no approved FDA drugs to combat mitochondrial dysfunction. The goal of my research program is to produce a more complete understanding of mitochondrial biology by systematically establishing the functions of orphan mitochondrial proteins and their roles within disease‐related processes. We begin by devising multi‐dimensional analyses designed to make new connections between these proteins and established pathways and processes. We then employ mechanistic and structural approaches to define the functions of select proteins at biochemical depth. This ‘systems biochemistry’ strategy is helping us address outstanding biological questions, including: Which orphan mitochondrial proteins fulfill missing steps in classic mitochondrial processes, such as the biosynthesis of coenzyme Q and the assembly of complex I, and how do they operate mechanistically? And, which resident signaling proteins direct the post‐translational regulation of mitochondrial activities? In answering these and other questions, we aim to help transform the mitochondrial proteome from a component list into a metabolic circuitry of connected functions, and to establish the biochemical underpinnings of mitochondrial dysfunction in human disease.Support or Funding InformationNIH R01DK098672 NIH R35GM131795 NIH R01GM115591 NIH P41GM108538

Mitochondrial-targeted methionine sulfoxide reductase overexpression increases the production of oxidative stress in mitochondria from skeletal muscle.

Objective:Mitochondrial dysfunction comprises part of the etiology of myriad health issues, particularly those that occur with advancing age. Methionine sulfoxide reductase A (MsrA) is a ubiquitous protein oxidation repair enzyme that specifically and catalytically reduces a specific epimer of oxidized methionine: methionine sulfoxide. In this study, we tested the ways in which mitochondrial bioenergetic functions are affected by increasing MsrA expression in different cellular compartments.Methods:In this study, we tested the function of isolated mitochondria, including free radical generation, ATP production, and respiration, from the skeletal muscle of two lines of transgenic mice with increased MsrA expression: mitochondria-targeted MsrA overexpression or cytosol-targeted MsrA overexpression.Results:Surprisingly, in the samples from mice with mitochondrial-targeted MsrA overexpression, we found dramatically increased free radical production though no specific defect in respiration, ATP production, or membrane potential. Among the electron transport chain complexes, we found the activity of complex I was specifically reduced in mitochondrial MsrA transgenic mice. In mice with cytosolic-targeted MsrA overexpression, we found no significant alteration made to any of these parameters of mitochondrial energetics.Conclusions:There is also a growing amount of evidence that MsrA is a functional requirement for sustaining optimal mitochondrial respiration and free radical generation. MsrA is also known to play a partial role in maintaining normal protein homeostasis by specifically repairing oxidized proteins. Our studies highlight a potential novel role for MsrA in regulating the activity of mitochondrial function through its interaction with the mitochondrial proteome.

Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift

Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

Exploring the HeLa Dark Mitochondrial Proteome.

In the framework of the Human Proteome Project initiative, we aim to improve mapping and characterization of mitochondrial proteome. In this work we implemented an experimental workflow, combining classical biochemical enrichments and mass spectrometry, to pursue a much deeper definition of mitochondrial proteome and possibly mine mitochondrial uncharacterized dark proteins. We fractionated in two compartments mitochondria enriched from HeLa cells in order to annotate 4230 proteins in both fraction by means of a multiple-enzyme digestion (trypsin, chymotrypsin and Glu-C) followed by mass spectrometry analysis using a combination of Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA). We detected 22 mitochondrial dark proteins not annotated for their function and we provide their relative abundance inside the mitochondrial organelle. Considering this work as a pilot study we expect that the same approach, in different biological system, could represent an advancement in the characterization of the human mitochondrial proteome providing uncharted ground to explore the mitonuclear phenotypic relationships. All spectra have been deposited to ProteomeXchange with PXD014201 and PXD014200 identifier.

The draft nuclear genome sequence and predicted mitochondrial proteome of Andalucia godoyi, a protist with the most gene-rich and bacteria-like mitochondrial genome

BackgroundComparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date.ResultsIn this study, we assembled the draft nuclear genome sequence for the jakobid Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont (animal and fungal) mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids.ConclusionsAs genome and mitochondrial proteome data have become more widely available, a strikingly punctuate phylogenetic distribution of different mitochondrial components has been revealed, emphasizing that the pathways of mitochondrial proteome evolution are likely complex and lineage-specific. Unraveling this complexity will require comprehensive comparative analyses of mitochondrial proteomes from a phylogenetically broad range of eukaryotes, especially protists. The systematic in silico approach described here offers a valuable adjunct to direct proteomic analysis (e.g., via mass spectrometry), particularly in cases where the latter approach is constrained by sample limitation or other practical considerations.

NcRNAs: New Players in Mitochondrial Health and Disease?

The regulation of mitochondrial proteome is unique in that its components have origins in both mitochondria and nucleus. With the development of OMICS technologies, emerging evidence indicates an interaction between mitochondria and nucleus based not only on the proteins but also on the non-coding RNAs (ncRNAs). It is now accepted that large parts of the non‐coding genome are transcribed into various ncRNA species. Although their characterization has been a hot topic in recent years, the function of the majority remains unknown. Recently, ncRNA species microRNA (miRNA) and long-non coding RNAs (lncRNA) have been gaining attention as direct or indirect modulators of the mitochondrial proteome homeostasis. These ncRNA can impact mitochondria indirectly by affecting transcripts encoding for mitochondrial proteins in the cytoplasm. Furthermore, reports of mitochondria-localized miRNAs, termed mitomiRs, and lncRNAs directly regulating mitochondrial gene expression suggest the import of RNA to mitochondria, but also transcription from the mitochondrial genome. Interestingly, ncRNAs have been also shown to hide small open reading frames (sORFs) encoding for small functional peptides termed micropeptides, with several examples reported with a role in mitochondria. In this review, we provide a literature overview on ncRNAs and micropeptides found to be associated with mitochondrial biology in the context of both health and disease. Although reported, small study overlap and rare replications by other groups make the presence, transport, and role of ncRNA in mitochondria an attractive, but still challenging subject. Finally, we touch the topic of their potential as prognosis markers and therapeutic targets.

Subcellular proteomics combined with bioenergetic phenotyping reveals protein biomarkers of respiratory insufficiency in the setting of proofreading-deficient mitochondrial polymerase

The mitochondrial mutator mouse is a well-established model of premature aging. In addition to accelerated aging, these mice develop hypertrophic cardiomyopathy at ~13 months of age, presumably due to overt mitochondrial dysfunction. Despite evidence of bioenergetic disruption within heart mitochondria, there is little information about the underlying changes to the mitochondrial proteome that either directly underly or predict respiratory insufficiency in mutator mice. Herein, nLC-MS/MS was used to interrogate the mitochondria-enriched proteome of heart and skeletal muscle of aged mutator mice. The mitochondrial proteome from heart tissue was then correlated with respiratory conductance data to identify protein biomarkers of respiratory insufficiency. The majority of downregulated proteins in mutator mitochondria were subunits of respiratory complexes I and IV, including both nuclear and mitochondrial-encoded proteins. Interestingly, the mitochondrial-encoded complex V subunits, were unchanged or upregulated in mutator mitochondria, suggesting a robustness to mtDNA mutation. Finally, the proteins most strongly correlated with respiratory conductance were PPM1K, NDUFB11, and C15orf61. These results suggest that mitochondrial mutator mice undergo a specific loss of mitochondrial complexes I and IV that limit their respiratory function independent of an upregulation of complex V. Additionally, the role of PPM1K in responding to mitochondrial stress warrants further exploration.

Causes and consequences of mitochondrial proteome size variation in animals

Despite a conserved set of core mitochondrial functions, animal mitochondrial proteomes show a large variation in size. We analyzed putative mechanisms behind and functional significance of this variation by performing comparative analysis of the experimentally-verified mitochondrial proteomes of four bilaterian animals (human, mouse, Caenorhabditis elegans, and Drosophila melanogaster) and two non-animal outgroups (Acanthamoeba castellanii and Saccharomyces cerevisiae). We found that of several factors affecting mitochondrial proteome size, evolution of novel mitochondrial proteins in mammals and loss of ancestral proteins in protostomes were the main contributors. Interestingly, the gain and loss of the N-terminal mitochondrial targeting signal was not a major factor in the proteome size evolution.

MASS SPECTROMETRY-BASED MITOCHONDRIAL PROTEOMICS IN HUMAN OVARIAN CANCERS.

The prominent characteristics of mitochondria are highly dynamic and regulatory, which have crucial roles in cell metabolism, biosynthetic, senescence, apoptosis, and signaling pathways. Mitochondrial dysfunction might lead to multiple serious diseases, including cancer. Therefore, identification of mitochondrial proteins in cancer could provide a global view of tumorigenesis and progression. Mass spectrometry-based quantitative mitochondrial proteomics fulfils this task by enabling systems-wide, accurate, and quantitative analysis of mitochondrial protein abundance, and mitochondrial protein posttranslational modifications (PTMs). Multiple quantitative proteomics techniques, including isotope-coded affinity tag, stable isotope labeling with amino acids in cell culture, isobaric tags for relative and absolute quantification, tandem mass tags, and label-free quantification, in combination with different PTM-peptide enrichment methods such as TiO2 enrichment of tryptic phosphopeptides and antibody enrichment of other PTM-peptides, increase flexibility for researchers to study mitochondrial proteomes. This article reviews isolation and purification of mitochondria, quantitative mitochondrial proteomics, quantitative mitochondrial phosphoproteomics, mitochondrial protein-involved signaling pathway networks, mitochondrial phosphoprotein-involved signaling pathway networks, integration of mitochondrial proteomic and phosphoproteomic data with whole tissue proteomic and transcriptomic data and clinical information in ovarian cancers (OC) to in-depth understand its molecular mechanisms, and discover effective mitochondrial biomarkers and therapeutic targets for predictive, preventive, and personalized treatment of OC. This proof-of-principle model about OC mitochondrial proteomics is easily implementable to other cancer types. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Listeria monocytogenes Exploits Mitochondrial Contact Site and Cristae Organizing System Complex Subunit Mic10 To Promote Mitochondrial Fragmentation and Cellular Infection.

Mitochondrial function adapts to cellular demands and is affected by the ability of the organelle to undergo fusion and fission in response to physiological and nonphysiological cues. We previously showed that infection with the human bacterial pathogen Listeria monocytogenes elicits transient mitochondrial fission and a drop in mitochondrion-dependent energy production through a mechanism requiring the bacterial pore-forming toxin listeriolysin O (LLO). Here, we performed quantitative mitochondrial proteomics to search for host factors involved in L. monocytogenes-induced mitochondrial fission. We found that Mic10, a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex, is significantly enriched in mitochondria isolated from cells infected with wild-type but not with LLO-deficient L. monocytogenes Increased mitochondrial Mic10 levels did not correlate with upregulated transcription, suggesting a posttranscriptional mechanism. We then showed that Mic10 is necessary for L. monocytogenes-induced mitochondrial network fragmentation and that it contributes to L. monocytogenes cellular infection independently of MICOS proteins Mic13, Mic26, and Mic27. In conclusion, investigation of L. monocytogenes infection allowed us to uncover a role for Mic10 in mitochondrial fission.IMPORTANCE Pathogenic bacteria can target host cell organelles to take control of key cellular processes and promote their intracellular survival, growth, and persistence. Mitochondria are essential, highly dynamic organelles with pivotal roles in a wide variety of cell functions. Mitochondrial dynamics and function are intimately linked. Our previous research showed that Listeria monocytogenes infection impairs mitochondrial function and triggers fission of the mitochondrial network at an early infection stage, in a process that is independent of the presence of the main mitochondrial fission protein Drp1. Here, we analyzed how mitochondrial proteins change in response to L. monocytogenes infection and found that infection raises the levels of Mic10, a mitochondrial inner membrane protein involved in formation of cristae. We show that Mic10 is important for L. monocytogenes-dependent mitochondrial fission and infection of host cells. Our findings thus offer new insight into the mechanisms used by L. monocytogenes to hijack mitochondria to optimize host infection.

Omics Integration for Mitochondria Systems Biology.

Significance: Elucidation of the central importance of mitophagy in homeostasis of cells and organisms emphasizes that mitochondrial functions extend far beyond short-term needs for energy production. In mitochondria systems biology, the mitochondrial genome, proteome, and metabolome operate as a functional network in coordination of cell activities. Organization occurs through subnetworks that are interconnected by membrane potential, transport activities, allosteric and cooperative interactions, redox signaling mechanisms, rheostatic control by post-translational modifications, and metal ion homeostasis. These subnetworks enable use of varied energy precursors, defense against environmental stressors, and macromolecular rewiring to titrate energy production, biosynthesis, and detoxification according to cell-specific needs. Rewiring mechanisms, termed mitochondrial reprogramming, enhance fitness to respond to metabolic resources and challenges from the environment. Maladaptive responses can cause cell death. Maladaptive rewiring can cause disease. In cancer, adaptive rewiring can interfere with effective treatment. Recent Advances: Many recent advances have been facilitated by the development of new omics tools, which create opportunities to use data-driven analysis of omics data to address these complex adaptive and maladaptive mechanisms of mitochondrial reprogramming in human disease. Critical Issues: Application of omics integration to model systems reveals a critical role for metal ion homeostasis broadly impacting mitochondrial reprogramming. Importantly, data show that trans-omics associations are more robust and biologically relevant than single omics associations. Future Directions: Application of omics integration to mitophagy research creates new opportunities to link the complex, interactive functions of mitochondrial form and function in mitochondria systems biology.

Identification of ALDH6A1 as a Potential Molecular Signature in Hepatocellular Carcinoma via Quantitative Profiling of the Mitochondrial Proteome.

Various liver diseases, including hepatocellular carcinoma (HCC), have been linked to mitochondrial dysfunction, reduction of reactive oxygen species (ROS), and elevation of nitric oxide (NO). In this study, we subjected the human liver mitochondrial proteome to extensive quantitative proteomic profiling analysis and molecular characterization to identify potential signatures indicative of cancer cell growth and progression. Sequential proteomic analysis identified 2452 mitochondrial proteins, of which 1464 and 2010 were classified as nontumor and tumor (HCC) mitochondrial proteins, respectively, with 1022 overlaps. Further metabolic mapping of the HCC mitochondrial proteins narrowed our biological characterization to four proteins, namely, ALDH4A1, LRPPRC, ATP5C1, and ALDH6A1. The latter protein, a mitochondrial methylmalonate semialdehyde dehydrogenase (ALDH6A1), was most strongly suppressed in HCC tumor regions (∼10-fold decrease) in contrast to LRPPRC (∼6-fold increase) and was predicted to be present in plasma. Accordingly, we selected ALDH6A1 for functional analysis and engineered Hep3B cells to overexpress this protein, called ALDH6A1-O/E cells. Since ALDH6A1 is predicted to be involved in mitochondrial respiration, we assessed changes in the levels of NO and ROS in the overexpressed cell lines. Surprisingly, in ALDH6A1-O/E cells, NO was decreased nearly 50% but ROS was increased at a similar level, while the former was restored by treatment with S-nitroso-N-acetyl-penicillamine. The lactate levels were also decreased relative to control cells. Propidium iodide and Rhodamine-123 staining suggested that the decrease in NO and increase in ROS in ALDH6A1-O/E cells could be caused by depolarization of the mitochondrial membrane potential (ΔΨ). Taken together, our results suggest that hepatic neoplastic transformation appears to suppress the expression of ALDH6A1, which is accompanied by a respective increase and decrease in NO and ROS in cancer cells. Given the close link between ALDH6A1 suppression and abnormal cancer cell growth, this protein may serve as a potential molecular signature or biomarker of hepatocarcinogenesis and treatment responses.