Michael Ryan

Low mutant load of mitochondrial DNA G13513A mutation can cause Leigh's disease

Publisher: Wiley

Date: 11-08-2003

DOI: 10.1002/ANA.10687

Biallelic pathogenic variants in COX11 are associated with an infantile‐onset mitochondrial encephalopathy

Publisher: Hindawi Limited

Date: 07-09-2022

DOI: 10.1002/HUMU.24453

Abstract: Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q

Sideroflexin 4 is a complex I assembly factor that interacts with the MCIA complex and is required for the assembly of the ND2 module

Publisher: Proceedings of the National Academy of Sciences

Date: 25-03-2022

DOI: 10.1073/PNAS.2115566119

Abstract: Mitochondria are double-membraned eukaryotic organelles that house the proteins required for generation of ATP, the energy currency of cells. ATP generation within mitochondria is performed by five multisubunit complexes (complexes I to V), the assembly of which is an intricate process. Mutations in subunits of these complexes, or the suite of proteins that help them assemble, lead to a severe multisystem condition called mitochondrial disease. We show that SFXN4, a protein that causes mitochondrial disease when mutated, assists with the assembly of complex I. This finding explains why mutations in SFXN4 cause mitochondrial disease and is surprising because SFXN4 belongs to a family of amino acid transporter proteins, suggesting that it has undergone a dramatic shift in function through evolution.

Impaired folding of the mitochondrial small TIM chaperones induces clearance by the i-AAA protease

Publisher: Elsevier BV

Date: 12-2012

DOI: 10.1016/J.JMB.2012.09.019

Abstract: The intermembrane space of mitochondria contains a dedicated chaperone network-the small translocase of the inner membrane (TIM) family-for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX(3)C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no in idual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1).

Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

Publisher: Cold Spring Harbor Laboratory

Date: 26-07-2020

DOI: 10.1101/2020.07.26.222158

Abstract: Caveolae-associated protein 3 (cavin3), a putative tumor suppressor protein, is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays, we show a direct interaction between BRCA1 and cavin3. Association of BRCA1 and cavin3 occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Supporting a role in DNA repair, cavin3-deficient cells were sensitized to the effects of PARP inhibition, which compromises DNA repair, and showed reduced recruitment of the BRCA1 A-complex to UV DNA damage foci. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. We conclude that cavin3 functions together with BRCA1 in multiple pathways that contribute to tumorigenesis.

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

Publisher: Wiley

Date: 25-05-2020

DOI: 10.1111/CGE.13773

Deficiency in apoptosis-inducing factor recapitulates chronic kidney disease via aberrant mitochondrial homeostasis

Publisher: American Diabetes Association

Date: 28-01-2016

DOI: 10.2337/DB15-0864

Abstract: Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease.

Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathoge

Publisher: Wiley

Date: 15-12-2003

DOI: 10.1046/J.1432-1033.2003.03936.X

Abstract: The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer's disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N- and C-terminal fragments in association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1 proteins. Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin 1 complex mass, stability and association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1. Solubilization of mouse or human brain membranes with dodecyl-d-maltoside produced a 360-kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X-100 and SDS, but stable to reducing agent. Addition of 5 M urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and presenilin enhancer-2 were detected in the presenilin 1 complex following BN/PAGE, electroelution and second-dimension analysis. Anterior pharynx defective-1 was detected as an 18-kDa form and 9-kDa C-terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36-kDa band after presenilin 1 complex second-dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1-reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.

Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I

Publisher: Oxford University Press (OUP)

Date: 12-02-2015

DOI: 10.1093/HMG/DDV058

Abstract: Human mitochondrial complex I is the largest enzyme of the respiratory chain and is composed of 44 different subunits. Complex I subunits are encoded by both nuclear and mitochondrial (mt) DNA and their assembly requires a number of additional proteins. FAD-dependent oxidoreductase domain-containing protein 1 (FOXRED1) was recently identified as a putative assembly factor and FOXRED1 mutations in patients cause complex I deficiency however, its role in assembly is unknown. Here, we demonstrate that FOXRED1 is involved in mid-late stages of complex I assembly. In a patient with FOXRED1 mutations, the levels of mature complex I were markedly decreased, and a smaller ∼475 kDa subcomplex was detected. In the absence of FOXRED1, mtDNA-encoded complex I subunits are still translated and transiently assembled into a late stage ∼815 kDa intermediate but instead of transitioning further to the mature complex I, the intermediate breaks down to an ∼475 kDa complex. As the patient cells contained residual assembled complex I, we disrupted the FOXRED1 gene in HEK293T cells through TALEN-mediated gene editing. Cells lacking FOXRED1 had ∼10% complex I levels, reduced complex I activity, and were unable to grow on galactose media. Interestingly, overexpression of FOXRED1 containing the patient mutations was able to rescue complex I assembly. In addition, FOXRED1 was found to co-immunoprecipitate with a number of complex I subunits. Our studies reveal that FOXRED1 is a crucial component in the productive assembly of complex I and that mutations in FOXRED1 leading to partial loss of function cause defects in complex I biogenesis.

Caveolae respond to acute oxidative stress through membrane lipid peroxidation, cytosolic release of CAVIN1, and downstream regulation of NRF2

Publisher: Research Square Platform LLC

Date: 22-10-2021

DOI: 10.21203/RS.3.RS-963423/V1

Abstract: Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation (LPO) and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to LPO-induced ferroptosis. Thus, caveolae, via LPO and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response.

Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

Publisher: eLife Sciences Publications, Ltd

Date: 18-06-2021

DOI: 10.7554/ELIFE.61407

Abstract: Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions.

SILAC-based complexome profiling dissects the structural organization of the human respiratory supercomplexes in SCAFIKO cells

Publisher: Elsevier BV

Date: 07-2021

DOI: 10.1016/J.BBABIO.2021.148414

Abstract: The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the in idual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of in idual MRC complexes III and IV in the minor SC III

Biochemical and molecular diagnosis of mitochondrial respiratory chain disorders

Publisher: Elsevier BV

Date: 12-2004

DOI: 10.1016/J.BBABIO.2004.08.006

Mitochondrial OXPHOS complex assembly lines

Publisher: Springer Science and Business Media LLC

Date: 16-04-2018

DOI: 10.1038/S41556-018-0098-Z

Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia - MLASA syndrome

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.AJHG.2010.06.001

Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.CMET.2011.07.010

Loss of BIM increases mitochondrial oxygen consumption and lipid oxidation, reduces adiposity and improves insulin sensitivity in mice

Publisher: Springer Science and Business Media LLC

Date: 20-10-2018

DOI: 10.1038/CDD.2017.168

Identification of new channels by systematic analysis of the mitochondrial outer membrane

Publisher: Rockefeller University Press

Date: 15-09-2017

DOI: 10.1083/JCB.201706043

Abstract: The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.

Mutations in the gene encoding C8orf38 block complex i assembly by inhibiting production of the mitochondria-encoded subunit ND1

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1016/J.JMB.2011.10.012

Abstract: The assembly of complex I (NADH-ubiquinone oxidoreductase) is a complicated process, requiring the integration of 45 subunits encoded by both nuclear and mitochondrial DNAs into a structure of approximately 1 MDa. A number of "assembly factors" that aid complex I biogenesis have recently been described, including C8orf38. This protein was identified as an assembly factor by its evolutionary conservation in organisms containing complex I and by a C8orf38 mutation in a patient presenting with Leigh syndrome and isolated complex I deficiency. In this report, we have undertaken the characterization of C8orf38 and its role in complex I assembly. Analysis of mitochondria from fibroblasts of a patient harboring a C8orf38 mutation showed almost undetectable levels of steady-state complex I and defective biogenesis of the mtDNA-encoded subunit ND1. Complementation with wild-type C8orf38 restored the levels of both ND1 and complex I, confirming the C8orf38 mutation as the cause of the complex I defect in the patient. In the absence of ND1 in patient cells, early- and mid-stage intermediate complexes were still formed however, assembly of late-stage intermediates was impaired, indicating a convergence point in the assembly process. While C8orf38 appears to behave at a step in complex I biogenesis similar to that of the assembly factor C20orf7, complementation studies showed that both proteins are required for ND1 synthesis/stabilization. We conclude that C8orf38 is a crucial factor required for the translation and/or integration of ND1 into an early-stage assembly intermediate and that mutation of C8orf38 disrupts the initial stages of complex I biogenesis.

Next-generation sequencing in molecular diagnosis: NUBPL mutations highlight the challenges of variant detection and interpretation

Publisher: Hindawi Limited

Date: 22-12-2012

DOI: 10.1002/HUMU.21654

Abstract: Next-generation sequencing (NGS) is transitioning from being a research tool to being used in routine genetic diagnostics, where a major challenge is distinguishing which of many sequence variants in an in idual are truly pathogenic. We describe some limitations of in silico analyses of NGS data that emphasize the need for experimental confirmation. Using NGS, we recently identified an apparently homozygous missense mutation in NUBPL in a patient with mitochondrial complex I deficiency. Causality was established via lentiviral correction studies with wild-type NUBPL cDNA. NGS data, however, provided an incomplete understanding of the genetic abnormality. We show that the maternal allele carries an unbalanced inversion, while the paternal allele carries a branch-site mutation in addition to the missense mutation. We demonstrate that the branch-site mutation, which is present in approximately one of 120 control chromosomes, likely contributes to pathogenicity and may be one of the most common autosomal mutations causing mitochondrial dysfunction. Had these analyses not been performed following NGS, the original missense mutation may be incorrectly annotated as pathogenic and a potentially common pathogenic variant not detected. It is important that locus-specific databases contain accurate information on pathogenic variation. NGS data, therefore, require rigorous experimental follow-up to confirm mutation pathogenicity.

COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2

Publisher: Oxford University Press (OUP)

Date: 09-07-2015

DOI: 10.1093/HMG/DDV265

Abstract: Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.

A patient with homozygous nonsense variants in two Leigh syndrome disease genes: Distinguishing a dual diagnosis from a hypomorphic protein‐truncating variant

Publisher: Hindawi Limited

Date: 13-04-2019

DOI: 10.1002/HUMU.23753

Mitochondrial fusion: Reaching the end of mitofusin’s tether

Publisher: Rockefeller University Press

Date: 23-11-2016

DOI: 10.1083/JCB.201611048

Abstract: In this issue, Qi et al. (2016. J. Cell Biol. 0.1083/jcb.201609019) provide structural insights into the mechanisms of mitochondrial outer membrane fusion by investigating the structure of mitofusin 1 (MFN1). This work proposes a new model to explain the important and elusive process of MFN-mediated mitochondrial fusion.

Resolving mitochondrial cristae: introducing a new model into the fold

Publisher: EMBO

Date: 22-06-2020

DOI: 10.15252/EMBJ.2020105714

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Publisher: Public Library of Science (PLoS)

Date: 19-05-2016

DOI: 10.1371/JOURNAL.PGEN.1006033

Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context

Publisher: Elsevier BV

Date: 12-2021

DOI: 10.1016/J.CMET.2021.11.001

Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission

Publisher: Springer Science and Business Media LLC

Date: 07-12-2018

DOI: 10.1038/S41467-018-07543-W

Abstract: Dynamin-related protein 1 (Drp1) is essential for mitochondrial and peroxisomal fission. Recent studies propose that Drp1 does not sever but rather constricts mitochondrial membranes allowing dynamin 2 (Dnm2) to execute final scission. Here, we report that unlike Drp1, Dnm2 is dispensable for peroxisomal and mitochondrial fission, as these events occurred in Dnm2 knockout cells. Fission events were also observed in mouse embryonic fibroblasts lacking Dnm1, 2 and 3. Using reconstitution experiments on preformed membrane tubes, we show that Drp1 alone both constricts and severs membrane tubes. Scission required the membrane binding, self-assembling and GTPase activities of Drp1 and occurred on tubes up to 250 nm in radius. In contrast, Dnm2 exhibited severely restricted fission capacity with occasional severing of tubes below 50 nm in radius. We conclude that Drp1 has both membrane constricting and severing abilities and is the dominant dynamin performing mitochondrial and peroxisomal fission.

Inhibition of Bak activation by VDAC2 is dependent on the Bak transmembrane anchor

Publisher: Elsevier BV

Date: 11-2010

DOI: 10.1074/JBC.M110.159301

Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6

Publisher: Life Science Alliance, LLC

Date: 12-09-2019

DOI: 10.26508/LSA.201900458

Abstract: Assembly factors play key roles in the biogenesis of many multi-subunit protein complexes regulating their stability, activity, and the incorporation of essential cofactors. The human assembly factor Coa6 participates in the biogenesis of the Cu A site in complex IV (cytochrome c oxidase, COX). Patients with mutations in Coa6 suffer from mitochondrial disease due to complex IV deficiency. Here, we present the crystal structures of human Coa6 and the pathogenic W59C Coa6-mutant protein. These structures show that Coa6 has a 3-helical bundle structure, with the first 2 helices tethered by disulfide bonds, one of which likely provides the copper-binding site. Disulfide-mediated oligomerization of the W59C Coa6 protein provides a structural explanation for the loss-of-function mutation.

Mitofusins 'bridge' the gap between oxidative stress and mitochondrial hyperfusion

Publisher: EMBO

Date: 11-09-2012

DOI: 10.1038/EMBOR.2012.132

Splitting up the powerhouse: structural insights into the mechanism of mitochondrial fission

Publisher: Springer Science and Business Media LLC

Date: 10-06-2015

DOI: 10.1007/S00018-015-1950-Y

Abstract: Mitochondria are dynamic organelles whose shape is regulated by the opposing processes of fission and fusion, operating in conjunction with organelle distribution along the cytoskeleton. The importance of fission and fusion homeostasis has been highlighted by a number of disease states linked to mutations in proteins involved in regulating mitochondrial morphology, in addition to changes in mitochondrial dynamics in Alzheimer's, Huntington's and Parkinson's diseases. While a number of mitochondrial morphology proteins have been identified, how they co-ordinate to assemble the fission apparatus is not clear. In addition, while the master mediator of mitochondrial fission, dynamin-related protein 1, is conserved throughout evolution, the adaptor proteins involved in its mitochondrial recruitment are not. This review focuses on our current understanding of mitochondrial fission and the proteins that regulate this process in cell homeostasis, with a particular focus on the recent mechanistic insights based on protein structures.

Mitochondria: Organization of Respiratory Chain Complexes Becomes Cristae-lized

Publisher: Elsevier BV

Date: 11-2013

DOI: 10.1016/J.CUB.2013.09.035

Abstract: For over 100 years mitochondria have been known for their distinctive morphology featuring elaborately folded cristae, and their role as 'the powerhouse of the cell'. New research shows that these two characteristics are more dependent on each other than previously thought.

A founder mutation in PET100 causes isolated complex IV deficiency in lebanese individuals with Leigh syndrome

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1016/J.AJHG.2013.12.015

Mapping biological composition through quantitative phase and absorption X-ray ptychography

Publisher: Springer Science and Business Media LLC

Date: 28-10-2014

DOI: 10.1038/SREP06796

Recent advances into the understanding of mitochondrial fission

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.BBAMCR.2012.05.002

Abstract: Mitochondria exist as a highly dynamic tubular network, and their morphology is governed by the delicate balance between frequent fusion and fission events, as well as by interactions with the cytoskeleton. Alterations in mitochondrial morphology are associated with changes in metabolism, cell development and cell death, whilst several human pathologies have been associated with perturbations in the cellular machinery that coordinate these processes. Mitochondrial fission also contributes to ensuring the proper distribution of mitochondria in response to the energetic requirements of the cell. The master mediator of fission is Dynamin related protein 1 (Drp1), which polymerises and constricts mitochondria to facilitate organelle ision. The activity of Drp1 at the mitochondrial outer membrane is regulated through post-translational modifications and interactions with mitochondrial receptor and accessory proteins. This review will concentrate on recent advances made in delineating the mechanism of mitochondrial fission, and will highlight the importance of mitochondrial fission in health and disease. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.

Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1016/J.MITO.2012.07.084

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy

Publisher: Proceedings of the National Academy of Sciences

Date: 02-04-2012

DOI: 10.1073/PNAS.1113987109

Abstract: Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6 gt/gt mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6 gt/gt mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6 gt/gt mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6 gt/gt hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6 gt/gt mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6 gt/gt isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of in iduals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches.

De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency

Publisher: Wiley

Date: 12-2003

DOI: 10.1002/ANA.10787

Abstract: Both nuclear and mitochondrial DNA mutations can cause energy generation disorders. Respiratory chain complex I deficiency is the most common energy generation disorder and a frequent cause of infantile mitochondrial encephalopathies such as Leigh's disease and lethal infantile mitochondrial disease. Most such cases have been assumed to be caused by nuclear gene defects, but recently an increasing number have been shown to be caused by mutations in the mitochondrially encoded complex I subunit genes ND4, ND5, and ND6. We report the first four cases of infantile mitochondrial encephalopathies caused by mutations in the ND3 subunit gene. Three unrelated children have the same novel heteroplasmic mutation (T10158C), only the second mutation reported in ND3, and one has the previously identified T10191C mutation. Both mutations cause disproportionately greater reductions in enzyme activity than in the amount of fully assembled complex I, suggesting the ND3 subunit plays an unknown but important role in electron transport, proton pumping, or ubiquinone binding. Three cases appear to have a de novo mutation, with no mutation detected in maternal relatives. Mitochondrial DNA disease may be considerably more prevalent in the pediatric population than currently predicted and should be considered in patients with infantile mitochondrial encephalopathies and complex I deficiency.

Mitochondrial COA7 is a heme-binding protein involved in the early stages of complex IV assembly

Publisher: Cold Spring Harbor Laboratory

Date: 11-06-2021

DOI: 10.1101/2021.06.10.447992

Abstract: Cytochrome c oxidase assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked in patients to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here we show that the absence of COA7 leads to arrest of the complex IV assembly pathway at the initial step where the COX1 module is built, which requires incorporation of copper and heme cofactors. In solution, purified COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. Surprisingly, the crystal structure of COA7, determined to 2.4 Å resolution, reveals a ‘banana-shaped’ molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds, with a structure entirely distinct from proteins with characterized heme binding activities. We therefore propose a role for COA7 in heme binding/chaperoning in the mitochondrial intermembrane space, this activity being crucial for and providing a missing link in complex IV biogenesis. Assembly factors play key roles in the biogenesis of many mitochondrial protein complexes regulating their stability, activity and incorporation of essential cofactors. COA7 is a metazoan-specific assembly factor, the absence or mutation of which in humans accompanies complex IV assembly defects and neurological conditions. Here we report the crystal structure of COA7 to 2.4 Å resolution, revealing a ‘banana-shaped’ molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds. Characterization of pathogenic variants reveals significantly lower stabilities, correlating with the associated disease outcomes. Fascinatingly, COA7 binds heme with micromolar affinity, despite the fact that the protein structure does not resemble previously characterized heme-binding proteins. This provides a possible missing link for heme handling in the mitochondrial intermembrane space.

The Mitochondrial Acyl-carrier Protein Interaction Network Highlights Important Roles for LYRM Family Members in Complex I and Mitoribosome Assembly

Publisher: Elsevier BV

Date: 2020

DOI: 10.1074/MCP.RA119.001784

BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis

Publisher: American Association for the Advancement of Science (AAAS)

Date: 23-02-2018

DOI: 10.1126/SCIENCE.AAO6047

Abstract: Mitochondrial DNA (mtDNA) is a potent damage-associated molecular pattern that, if it reaches the cytoplasm or extracellular milieu, triggers innate immune pathways. mtDNA signaling has been implicated in a wide range of diseases however, the mechanisms of mtDNA release are unclear, and the process has not been observed in real time thus far. McArthur et al. used live-cell lattice light-sheet microscopy to look at mtDNA release during intrinsic apoptosis. Activation of the pro-death proteins BAK and BAX resulted in the formation of large macro-pores in the mitochondrial outer membrane. These massive holes caused the inner mitochondrial membrane to balloon out into the cytoplasm, resulting in mitochondrial herniation. This process allowed the contents of the mitochondrial matrix, including mtDNA, to escape into the cytoplasm. Science , this issue p. eaao6047

Optic atrophy?associated TMEM126A is an assembly factor for the ND4-module of mitochondrial complex I

Publisher: Proceedings of the National Academy of Sciences

Date: 20-04-2021

DOI: 10.1073/PNAS.2019665118

Abstract: Mitochondria are critical organelles that play a central role in energy conversion in cells. Mutations in genes encoding mitochondrial proteins can lead to disease. Here, we discover that the optic atrophy–associated mitochondrial disease gene TMEM126A plays a role in the assembly of the first enzyme of the respiratory chain, complex I. Critically, we show that TMEM126A interacts in a complex with newly synthesized ND4, a highly hydrophobic subunit of complex I that is encoded by mitochondrial DNA. Our findings expand the molecular machinery required to build complex I from its constituent subunits and elucidates the function of a gene mutated in mitochondrial disease.

Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria

Publisher: Springer New York

Date: 2016

DOI: 10.1007/978-1-4939-3040-1_9

Abstract: In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission.

Publisher: Rockefeller University Press

Date: 10-02-2014

DOI: 10.1083/JCB.201311014

Abstract: Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.

MiD49 and MiD51, new components of the mitochondrial fission machinery

Publisher: EMBO

Date: 21-04-2011

DOI: 10.1038/EMBOR.2011.54

Mitochondria – The energy powerhouses

Publisher: Elsevier BV

Date: 04-2018

DOI: 10.1016/J.SEMCDB.2017.09.038

Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit

Publisher: Springer Science and Business Media LLC

Date: 23-06-2022

DOI: 10.1038/S42003-022-03568-6

Abstract: Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10 E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance.

Mitochondrial fission protein Drp1 inhibition promotes cardiac mesodermal differentiation of human pluripotent stem cells

Publisher: Springer Science and Business Media LLC

Date: 05-03-2018

DOI: 10.1038/S41420-018-0042-9

Abstract: Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or M i-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with M i-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Publisher Correction: Metabolic characteristics of CD8+ T cell subsets in young and aged individuals are not predictive of functionality

Publisher: Springer Science and Business Media LLC

Date: 09-07-2020

DOI: 10.1038/S41467-020-17441-9

Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nicotinamide riboside attenuates age-associated metabolic and functional changes in hematopoietic stem cells.

Publisher: Springer Science and Business Media LLC

Date: 11-05-2021

DOI: 10.1038/S41467-021-22863-0

Abstract: With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, d ing of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

Metabolic characteristics of CD8+ T cell subsets in young and aged individuals are not predictive of functionality

Publisher: Springer Science and Business Media LLC

Date: 05-06-2020

DOI: 10.1038/S41467-020-16633-7

Abstract: Virtual memory T (T VM ) cells are antigen-naïve CD8 + T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (T MEM ) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of T VM cells and their altered functionality with age, here we investigate T VM cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of T VM , but not T MEM , cells and it increases with age in both subsets. The elevated SRC observed in aged mouse T VM cells and human CD8 + T cells from older in iduals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of T VM , but not T MEM , cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8 + T cells.

Mitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly

Publisher: Proceedings of the National Academy of Sciences

Date: 24-02-2022

DOI: 10.1073/PNAS.2110357119

Abstract: Assembly factors play key roles in the biogenesis of mitochondrial protein complexes, regulating their stabilities, activities, and incorporation of essential cofactors. Cytochrome c oxidase assembly factor 7 (COA7) is a metazoan-specific assembly factor, the absence or mutation of which in humans accompanies complex IV assembly defects and neurological conditions. Here, we report the crystal structure of COA7 to 2.4 Å resolution, revealing a banana-shaped molecule composed of five helix-turn-helix (α/α) repeats. COA7 binds heme with micromolar affinity, even though the protein structure does not resemble previously characterized heme-binding proteins. The heme-bound COA7 can redox cycle between oxidation states Fe(II) and Fe(III) and shows disulfide reductase activity toward copper binding assembly factors. We propose that COA7 functions to facilitate the biogenesis of the binuclear copper site (Cu A ) of complex IV.

Assembly factors of human mitochondrial complex I and their defects in disease

Publisher: Wiley

Date: 21-04-2010

DOI: 10.1002/IUB.335

Abstract: NADH-ubiquinone oxidoreductase (complex I) is a large, multimeric enzyme complex involved in the generation of ATP by oxidative phosphorylation. Complex I is comprised of 45 subunits which must be assembled together in a coordinated process to form the mature holoenzyme. In recent years, much progress has been made into understanding how complex I is assembled and the work provides potential insights into the biogenesis of other multisubunit membrane complexes. For complex I assembly to proceed effectively, a group of proteins termed "assembly factors" are required. A number of these assembly factors have now been identified and characterized however, their exact roles in complex I biogenesis are not yet fully understood. This review summarizes the current model of human complex I assembly and the roles played by different assembly factors at early, mid, and late assembly stages. Defects in assembly factors which disrupt complex I assembly and contribute to human disease pathogenesis will also be discussed.

Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high-intensity exercise training

Publisher: Wiley

Date: 23-04-2017

DOI: 10.1113/JP273950

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

Publisher: EMBO

Date: 20-07-2022

DOI: 10.15252/EMBJ.2022110784

Abstract: 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.

Mitochondrial protein import machineries and lipids: A functional connection

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.BBAMEM.2010.08.003

Abstract: Protein trafficking and translocation are essential processes in even the simplest living cells. The compartmentalisation within eukaryotic cells places a very high demand on the fidelity of protein trafficking and translocation, since a large percentage of the cell's protein complement is inserted into, or translocated across membranes. Indeed, most mitochondrial proteins are imported from the cytosol into the organelle and reach their final destination with the assistance of versatile translocation machineries. The first components involved in mitochondrial protein import were identified about 20years ago and over the last two decades many new factors and machineries have been brought to light. However, in spite of these discoveries we still have much to explore regarding the molecular mechanisms that distinguish the different mitochondrial import pathways. In particular, an open question that requires deeper exploration is the role of lipids and lipid modifying enzymes in this process. Mitochondrial biogenesis requires the coordinated synthesis and import of both proteins and phospholipids, however, these have typically been considered as distinct research fields. Recent findings have placed phospholipids at the forefront of research dealing with mitochondrial biogenesis, in particular their role in the regulation of mitochondrial transport machineries. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Publisher: Elsevier BV

Date: 08-2017

DOI: 10.1016/J.MOLCEL.2017.06.014

Abstract: Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

FunRich: An open access standalone functional enrichment and interaction network analysis tool

Publisher: Wiley

Date: 17-06-2015

DOI: 10.1002/PMIC.201400515

Abstract: As high-throughput techniques including proteomics become more accessible to in idual laboratories, there is an urgent need for a user-friendly bioinformatics analysis system. Here, we describe FunRich, an open access, standalone functional enrichment and network analysis tool. FunRich is designed to be used by biologists with minimal or no support from computational and database experts. Using FunRich, users can perform functional enrichment analysis on background databases that are integrated from heterogeneous genomic and proteomic resources (>1.5 million annotations). Besides default human specific FunRich database, users can download data from the UniProt database, which currently supports 20 different taxonomies against which enrichment analysis can be performed. Moreover, the users can build their own custom databases and perform the enrichment analysis irrespective of organism. In addition to proteomics datasets, the custom database allows for the tool to be used for genomics, lipidomics and metabolomics datasets. Thus, FunRich allows for complete database customization and thereby permits for the tool to be exploited as a skeleton for enrichment analysis irrespective of the data type or organism used. FunRich (www.funrich.org) is user-friendly and provides graphical representation (Venn, pie charts, bar graphs, column, heatmap and doughnuts) of the data with customizable font, scale and color (publication quality).

OXA 1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect

Publisher: EMBO

Date: 10-09-2018

DOI: 10.15252/EMMM.201809060

High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production

Publisher: Springer Science and Business Media LLC

Date: 13-04-2023

DOI: 10.1038/S41467-023-37780-7

Abstract: Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model ( Pf4-Srsf3 Δ/Δ ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.

Optic Atrophy-associated TMEM126A is an assembly factor for the ND4-module of Mitochondrial Complex I

Publisher: Cold Spring Harbor Laboratory

Date: 18-09-2020

DOI: 10.1101/2020.09.18.303255

Abstract: Mitochondrial disease is a debilitating condition with a erse genetic aetiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labelling interaction studies reveal that TMEM126A associates with the newly synthesised mtDNA-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. Importantly, we clarify that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I, helping to explain the differences in disease aetiology observed between these two genes.

The constriction and scission machineries involved in mitochondrial fission

Publisher: The Company of Biologists

Date: 2017

DOI: 10.1242/JCS.199562

Abstract: A key event in the evolution of eukaryotic cells was the engulfment of an aerobic bacterium by a larger anaerobic archaebacterium, leading to a close relationship between the host and the newly formed endosymbiont. Mitochondria, originating from this event, have evolved to be the main place of cellular ATP production. Maintaining elements of their independence, mitochondria undergo growth and ision in the cell, thereby ensuring that new daughter cells inherit a mitochondrial complement. Mitochondrial ision is also important for other processes, including quality control, mitochondrial (mt)DNA inheritance, transport and cell death. However, unlike bacterial fission, which uses a dynamin-related protein to constrict the membrane at its inner face, mitochondria use dynamin and dynamin-related proteins to constrict the outer membrane from the cytosolic face. In this Review, we summarize the role of proteins from the dynamin superfamily in mitochondrial ision. This includes recent findings highlighting that dynamin-2 (Dnm2) is involved in mitochondrial scission, which led to the reappraisal of the role of dynamin-related protein 1 (Drp1 also known as Dnm1l) and its outer membrane adaptors as components of the mitochondrial constriction machinery along with ER components and actin.

HIGD2A is Required for Assembly of the COX3 Module of Human Mitochondrial Complex IV

Publisher: Elsevier BV

Date: 07-2020

DOI: 10.1074/MCP.RA120.002076

Function and regulation of the divisome for mitochondrial fission

Publisher: Springer Science and Business Media LLC

Date: 03-02-2021

DOI: 10.1038/S41586-021-03214-X

Abstract: Mitochondria form dynamic networks in the cell that are balanced by the flux of iterative fusion and fission events of the organelles. It is now appreciated that mitochondrial fission also represents an end-point event in a signalling axis that allows cells to sense and respond to external cues. The fission process is orchestrated by membrane-associated adaptors, influenced by organellar and cytoskeletal interactions and ultimately executed by the dynamin-like GTPase DRP1. Here we invoke the framework of the 'mitochondrial isome', which is conceptually and operationally similar to the bacterial cell- ision machinery. We review the functional and regulatory aspects of the mitochondrial isome and, within this framework, parse the core from the accessory machinery. In so doing, we transition from a phenomenological to a mechanistic understanding of the fission process.

Caveolae respond to acute oxidative stress through membrane lipid peroxidation, cytosolic release of CAVIN1, and downstream regulation of NRF2

Publisher: Cold Spring Harbor Laboratory

Date: 09-06-2021

DOI: 10.1101/2021.06.09.447684

Abstract: Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response.

The regulation of mitochondrial morphology: Intricate mechanisms and dynamic machinery

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1016/J.CELLSIG.2011.05.021

Abstract: Mitochondria typically form a reticular network radiating from the nucleus, creating an interconnected system that supplies the cell with essential energy and metabolites. These mitochondrial networks are regulated through the complex coordination of fission, fusion and distribution events. While a number of key mitochondrial morphology proteins have been identified, the precise mechanisms which govern their activity remain elusive. Moreover, post translational modifications including ubiquitination, phosphorylation and sumoylation of the core machinery are thought to regulate both fusion and ision of the network. These proteins can undergo several different modifications depending on cellular signals, environment and energetic demands of the cell. Proteins involved in mitochondrial morphology may also have dual roles in both dynamics and apoptosis, with regulation of these proteins under tight control of the cell to ensure correct function. The absolute reliance of the cell on a functional mitochondrial network is highlighted in neurons, which are particularly vulnerable to any changes in organelle dynamics due to their unique biochemical requirements. Recent evidence suggests that defects in the shape or distribution of mitochondria correlate with the progression of neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's disease. This review focuses on our current understanding of the mitochondrial morphology machinery in cell homeostasis, apoptosis and neurodegeneration, and the post translational modifications that regulate these processes.

Proteomic and metabolomic analyses of mitochondrial complex I-deficient mouse model generated by spontaneous B2 short interspersed nuclear element (SINE) insertion into NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) gene

Publisher: Elsevier BV

Date: 06-2012

DOI: 10.1074/JBC.M111.327601

Dissecting the Roles of Mitochondrial Complex I Intermediate Assembly (MCIA) Complex Factors in the Biogenesis of Complex I

Publisher: Cold Spring Harbor Laboratory

Date: 17-10-2019

DOI: 10.1101/808311

Abstract: Mitochondrial Complex I harbors 7 mitochondrial and 38 nuclear-encoded subunits. Its biogenesis requires the assembly and integration of distinct intermediate modules, mediated by numerous assembly factors. The Mitochondrial Complex I Intermediate Assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module. The role of the MCIA complex and the involvement of other proteins in the biogenesis of this module is unclear. Cell knockout studies reveal that while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centred on ACAD9. We also identify TMEM186 and COA1 as bona fide components of the MCIA complex with loss of either resulting in in MCIA complex defects and reduced complex I assembly. TMEM186 enriches with newly translated ND3, while COA1 enriches with ND2. Our findings provide new functional insights into the essential nature of the MCIA complex in complex I assembly.

Mitochondrial dynamics in health and disease

Publisher: Wiley

Date: 04-2021

DOI: 10.1002/1873-3468.14077

Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.AJHG.2018.08.013

Hydrogen Production from Methane Cracking in Dielectric Barrier Discharge Catalytic Plasma Reactor using a Nanocatalyst

Publisher: MDPI AG

Date: 10-11-2020

DOI: 10.20944/PREPRINTS202011.0316.V1

Abstract: The study experimentally investigated a novel approach for producing hydrogen from methane cracking in dielectric barrier discharge catalytic plasma reactor using a nanocatalyst. Plasma-catalytic methane (CH4) cracking was undertaken in a dielectric barrier discharge (DBD) catalytic plasma reactor using Ni/MgAl2O4. The Ni/MgAl2O4 was synthesised through co-precipitation followed customised hydrothermal method. The physicochemical properties of the catalyst were examined using X-ray diffraction (XRD), scanning electron microscopy - energy dispersive X-ray spectrometry (SEM-EDX) and thermogravimetric analysis (TGA). The Ni/MgAl2O4 shows a porous structure spinel MgAl2O4 and thermal stability. In the catalytic-plasma methane cracking, the Ni/MgAl2O4 shows 80% of the maximum conversion of CH4 with H2 selectivity 75%. Furthermore, the stability of the catalyst was encouraging 16 hours with CH4 conversion above 75%, and the selectivity of H2 was above 70%. This is attributed to the synergistic effect of the catalyst and plasma. The plasma-catalytic CH4 cracking is a promising technology for the simultaneous H2 and carbon nanotubes (CNTs) production for energy storage applications.

Analysis of ER-mitochondria contacts using correlative fluorescence microscopy and soft X-ray tomography of mammalian cells

Publisher: The Company of Biologists

Date: 2015

DOI: 10.1242/JCS.169136

Abstract: Mitochondrial fission is important for organelle transport, quality control and apoptosis. Changes to the fission process can manifest itself in a wide variety of neurological diseases. In mammals, mitochondrial fission is executed by the GTPase dynamin-related protein 1 (Drp1) that oligomerizes around mitochondria and constricts the organelle. The mitochondrial outer membrane proteins Mff, MiD49 and MiD51 are involved in mitochondrial fission by recruiting Drp1 from the cytosol to the organelle surface. In addition, ER tubules have been shown to wrap around and constrict mitochondria prior to a fission event. Up to now, the presence of MiD49/51 at ER-mitochondrial ision (ERMD) foci has not been established. Here we incorporate confocal live cell imaging with novel correlative cryogenic fluorescence microscopy and soft x-ray tomography to link MiD49/51 and ER involvement in mitochondrial fission. We gain further insight into this complex process and characterize the 3D structure of ER-mitochondria contact sites.

VDAC2 enables BAX to mediate apoptosis and limit tumor development

Publisher: Springer Science and Business Media LLC

Date: 26-11-2018

DOI: 10.1038/S41467-018-07309-4

Abstract: Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.

Mitochondrial dysfunction in a novel form of autosomal recessive ataxia

Publisher: Elsevier BV

Date: 05-2013

DOI: 10.1016/J.MITO.2012.11.006

Abstract: Defects in the recognition and/or repair of damage to DNA are responsible for a sub-group of autosomal recessive ataxias. Included in this group is a novel form of ataxia with oculomotor apraxia characterised by sensitivity to DNA damaging agents, a defect in p53 stabilisation, oxidative stress and resistance to apoptosis. We provide evidence here that the defect in this patient's cells is at the level of the mitochondrion. Mitochondrial membrane potential was markedly reduced in cells from the patient and ROS levels were elevated. This was accompanied by lipid peroxidation of mitochondrial proteins involved in electron transport and RNA synthesis. However, no gross changes or alteration in composition or activity of mitochondrial electron transport complexes was evident. Sequencing of mitochondrial DNA revealed a mutation, I349T, in the mitochondrial cytochrome b gene. These results describe a patient with an apparently novel form of AOA characterised by a defect at the level of the mitochondrion.

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

Publisher: EMBO

Date: 05-06-2023

DOI: 10.15252/EMBR.202256430

Abstract: 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.

Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission.

Publisher: The Company of Biologists

Date: 2016

DOI: 10.1242/JCS.185165

Abstract: Cytosolic Dynamin-related protein 1 (Drp1) is required for both mitochondrial and peroxisomal fission. Drp1-dependent ision of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking adaptors MiD49, MiD51, MFF and Fis1. Increased mitochondrial connectivity was observed following loss of in idual adaptors while this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49/MiD51 showing a more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we find that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

Monash University

Location: Australia

View Organisation

Discovery Projects - Grant ID: DP0208309

Start Date: 2002

End Date: 01-2005

Amount: $180,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0342596

Start Date: 2003

End Date: 01-2006

Amount: $330,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0771309

Start Date: 2007

End Date: 12-2009

Amount: $369,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP220103559

Start Date: 02-2022

End Date: 02-2025

Amount: $540,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0775758

Start Date: 2007

End Date: 12-2007

Amount: $587,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0775708

Start Date: 2007

End Date: 12-2007

Amount: $289,680.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0560722

Start Date: 2005

End Date: 12-2006

Amount: $512,744.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE0561787

Start Date: 12-2005

End Date: 12-2014

Amount: $16,700,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160102176

Start Date: 2016

End Date: 12-2018

Amount: $433,300.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1094973

Start Date: 2010

End Date: 12-2012

Amount: $328,348.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0454399

Start Date: 02-2004

End Date: 06-2008

Amount: $41,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP190103068

Start Date: 02-2019

End Date: 02-2022

Amount: $405,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP220102030

Start Date: 05-2022

End Date: 05-2025

Amount: $550,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE220100138

Start Date: 05-2022

End Date: 12-2022

Amount: $800,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100037

Start Date: 02-2012

End Date: 12-2012

Amount: $350,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100152

Start Date: 01-2012

End Date: 12-2012

Amount: $360,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989920

Start Date: 2009

End Date: 12-2009

Amount: $750,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100001

Start Date: 2018

End Date: 12-2018

Amount: $345,475.00

Funder: Australian Research Council