Biochemistry and Pharmacology - Research Publications
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ItemNo Preview AvailableMitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins(SPRINGERNATURE, 2023-03)Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.
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ItemMitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly(NATL ACAD SCIENCES, 2022-02-25)Cytochrome c oxidase (COX) 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 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 loss of COA7 blocks complex IV assembly after the initial step where the COX1 module is built, progression from which requires the incorporation of copper and addition of the COX2 and COX3 modules. 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. COA7 interacts transiently with the copper metallochaperones SCO1 and SCO2 and catalyzes the reduction of disulfide bonds within these proteins, which are crucial for copper relay to COX2. COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. We therefore propose that COA7 is a heme-binding disulfide reductase for regenerating the copper relay system that underpins complex IV assembly.
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ItemApplying Sodium Carbonate Extraction Mass Spectrometry to Investigate Defects in the Mitochondrial Respiratory Chain(FRONTIERS MEDIA SA, 2022-03-01)Mitochondria are complex organelles containing 13 proteins encoded by mitochondrial DNA and over 1,000 proteins encoded on nuclear DNA. Many mitochondrial proteins are associated with the inner or outer mitochondrial membranes, either peripherally or as integral membrane proteins, while others reside in either of the two soluble mitochondrial compartments, the mitochondrial matrix and the intermembrane space. The biogenesis of the five complexes of the oxidative phosphorylation system are exemplars of this complexity. These large multi-subunit complexes are comprised of more than 80 proteins with both membrane integral and peripheral associations and require soluble, membrane integral and peripherally associated assembly factor proteins for their biogenesis. Mutations causing human mitochondrial disease can lead to defective complex assembly due to the loss or altered function of the affected protein and subsequent destabilization of its interactors. Here we couple sodium carbonate extraction with quantitative mass spectrometry (SCE-MS) to track changes in the membrane association of the mitochondrial proteome across multiple human knockout cell lines. In addition to identifying the membrane association status of over 840 human mitochondrial proteins, we show how SCE-MS can be used to understand the impacts of defective complex assembly on protein solubility, giving insights into how specific subunits and sub-complexes become destabilized.
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ItemHigh-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content(NATURE PORTFOLIO, 2021-12-03)Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our findings suggest enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training-induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous findings.
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ItemNo Preview AvailableOptic atrophy?associated TMEM126A is an assembly factor for the ND4-module of mitochondrial complex I(NATL ACAD SCIENCES, 2021-04-27)Mitochondrial disease is a debilitating condition with a diverse genetic etiology. 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-labeling interaction studies reveal that TMEM126A associates with the newly synthesized mitochondrial DNA (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. In addition, we find that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I.
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ItemHIGD2A is Required for Assembly of the COX3 Module of Human Mitochondrial Complex IV(American Society for Biochemistry and Molecular Biology, 2020-07-01)Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. Several assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. Although in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.
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ItemNo Preview AvailableAbnormalities of mitochondrial dynamics and bioenergetics in neuronal cells from CDKL5 deficiency disorder(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2021-07)CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by pathogenic variants in the Cyclin-dependent kinase-like 5 (CDKL5) gene, resulting in dysfunctional CDKL5 protein. It predominantly affects females and causes seizures in the first few months of life, ultimately resulting in severe intellectual disability. In the absence of targeted therapies, treatment is currently only symptomatic. CDKL5 is a serine/threonine kinase that is highly expressed in the brain, with a critical role in neuronal development. Evidence of mitochondrial dysfunction in CDD is gathering, but has not been studied extensively. We used human patient-derived induced pluripotent stem cells with a pathogenic truncating mutation (p.Arg59*) and CRISPR/Cas9 gene-corrected isogenic controls, differentiated into neurons, to investigate the impact of CDKL5 mutation on cellular function. Quantitative proteomics indicated mitochondrial defects in CDKL5 p.Arg59* neurons, and mitochondrial bioenergetics analysis confirmed decreased activity of mitochondrial respiratory chain complexes. Additionally, mitochondrial trafficking velocity was significantly impaired, and there was a higher percentage of stationary mitochondria. We propose mitochondrial dysfunction is contributing to CDD pathology, and should be a focus for development of targeted treatments for CDD.
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ItemNo Preview AvailableIntact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias(AMER SOC HEMATOLOGY, 2021-05-20)Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.
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ItemDissecting the Roles of Mitochondrial Complex I Intermediate Assembly Complex Factors in the Biogenesis of Complex I(CELL PRESS, 2020-04-21)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 centered on ACAD9. We also identify TMEM186 and COA1 as bona fide components of the MCIA complex with loss of either resulting in MCIA complex defects and reduced complex I assembly. TMEM186 enriches with newly translated ND3, and COA1 enriches with ND2. Our findings provide new functional insights into the essential nature of the MCIA complex in complex I assembly.
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ItemMetabolic remodeling of dystrophic skeletal muscle reveals biological roles for dystrophin and utrophin in adaptation and plasticity(ELSEVIER, 2021-03)OBJECTIVES: Preferential damage to fast, glycolytic myofibers is common in many muscle-wasting diseases, including Duchenne muscular dystrophy (DMD). Promoting an oxidative phenotype could protect muscles from damage and ameliorate the dystrophic pathology with therapeutic relevance, but developing efficacious strategies requires understanding currently unknown biological roles for dystrophin and utrophin in dystrophic muscle adaptation and plasticity. METHODS: Combining whole transcriptome RNA sequencing and mitochondrial proteomics with assessments of metabolic and contractile function, we investigated the roles of dystrophin and utrophin in fast-to-slow muscle remodeling with low-frequency electrical stimulation (LFS, 10 Hz, 12 h/d, 7 d/wk, 28 d) in mdx (dystrophin null) and dko (dystrophin/utrophin null) mice, two established preclinical models of DMD. RESULTS: Novel biological roles in adaptation were demonstrated by impaired transcriptional activation of estrogen-related receptor alpha-responsive genes supporting oxidative phosphorylation in dystrophic muscles. Further, utrophin expression in dystrophic muscles was required for LFS-induced remodeling of mitochondrial respiratory chain complexes, enhanced fiber respiration, and conferred protection from eccentric contraction-mediated damage. CONCLUSIONS: These findings reveal novel roles for dystrophin and utrophin during LFS-induced metabolic remodeling of dystrophic muscle and highlight the therapeutic potential of LFS to ameliorate the dystrophic pathology and protect from contraction-induced injury with important implications for DMD and related muscle disorders.