Biochemistry and Pharmacology - Research Publications
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ItemNo Preview AvailableProteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins(CELL PRESS, 2022-12-01)The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.
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ItemNo Preview AvailableTwo independent respiratory chains adapt OXPHOS performance to glycolytic switch(CELL PRESS, 2022-11-01)The structural and functional organization of the mitochondrial respiratory chain (MRC) remains intensely debated. Here, we show the co-existence of two separate MRC organizations in human cells and postmitotic tissues, C-MRC and S-MRC, defined by the preferential expression of three COX7A subunit isoforms, COX7A1/2 and SCAFI (COX7A2L). COX7A isoforms promote the functional reorganization of distinct co-existing MRC structures to prevent metabolic exhaustion and MRC deficiency. Notably, prevalence of each MRC organization is reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC). Under oxidative conditions, the C-MRC is bioenergetically more efficient, whereas the S-MRC preferentially maintains oxidative phosphorylation (OXPHOS) upon metabolic rewiring toward glycolysis. We show a link between the metabolic signatures converging at the PDC and the structural and functional organization of the MRC, challenging the widespread notion of the MRC as a single functional unit and concluding that its structural heterogeneity warrants optimal adaptation to metabolic function.
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ItemSideroflexin 4 is a complex I assembly factor that interacts with the MCIA complex and is required for the assembly of the ND2 module(NATL ACAD SCIENCES, 2022-03-29)SignificanceMitochondria 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.
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ItemMitochondrial microproteins link metabolic cues to respiratory chain biogenesis(CELL PRESS, 2022-08-16)Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.
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ItemPremature Ovarian Insufficiency in CLPB Deficiency: Transcriptomic, Proteomic and Phenotypic Insights(ENDOCRINE SOC, 2022-11-25)CONTEXT: Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes. OBJECTIVE: We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts. METHODS: We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene. RESULTS: We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment. CONCLUSION: A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities.
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ItemAIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5(WILEY, 2022-09-01)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.
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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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ItemBiallelic Variants in PYROXD2 Cause a Severe Infantile Metabolic Disorder Affecting Mitochondrial Function(MDPI, 2022-01)Pyridine Nucleotide-Disulfide Oxidoreductase Domain 2 (PYROXD2; previously called YueF) is a mitochondrial inner membrane/matrix-residing protein and is reported to regulate mitochondrial function. The clinical importance of PYROXD2 has been unclear, and little is known of the protein's precise biological function. In the present paper, we report biallelic variants in PYROXD2 identified by genome sequencing in a patient with suspected mitochondrial disease. The child presented with acute neurological deterioration, unresponsive episodes, and extreme metabolic acidosis, and received rapid genomic testing. He died shortly after. Magnetic resonance imaging (MRI) brain imaging showed changes resembling Leigh syndrome, one of the more common childhood mitochondrial neurological diseases. Functional studies in patient fibroblasts showed a heightened sensitivity to mitochondrial metabolic stress and increased mitochondrial superoxide levels. Quantitative proteomic analysis demonstrated decreased levels of subunits of the mitochondrial respiratory chain complex I, and both the small and large subunits of the mitochondrial ribosome, suggesting a mitoribosomal defect. Our findings support the critical role of PYROXD2 in human cells, and suggest that the biallelic PYROXD2 variants are associated with mitochondrial dysfunction, and can plausibly explain the child's clinical presentation.
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ItemOligonucleotide correction of an intronic TIMMDC1 variant in cells of patients with severe neurodegenerative disorder(NATURE PORTFOLIO, 2022-01-28)TIMMDC1 encodes the Translocase of Inner Mitochondrial Membrane Domain-Containing protein 1 (TIMMDC1) subunit of complex I of the electron transport chain responsible for ATP production. We studied a consanguineous family with two affected children, now deceased, who presented with failure to thrive in the early postnatal period, poor feeding, hypotonia, peripheral neuropathy and drug-resistant epilepsy. Genome sequencing data revealed a known, deep intronic pathogenic variant TIMMDC1 c.597-1340A>G, also present in gnomAD (~1/5000 frequency), that enhances aberrant splicing. Using RNA and protein analysis we show almost complete loss of TIMMDC1 protein and compromised mitochondrial complex I function. We have designed and applied two different splice-switching antisense oligonucleotides (SSO) to restore normal TIMMDC1 mRNA processing and protein levels in patients' cells. Quantitative proteomics and real-time metabolic analysis of mitochondrial function on patient fibroblasts treated with SSOs showed restoration of complex I subunit abundance and function. SSO-mediated therapy of this inevitably fatal TIMMDC1 neurologic disorder is an attractive possibility.