Diverse genes involved in distinct mitochondrial processes cause disease

Mitochondrial Disease

Defects in oxidative phosphorylation (OXPHOS) result in human mitochondrial disease and affect ~1/5,000 live births. Onset can be at any age, but severe childhood disease is common and symptoms regularly involve neurological and muscular disease. Defects in respiratory chain Complex I is the most common mitochondrial disease, and results in multi-system disorders. Complex I contains 44 different protein subunits, with 7 subunits encoded by mitochondrial genes and the rest by nuclear genes. Complex I failure leads to defects in aerobic respiration with elevated lactic acid and ketone bodies. Complex I is also known to generate reactive oxygen species, an important contributor to many mitochondrial pathologies.

In collaboration with the Murdoch Childrens Research Institute at the Royal Children's Hospital, we analyse cells from patients for defects in assembly of the respiratory chain, in particular Complex I. We complement our patient studies by making knockouts in cell lines (using CRISPR/Cas9) and performing cellular, biochemical and proteomic approaches to understand the function of specific genes involved in OXPHOS

 

  Mitochondrial oxidative phosphorylation and protein complexes

Recent publications:

Stroud DA, Maher MJ, Lindau C, Vögtle FN, Frazier AE, Surgenor E, Mountford H, Singh AP, Bonas M, Oeljeklaus S, Warscheid B, Meisinger C, Thorburn DR, Ryan MT. COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2. Hum Mol Genet. (2015) 24(19):5404-15. Pubmed

Formosa LE, Mimaki M, Frazier AE, McKenzie M, Stait TL, Thorburn DR, Stroud DA, Ryan MT. Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I. Hum Mol Genet. (2015) 24(10):2952-65 Pubmed

Lim SC, Smith KR, Stroud DA, Compton AG, Tucker EJ, Dasvarma A, Gandolfo LC, Marum JE, McKenzie M, Peters HL, Mowat D, Procopis PG, Wilcken B, Christodoulou J, Brown GK, Ryan MT, Bahlo M, Thorburn DR. A founder mutation in PET100 causes isolated complex IV deficiency in Lebanese individuals with Leigh syndrome. Am J Hum Genet. (2014) 94(2):209-22 Pubmed

Tucker EJ, Wanschers BF, Szklarczyk R, Mountford HS, Wijeyeratne XW, van den Brand MA, Leenders AM, Rodenburg RJ, Reljić B, Compton AG, Frazier AE, Bruno DL, Christodoulou J, Endo H, Ryan MT, Nijtmans LG, Huynen MA, Thorburn DR. Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression. PLoS Genet. 2013 ;9(12):e1004034 Pubmed

Stroud DA, Formosa LE, Wijeyeratne XW, Nguyen TN, Ryan MT. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I. J Biol Chem. 2013288(3):1685-90 Pubmed

Funding:

National Health & Medical Research Council Project Grants (2014-2016; 2016-2021)

Australian Mitochondrial Disease Foundation incubator grant (2016)

Links:

Australian Mitochondrial Disease Foundation

Mitochondrial Dynamics

Mitochondria form a network that is finely tuned to the needs of the cell. Changes in mitochondrial fission and fusion are important events in cell signalling and development. However unscripted changes in the mitochondrial network can be pathological. Increased mitochondrial fission also appears to be a hallmark of disease states including Huntington's and Parkinson's and during apoptosis.

We have identified two new players of mitochondrial fission, MiD49 and MiD51, involved in recruiting the master fission mediator Dynamin related protein 1 (Drp1) to mitochondria. Using imaging, cell biology and structural biology, we are working out how the MiD proteins and other members of the morphology machinery regulate mitochondrial dynamics at the cellular and molecular level. Our findings are being used to determine how fission and fusion events may be controlled in disease states.

 

Recent publications:

Osellame LD, Singh AP, Stroud DA, Palmer CS, Stojanovski D, Ramachandran R, Ryan MT. Cooperative and independent roles of Drp1 adaptors Mff and MiD49/51 in mitochondrial fission. J Cell Sci. 2016 Apr 12. pii: jcs.185165. [Epub ahead of print] Pubmed

Elgass KD, Smith EA, LeGros MA, Larabell CA, Ryan MT. Analysis of ER-mitochondria contacts using correlative fluorescence microscopy and soft X-ray tomography of mammalian cells. J Cell Sci. 2015 Aug 1;128(15):2795-804. Pubmed

Richter V, Palmer CS, Osellame LD, Singh AP, Elgass K, Stroud DA, Sesaki H, Kvansakul M, Ryan MT. Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission. J Cell Biol. 2014 Feb 17;204(4):477-86. Pubmed

Palmer CS, Elgass KD, Parton RG, Osellame LD, Stojanovski D, Ryan MT. Adaptor proteins MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and are specific for mitochondrial fission. J Biol Chem. 2013 Sep 20;288(38):27584-93. doi: 10.1074/jbc.M113.479873. Epub 2013 Aug 6. Pubmed

Palmer CS, Osellame LD, Laine D, Koutsopoulos OS, Frazier AE, Ryan MT. MiD49 and MiD51, new components of the mitochondrial fission machinery. EMBO Rep. 2011 Jun;12(6):565-73. Pubmed

Funding:

Australian Research Council Discovery Grant (2016-2018)

Functional analysis of the human mitochondrial proteome

While we understand many aspects of mitochondrial function, we still do not fully appreciate the roles played by many individual proteins present in the organelle. Of the ~1300 proteins in human mitochondria, about 30% have no known function while another 30% have been assigned functions based on sequence similarity. We are coupling gene-editing techniques (TALENs and CRISPRs) to generate gene knockouts in cultured human cells and investigating how a protein's loss impacts mitochondria and the cell. This work is being coupled with state-of-the-art proteomic and bioinformatic approaches to determine how the cell changes its protein make-up to adjust to these changes. In our approach to functionalise the human mitochondrial proteome we are making new and important discoveries into fundamental aspects related to mitochondrial protein function.

 

Recent publications:

Stroud DA, Surgenor EE, Formosa LE, Reljic B, Frazier AE, Dibley MG, Osellame LD, Stait T, Beilharz TH, Thorburn DR, Salim A &  Ryan MT. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature (14 September 2016) | doi:10.1038/nature19754 Pubmed http://rdcu.be/kkQg

Osellame LD, Singh AP, Stroud DA, Palmer CS, Stojanovski D, Ramachandran R, Ryan MT. Cooperative and independent roles of Drp1 adaptors Mff and MiD49/51 in mitochondrial fission. J Cell Sci. 2016 Apr 12. pii: jcs.185165. [Epub ahead of print] Pubmed

Stroud DA, Maher MJ, Lindau C, Vögtle FN, Frazier AE, Surgenor E, Mountford H, Singh AP, Bonas M, Oeljeklaus S, Warscheid B, Meisinger C, Thorburn DR, Ryan MT. COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2. Hum Mol Genet. (2015) 24(19):5404-15. Pubmed

Formosa LE, Mimaki M, Frazier AE, McKenzie M, Stait TL, Thorburn DR, Stroud DA, Ryan MT. Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I. Hum Mol Genet. (2015) 24(10):2952-65 Pubmed

Stroud DA, Formosa LE, Wijeyeratne XW, Nguyen TN, Ryan MT. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I. J Biol Chem. 2013288(3):1685-90 Pubmed