Research in this laboratory focuses on the mitochondrion. This organelle is critical to homeostasis of the cell, by virtue of providing most of the energy for cellular processes, and by involvement in other metabolic pathways. Defects in enzymes of energy metabolism, particularly mutations in the components of oxidative phosphorylation, are involved in a number of diseases, including a wide variety of early-onset genetic disorders such as Leigh's disease, MELAS and MERRF, which in total affect around 1/5000 of the population
Altered mitochondrial functioning is also a feature of some of the most common late-onset diseases affecting humans, including Alzheimer's and Parkinson's diseases as well as non-insulin dependent diabetes (NIDDM).
It is thought that these very common disorders result from a combination of a genetic predisposition along with accumulation of environmental insults that cause a long-term increased oxidative stress on cells. Free radical production as a consequence of oxidative stress occurs predominantly within mitochondria and much of the oxidative damage caused by these highly reactive species is to mtDNA and the proteins of OXPHOS. The result is a cycle of increased mitochondrial dysfunction causing increased oxidative damage until the cellular energy supply falls below the threshold for cellular survival. The resulting cell death, called apoptosis, is itself controlled by mitochondrial proteins further emphasizing the critical role of the organelle in cell survival.
Our major research projects at present are focused toward providing simple, rapid tests for detection of mitochondrial dysfunction that can be used for diagnosis of both early- and late-onset mitochondrial disorders.
We have developed a simple immunohistochemical test that identifies which of the five OXPHOS complexes are altered in early-onset genetic mitochondrial diseases. Also, we have developed a test for carriers of pyruvate dehydrogenase deficiency, an X-linked disease due to mutations in this mitochondrial enzyme that presents with many similarities to OXPHOS disorders. Ongoing work is aimed at approaches to detecting diseases such as mitochondrial DNA depletion syndrome, in which mtDNA replication is altered.
A second major emphasis is to develop tests for the late-onset mitochondrial diseases that appear to be a consequence of oxidative damage, including Alzheimer's and Parkinson's diseases. Approaches have been developed in which all five OXPHOS complexes are purified by immunocapture from very small amounts of cell culture or biopsy material, thereby allowing subsequent analysis of accrued oxidative damage. Present studies include work to identify hot spots for reaction of hydroxyl free radicals and peroxynitrite within each of the OXPHOS proteins using proteomics. Once these sites have been identified, a search will be initiated for the same modifications in animal models and cell lines, as well as patients with Alzheimer's and Parkinson's diseases.
Murray J., S. Yonally, R. Aggeler, M.F. Marusich, and R.A. Capaldi. (2005) Focused proteomics: towards a high throughput monoclonal antibody-based resolution of proteins for diagnosis of mitochondrial diseases. Biochem Biophys Acta 1659:206-11.
Murray J., M.F. Marusich, R.A. Capaldi, and R. Aggeler (2004) Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain. Electrophoresis 25:2520-5.
Hanson G.T., R. Aggeler, D. Oglesbee, M. Cannon, R.A. Capaldi, R.Y. Tsien, and S.J. Remington (2004) Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators. J Biol Chem 279:13044-53.
Rossignol R., R. Gilkerson, R. Aggeler, K. Yamagata, S.J. Remington, and R.A. Capaldi (2004) Engergy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Res 64:985-93.
Lib M., A. Rodriguez-Mari, M.F. Marusich, and R.A. Capaldi (2003) Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex. Anal Biochem 314:121-7.
Taylor S.W., E. Fahy, B. Zhang, G.M. Glenn, D.E. Warnock, S. Wiley, A.N. Murphy, S.P. Gaucher, R.A. Capaldi, B.W. Gibson, and S.S. Ghosh (2003) Characterization of the human heart mitochondrial proteome. Nat Biotechnol 21:281-6.
Murray J. , B. Zhang, S.W. Taylor, D. Oglesbee, E. Fahy, M.F. Marusich, S.S. Ghosh, and R.A. Capaldi (2003) The subunit composition of the human NADH dehydrogenase obtained by rapid one-step immunopurification. J Biol Chem 278:13619-22.
Murray J., S.W. Taylor, B. Zhang, S.S Ghosh, and R.A Capaldi (2003) Oxidative damage to mitochondrial complex I due to peroxynitrite; Identification of reactive tyrosines by mass spectrometry. J Biol Chem 278:37223-30.
Gilkerson R.W., J. M. Selker, and R.A. Capaldi (2003) The cristal membrane of mitochondria is the principal site of oxidative phosphorylation. FEBS Lett 546:355-8.
Schulenberg B., R. Aggeler, J.M. Beechem, R.A. Capaldi, and W.F. Patton (2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J Biol Chem 278:27251-5.
Taylor S.W., E. Fahy, J. Murray, R.A. Capaldi, ans S.S. Ghosh (2003) Oxidative post-translational modification of tryptophan residues in cardiac mitochondrial proteins. J Biol Chem 278:19587-90.
Capaldi, R.A and R. Aggeler (2002) Mechanism of the F1F0-type ATP synthase: a biological rotary motor. Trends Biol Sci 27:154-60.
Taylor S.W., D.E. Warnock,G.M. Glenn, B. Zhang, E. Fahy, S.P. Gaucher, R.A. Capaldi, B.W. Gibson, and S.S. Ghosh. (2002) An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and 1D PAGE on highly purified human heart mitochondria. J Proteome Res 1:451-8.
Knowles M.K., M.G. Guenza, R.A. Capaldi, and A.H. Marcus. (2002) Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells. PNAS 99:14772-7.
Murray J., R. Gilkerson, and R.A. Capaldi. (2002) Quantitative proteomics: the copy number of pyruvate dehydrogenase is more than 10(2)-fold lower than that of complex III in human mitochondria. FEBS Lett 529:173-8.
Margineantu, D.H., W.G., Cox, L. Sundell, S.W. Sherwood, J.M. Beechem, and R.A. Capaldi (2002) Cell cycle dependent morphology changes and associated mtDNA redistribution in mitochondria of human cell lines. Mitochondrion:425-35.
Margineantu, D.H., R.M. Brown, G.K. Brown, A.H. Marcus, and R.A. Capaldi (2002) Heterogeneous distribution of pyruvate dehydrogenase in the matrix of mitochondria. Mitochondrion 1:327-38.
Hanson B.J., R.A. Capaldi, M.F. Marusich, and S.W. Sherwood (2002) An immunocytochemical approach to detection of mitochondrial disorders. J Hist Cytochem 50:1281-8.
Lib, M.Y., R.M. Brown, G.K. Brown, M.F. Marusich, and R.A. Capaldi (2002) Detection of pyruvate dehydrogenase complex deficiencies in females by an immunohistochemical quantitation of mosaicism in patient fibroblast cell lines. J Hist Cytochem 50:877-84.
Hanson, B.J., B. Schulenberg, W.F. Patton, and R.A. Capaldi (2001) A novel subfractionation approach for mitochondrial proteins: a three-dimensional mitochondrial proteome map. Electophoresis 22:950-9.
Garcia, J.J., I.Ogilvie, B.H. Robinson, and R.A. Capaldi (2000) Structure functioning and assembly of the ATP syntase in cells from patients with the T8993G mitochondrial DNA mutation. Comparison with the enzyme in Rho cell completely lacking mtDNA. J Biol Chem 275:11075.