Catherine Clarke Summary

Introduction
Ubiquinone (coenzyme Q or Q) functions in cells as a redox-active coenzyme of mitochondrial and plasma membrane electron transport, as well as an essential lipid soluble antioxidant. Human dietary supplementation with Q appears to have beneficial effects in slowing the progression of neuro- and muscle-degenerative diseases. However, at least in lower animal species, such as the nematode Caenorhabditis elegans, dietary Q can also contribute to a shortened lifespan. These apparently disparate effects may reflect the potential of Q to contribute to the generation of reactive oxygen species. Cells are capable of synthesizing Q, but much remains to be learned about the sites of its synthesis, mechanisms of inter- and intra-cellular transport, and the regulation and enzymology of its biosynthesis. The goals of my research are to characterize the Coq polypeptides responsible for production of Q and to determine how their activity can be modulated for optimal health.

Research Overview
My research focuses on understanding the physiological roles of Q at the molecular level. Our studies have centered on elucidating the gene-enzyme relationships of Q biosynthesis, characterizing the polypeptide components responsible for the production of Q, and investigating the in vivo functions of Q/QH₂. Recently we have turned to the nematode Caenorhabditis elegans as an excellent model for genetic studies of the aging process. Gene mutations that increase life span have been identified in C. elegans that have homologs in vertebrates and appear to act by highly conserved mechanisms, and in pathways that parallel those present in humans.

Elucidating Q biosynthesis
Our research takes advantage of the availability of respiratory defective coq mutants of the yeast Saccharomyces cerevisiae auxotrophic for Q. The yeast coq mutants provide for the isolation and characterization of the COQ genes and their products from both yeast and animals. Yeast is an ideal organism for these studies because mutants completely lacking Q can remain viable through their ability to grow by fermentation. Synthetic analogs of Q intermediates provide reagents that serve both as standards in the isolation and characterization of Q intermediates, and as substrates for in vitro assays of enzyme activities. Thus, the experimental approach employs a combination of molecular genetics, chemistry and biochemistry to delineate the eukaryotic biosynthetic steps responsible for the production of Q, as well as to investigate the function of Q/QH₂ as an antioxidant. We now have isolated ten yeast COQ genes that complement the corresponding coq respiratory defective mutants. We have thus defined a minimal cohort of the potential polypeptide components in Q biosynthesis. My laboratory has contributed a large part of this information. Nine of the Coq polypeptides are matrix proteins peripherally associated with the mitochondrial inner membrane. A growing body of genetic evidence indicates that certain of these Coq polypeptides, together with Q and/or Q intermediates, interact to form a mitochondrial Q biosynthetic complex.

Q and aging
Mutations in the C. elegans clk-1 gene result in an extended life span, slowed development and sluggish behavior. Homology of nematode clk-1 and yeast COQ7 genes suggested that the long-lived C. elegans clk-1 mutants are defective in Q biosynthesis. Normally nematodes are provided a diet of Q-replete E. coli. In the absence of dietary Q, the clk-1 mutants display their true phenotype – growth arrest in early development and sterility when emerging from the dauer stage. Our studies show that the rescue by dietary Q₈ depends upon the uptake and transport of dietary Q₈ to mitochondria within the nematode. Our recent work shows that the rescue is mediated by Q₈, Q₉ or Q₁₀ isoforms

These results suggest that clk-1 is essential for Q biosynthesis, and that the aging and developmental phenotypes previously described may be attributed to Q levels. Our recent studies indicate that both mean and maximum life span of wild-type nematodes fed Q-less diets is extended 60%. This life span extension is quite robust and is observed in all Age mutants tested so far, when transferred to the Q-less diet as adults. Our current studies are aimed towards defining the metabolic alterations resulting from dietary Q, and should help determine how diet/environment and genotype interact to change longevity. Based on the strong conservation of Q biosynthesis and function, our findings should be very relevant to aging in other organisms.

44. Jonassen, T., Larsen, P. L. and Clarke, C. F. (2001) "A dietary source of coenzyme Q is essential for growth of long-lived Caenorhabditis elegans clk-1 mutants." Proc. Natl. Acad. Sci. U.S.A. 98, 421-426.

45. Do, T. Q., Hsu, A. Y., Jonassen, T., Lee, P. T., and Clarke, C. F. (2001) "A defect in coenzyme Q biosynthesis is responsible for the respiratory deficiency in Saccharomyces cerevisiae abc1 mutants." J. Biol. Chem. 276, 18161-18168.

46. Belogrudov, G. I., Lee, P. T., Jonassen, T., Hsu, A. Y., Gin, P., and Clarke, C. F. (2001) "Yeast COQ4 encodes a mitochondrial protein required for coenzyme Q biosynthesis." Arch. Biochem. Biophys. 392, 48-58.

47. Larsen, P. L. and Clarke, C. F. (2002) "Extension of Life-Span in Caenorhabditis elegans by a diet lacking coenzyme Q." Science, 295-123.

48. Santos-Ocana, C., Do, T. Q., Padilla, S., Navas, P. and Clarke, C. F. (2002) "Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants." J. Biol. Chem. 277, 10973-10981.

49. Jonassen, T., Marbois, B. N., Faull, K. F., Clarke, C. F. and Larsen, P. L. (2002) "Development and fertility in C. elegans clk-1 mutants depends upon transport of dietary coenzyme Q8 to mitochondria. " J. Biol. Chem. 277, 45020-45027.

50. Gin, P. Hsu, A.Y., Rothman, S., Jonassen, T., Lee, P.T., Tzagoloff, A. and Clarke, C.F. (2003) "The Saccharomyces cerevisiae COQ6 gene encodes a flavin dependent monooxygenase required for coenzyme Q biosynthesis." J. Biol. Chem. 278, 25308-25316.

51. Jonassen, T., Davis, D. E., Larsen, P. L., and Clarke, C. F. (2003) "Reproductive fitness and quinonecontent of C. elegans clk-1 mutants fed coenzyme Q isoforms of varying length." J. Biol. Chem. 278, 51735-51742.

52. Baba, S.W., Belogrudov, G.I., Lee, J.C., Lee, P.T., Strahan, J., Shepherd, J.N., Clarke, C.F. (2004) "Yeast coq5 C-methyltransferase is required for stability of other polypeptides involved in coenzyme q biosynthesis." J. Biol Chem. Mar 12;279(11):10052-9.

53. Esteves, T. C., Echtay, K. S., Jonassen, T., Clarke C. F., and Brand, M. D. (2004) "Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria". Biochem. J. 379, 309-315.

54. Hsieh, E. J., Dinoso, J. B., and Clarke, C. F. (2004) "A tRNA-TRP gene mediates the suppression of cbs2-223 previously attributed to ABC1/COQ8." Biochem. Biophys. Res. Commun. 317, 648-653.

55. Padilla, S., Jonassen, T., Jimenez-Hidalgo, M. J., Fernandez-Ayala, D. J. M., Lopez-Lluch, G. M., Marbois, B., Navas, P., Clarke, C. F. and Santos-Ocana, C. (2004) "Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration in Saccharomyces cervisiase and lacks antioxidant activity". J. Biol. Chem. 279, 25995-26004.

56. Dyall, S.D., Yan, W., Delgadillo-Correa, M.G., Lunceford, A., Loo, J.A., Clarke, C.F., Johnson, P.J. (2004) "Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex." Nature. Oct 28;431(7012):1103-7.

57. Gin, P., Clarke, C.F. (2004) "Genetic evidence for a multi-subunit complex in coenzyme Q biosynthesis in yeast and the role of the Coq1 hexaprenyl diphosphate synthase." J Biol Chem. Jan 28;280(4):2676-81.

58. Marbois, B., Gin, P., Faull, K.F., Poon, W.W., Lee, P.T., Strahan, J., Shepherd, J.N., Clarke, C.F. (2005) "Coq3 and Coq4 define a polypeptide complex in yeast mitochondria for the biosynthesis of coenzyme Q." J Biol Chem. May 27;280(21):20231-8.

59. Johnson, A., Gin, P., Marbois, B.N., Hsieh, E.J., Wu, M., Barros, M.H., Clarke, C.F., Tzagoloff, A. (2005) "COQ9, a New Gene Required for the Biosynthesis of Coenzyme Q in Saccharomyces cerevisiae." J Biol Chem. Sep 9;280(36):31397-404.

60. Barros, M. H., Johnson, A., Gin, P., Marbois, B. N., Clarke, C. F. and Tzagoloff, A. (2005) “The Saccharomyces cerevisiae COQ10 gene encodes a START domain protein required for function of coenzyme Q in respiration.” J. Biol. Chem. 280, 42627-42635.

61. Tran, U. C., Marbois, B., Gin, P., Gulmezian, M., Jonassen, T., and Clarke, C. F. (2006) “Complementation of Saccharomyces cerevisiae coq7 mutants by mitochondrial targeting of the Escherichia coli UbiF polypeptide: two functions of yeast Coq7 polypeptide in coenzyme Q biosynthesis.” J. Biol. Chem. 281, 16401-16409.

62. Gulmezian, M., Zhang, H., Javor, G. T., and Clarke, C. F. (2006) “Genetic Evidence for an interaction of the UbiG O-methyltransferase with UbiX in Escherichia coli coenzyme Q biosynthesis.” J. Bacteriol. 188, 6435-6439.

63. Hsieh, E. J., Gin, P., Gulmezian, M., Tran, U. C., Saiki, R., Marbois, B. N. and Clarke, C. F. (2007) “Saccharomyces cerevisiae Coq9 polypeptide is a subunit of the mitochondrial coenzyme Q biosynthetic complex.” Arch. Biochem. Biophys. 463, 19 -26.

64. Tran, U. C., and Clarke, C. F. (2007) “Endogenous synthesis of coenzyme Q in eukaryotes.” Mitochondrion. 7S, S62 -S71.

65. Gulmezian, M., Hyman, K. R., Marbois, B. N., Clarke, C. F. and Javor, G. T. (2007) “The role of UbiX in Escherichia coli coenzyme Q biosynthesis.” Arch. Biochem. Biophys. 467, 144 -153.

66. Peng M, Falk MJ, Haase VH, King R, Polyak E, Selak M, Yudkoff M, Hancock WW, Meade R, Saiki R, Lunceford AL, Clarke CF, L Gasser D. "Primary coenzyme Q deficiency in Pdss2 mutant mice causes isolated renal disease." PLoS Genet. 2008 Apr 25;4(4):e1000061. PMID: 18437205 [PubMed - in process].

67. Kiebish MA, Han X, Cheng H, Lunceford A, Clarke CF, Moon H, Chuang JH, Seyfried TN. " Liipidomic analysis and electron transport chain activities in C57BL/6J mouse brain mitochondria." J Neurochem. 2008 Apr 17. [Epub ahead of print] PMID: 18373617 [PubMed - as supplied by publisher].

68. Saiki R, Lunceford AL, Bixler T, Dang P, Lee W, Furukawa S, Larsen PL, Clarke CF. "Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q." Aging Cell. 2008 Mar 26; 7:291-304. PMID: 18267002 [PubMed - as supplied by publisher].

Researchers
Dr. Grigory Belogrudov	Department of Physiology, UCLA School of Medicine
Dr. Deborah Berthold	Research Associate, University of Illinois Champagne-Urbana
Dr. Jenna Hutton	Research Associate, University of Rutgers
Dr. Pam Larsen	University of Texas Health Sciences Center at San Antonio, Dept. of Cellular and Structural Biology
Postdoctoral Fellows
Dr. Ryoichi Saiki	B.A., M.S., & Ph.D. University of Shimane
Ph.D Students
Dr. Adam Lunceford	B.S., Brigham Young University, Ph.D., University of California, Los Angeles 2008
Dr. UyenPhuong C. Tran	B.S., University of California, Los Angeles, Ph.D., University of California, Los Angeles 2007
Dr. Melissa Gulmezian	B.A., University of California, Irvine, Ph.D., University of California, Los Angeles 2006
Dr. Thai Do	B.S., Ph.D., University of California, Los Angeles
Dr. Peter Gin	B.S., Ph.D. 2005, University of California, Los Angeles - Postdoctoral Researcher with Dr. Stephen Young, Dept. of Med. UCLA
Dr. Edward Hsieh	Ph.D. 2006, University of California, Los Angeles - Postdoctoral Fellow with Mike MacCoss at Univ. of Washington, Seattle
Dr. Tanya Jonassen	B.S., Ph.D., University of California, Los Angeles - Researcher - Ceres (Malibu, CA)
Dr. Wayne Poon	B.S., Ph.D. University of California, Los Angeles - Postdoctoral Fellow, University of California, Irvine
Master Students
Suzie Ward Baba
Diana Davis	B.S., North Carolina State University
Undergraduate Students
Jason Dinoso	Class of 2001, Biochemistry, University of California, Los Angeles
Nico Lee	B.S., M.S., Biochemistry, University of California, Los Angeles
Sabine Paterson	Class of 2001 - Biochemistry, University of California, Los Angeles
Dang, Peter	Class of 2006 - Biochemistry, University of California, Los Angeles - Masters student UCLA School of Dentistry