Edward E. McKee • Faculty • Department of Biological Sciences • University of Notre Dame

Mitochondrial Biogenesis, Bioenergetics, and Transport

Edward E. McKee

Associate Professor, Biochemistry and Molecular Biology, Indiana University School of Medicine / South Bend Center for Medical Education
Adjunct Associate Professor, Biological Sciences, University of Notre Dame
Adjunct Associate Professor Biochemistry and Molecular Biology Finch University of Health Sciences/The Chicago Medical School

Ph.D., Pennsylvania State University, Hershey Medical Center
Post-Doctoral: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder

e-mail | labpage

Research:

Mitochondria are sub-cellular power plants that consume oxygen and oxidizable substrates, and generate most of the energy of cells in the form of ATP. The biogenesis of these complex organelles requires the coordinated involvement of two genomic systems: the nuclear-cytoplasmic genomic system, responsible for the bulk of the organelle; and the mitochondrial genomic system, responsible for a limited, but essential set of proteins of the energy generating system. My laboratory is interested in the regulation of this biogenic process from the mitochondrial perspective, using purified, isolated, intact mitochondria from both ends of the evolutionary spectrum, yeast and mammalian tissues, particularly the rat heart. We are especially interested in the role of mitochondrial biogenesis in the acquired toxicity of drugs that may inhibit mitochondrial processes of DNA replication, transcription or translation. Antibiotics that function by inhibiting bacterial ribosomes sometimes also inhibit mitochondrial ribosomes and thus mitochondrial protein synthesis. Mitochondrial toxicity has clearly been implicated in hearing loss caused by amino glycosides, and bone marrow depression caused by chloramphenicol and the recently released Zyvox. Nucleoside analogs used as reverse transcriptase inhibitors in highly active antiretroviral therapy (HAART) often inhibit mitochondrial DNA replication. Because mitochondrial turnover is slow, typically problems associated with decreased levels of mitochondria ATP generation are slow to develop and are usually observed after long-term treatment. As a result, this type of toxicity is often overlooked in the early stages of drug development.

Past work has included:

1. Analysis of the relationship between mitochondrial rates of protein synthesis and mitochondrial metabolic processes demonstrating that energized mitochondria making endogenous ATP has the highest rates of mitochondrial protein synthesis.

2. Analysis of signal transduction and the effects of hormones and second messengers on mitochondrial protein synthesis and on protein phosphorylation in the mitochondria. Additions of several presumptive insulin second messengers have been shown to dramatically inhibit mitochondrial translation and phosphorylation of mitochondrial proteins. Drugs based on these compounds may be toxic to mitochondria.

3. Characterization of nucleotide and deoxynucleotide net transport across the inner mitochondrial membrane. Biogenesis requires the accumulation of molecules beside proteins. While most biochemistry texts suggest that molecules like GTP are not transported into the matrix, their presence in the mitochondrial matrix is uncontested. Our work has characterized transport of GTP into the mitochondrial matrix. We have shown that this ribo-nucleotide is transported only in a phosphorylated form. Neither the guanine nucleoside nor guanine is transported. We have also shown that IMP is not transported into the matrix and is not a precursor for mitochondrial GTP. On the other hand, the deoxy-ribo-nucleoside thymidine, but not thymine is transported into the matrix and converted to TTP. This work has been supported by grants from the American Heart Association (National, and Midwest Affiliate).

Our present projects include:

1. Mitochondrial Toxicity of Antibiotics: Dr. McKee has served as a consultant to Pharmacia and Upjohn and other drug companies on understanding the toxicity of oxazolidinone antibiotics (Zyvox) on the synthesis of mitochondria. With five years of grant support from Pharmacia and Upjohn, Dr. McKee’s laboratory demonstrated that members of this class of antibiotics specifically blocked the synthesis of proteins within the mitochondrion resulting in unwanted side effects. Studies showed that the effect of antibiotics on mitochondrial translation demonstrated an excellent correlation between inhibition of protein synthesis in isolated mitochondria and drug toxicity. This finding allowed Pharmacia and Upjohn in conjunction with Dr. McKee’s laboratory to design assays that would eliminate the consideration of drugs with this unwanted side effect.

2. Mitochondrial Toxicity of Anti-Viral Nucleoside Analog Reverse Transcriptase Inhibitors: There are nearly 1 million people in North America affected with HIV/AIDS and 42 million people worldwide. Present treatment of this worldwide scourge is a drug cocktail referred to as “highly active anti-retroviral therapy” or HAART. The cocktail consists of several different drugs and typically must be given over the lifetime of the patient. Unfortunately, long-term treatment with this cocktail is associated with serious to potentially lethal side effects that require that components of the cocktail be reduced or discontinued. These side effects appear to be directed at the synthesis of a sub-cellular organelle called the mitochondrion, which is the power plant of the cell. Dr. McKee’s laboratory is investigating the mechanism(s) that cause these anti-retroviral agents to be toxic to mitochondria, particularly heart mitochondria. Results from this work will provide information that may be beneficial to treating anti-retroviral drug-related side effects and will help drug companies to design anti-retroviral drugs that do not have this toxicity. This work is currently funded by a 5 year grant from the National Heart, Lung, and Blood Institute of The National Institute of Health to Dr. McKee. Preliminary data for this work was obtained with funding from the American Heart Association, Mid-West Affiliate.

Our present work has concentrated on AZT (azidothymidine, Zidovudine), one of the first drugs to used in the treatment of AIDS and still used in HAART. This compound appears to be preferentially toxic to the cardiovascular system. We have studied the transport and conversion of thymidine and AZT in isolated heart mitochondria and in the isolated perfused rat heart and have demonstrated that while thymidine is phosphorylated readily to TTP, AZT is phosphorylated only to AZT-MP in both systems (see below).

As a result, inhibition of the heart mitochondrial RNA polymerase by AZT-TP is unlikely to account for cardiac toxicity. Subsequently, we have shown that AZT is a potent inhibitor of the conversion of labeled thymidine to TMP and TTP in isolated heart mitochondria (see below left). Subsequently we have shown that AZT also inhibits the conversion of labeled thymidine to TTP in isolated intact perfused rat hearts (see below right). It is possible that a limiting TTP pool may be responsible for the inhibition of replication.

This project is funded by NIH grant # 1 R01 HL72710-01

Selected Publications:

Leung, A.L. and McKee, E.E. (1990) Mitochondrial protein synthesis during thyroxine-induced cardiac hypertrophy. Amer J. Physiol. 258: E511-E518.

McKee, E. E. and Grier, B. L. (1990) Insulin stimulates mitochondrial protein synthesis and respiration in isolated perfused rat heart. Amer. J. Physiol. 259: E413-E421.

Black-Schafer, C.L., McCourt, J.D., Poyton, R. O., and McKee, E.E. (1991) Mitochondrial gene expression in Saccharomyces cerevisiae. III. Proteolysis of nascent chains in isolated mitochondria optimized for protein synthesis. Biochem. J. 274: 199-205.

McKee, E.E. Mitochondrial gene expression in Saccharomyces cerevisiae. IV. Effect of yeast cytosol on mitochondrial protein synthesis, degradation, and respiration. (1994) Biochim. Biophys. Acta 1201: 235-244.

Poyton, R. O., K. A. Sevarino, E. E. McKee, D. J. M. Duhl, V. Cameron, and B. Goehring. Export of protein from mitochondria (1996) Advances in Molecular and Cell Biology 17: 245-277.

Poyton, R. O., G. Bellus, E. E. McKee, K. A. Sevarino, and B. Goehring (1996) In Organello mitochondrial protein and RNA synthesis. Methods in Enzymol. 264: 36-42.

McKee, E. E., Bentley, A. T. Smith, R.M., Jr., and Ciaccio, C. E. (1999) Origin of Guanine Nucloetides in Isolated Heart Mitochondria Biochem. Biophys. Res. Comm. 257: 466-472.

McKee, Edward E., Alice T. Bentley, Ronald M. Smith, Jr. Jonathan R. Kraas, and Christina E. Ciaccio (2000) Guanine Nucleotide Transport by Atractyloside Sensitive and Insensitive Carriers in Isolated Heart Mitochondria, American J. Physiology, Cell Physiology, 279: 1870-1879.

Abstracts:

McKee, E. E., Bentley, A. T., Kraas, J., and Gingerich, J. Uptake and phosphorylation of thymidine and AZT in isolated heart mitochondria. Mitochondrion 1: pp 96, 2001.

McKee, E. E., Bentley, A. T., and Gingerich, J. Inhibition of thymidine phosphorylation by AZT in isolated heart mitochondria. Keystone Symposia, Mitochondria and Pathogenesis, April 6-11, 2002, Copper Mountain, CO.