The line of inquiry proposed in this research program is based on the hypothesis that the mitochondrial ATP-dependent Lon protease monitors both the integrity and expression of the mitochondrial genome as well as the folding and assembly of nascent polypeptides. The goals of this proposal are to analyze the structure and function of yeast Lon and to identify mammalian and yeast proteins that functionally overlap with Lon. A combination of molecular biology, genetics, biochemistry and structural biology will be used to address the following aims: To analyze the role of Lon in regulating the mtDNA-protein interactions. The DNA-binding function of Lon is conserved from bacteria to humans, however the physiological importance of this activity is not understood. The investigator will identify the endogenous mtDNA sequences bound by yeast Lon, analyze the role of Lon in mtDNA-protein complexes called nucleoids and identify the protein(s) responsible for increased double-stranded DNA-binding in mitochondria that lack Lon. To identify and characterize mammalian and yeast proteins that have a shared function with Lon. Genetic complementation of a temperature-sensitive Lon mutant using mammalian libraries designed for expression in yeast will be carried out. The mammalian proteins identified will be characterized in mammalian cell systems. In addition, a genome-wide survey of transcript level changes in yeast deleted for the Lon gene will be conducted using oligonucleotide probe microarrays. To elucidate the structural dynamics and function of the homo-heptameric ring-shaped Lon protease using circular dichroism and high-resolution electron microscopy. Mitochondrial dysfunction has been implicated in a wide variety of degenerative disorders, aging and cancer. The best-characterized genetic mutations that lead to a loss of mitochondrial function are deletions, point mutations and base substitutions in mitochondrial DNA. Cellular responses to stress caused by oxidative damage and aging are likely to involve the action of ATP-dependent proteases which selectively degrade denatured, aggregated and unassembled proteins. Understanding the molecular details of how ATP-dependent proteases ensure cellular homeostasis by monitoring protein biogenesis as well as the integrity and expression of the mitochondrial genome, will provide insight into mechanisms that help to prevent mitochondrial dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061095-04
Application #
6636424
Study Section
Biochemistry Study Section (BIO)
Program Officer
Chin, Jean
Project Start
2000-05-01
Project End
2005-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
4
Fiscal Year
2003
Total Cost
$235,500
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Biochemistry
Type
Schools of Medicine
DUNS #
623946217
City
Newark
State
NJ
Country
United States
Zip Code
07107
Lu, Bin; Lee, Jae; Nie, Xiaobo et al. (2013) Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease. Mol Cell 49:121-32
Bernstein, Steven H; Venkatesh, Sundararajan; Li, Min et al. (2012) The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood 119:3321-9
Venkatesh, Sundararajan; Lee, Jae; Singh, Kamalendra et al. (2012) Multitasking in the mitochondrion by the ATP-dependent Lon protease. Biochim Biophys Acta 1823:56-66
Lee, Irene; Suzuki, Carolyn K (2008) Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates. Biochim Biophys Acta 1784:727-35
Granot, Zvi; Kobiler, Oren; Melamed-Book, Naomi et al. (2007) Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors. Mol Endocrinol 21:2164-77
Lee, Irene; Berdis, Anthony J; Suzuki, Carolyn K (2006) Recent developments in the mechanistic enzymology of the ATP-dependent Lon protease from Escherichia coli: highlights from kinetic studies. Mol Biosyst 2:477-83
Liu, Tong; Lu, Bin; Lee, Irene et al. (2004) DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein substrate. J Biol Chem 279:13902-10
Lu, Bin; Liu, Tong; Crosby, Juan A et al. (2003) The ATP-dependent Lon protease of Mus musculus is a DNA-binding protein that is functionally conserved between yeast and mammals. Gene 306:45-55