Unlike ethanol which is primarily oxidized in liver cytosol, acetaldehyde formed during the metabolism of ethanol is oxidized in the matrix space of the mitochondria by the action of aldehyde dehydrogenase. The nuclear coded enzyme, after being synthesized on free ribosomes, must be translocated into the mitochondria where it is processed to the mature protein by the action of a protease. The information for targeting and processing is located in an N-terminal extension of amino acids, called a leader sequence. The structure of the leader has been determined by 2DNMNR in a micellar milieu. Because of the inherent structural flexibility of the leader sequence, it is not known if this is the structure that is presented to each of the targets that binds the peptide. These are the outer membrane, the outer membrane receptor/translocator and the processing protease. It has been possible to identify some components of the import machinery using antigenic and cross-linking experiments. The search for additional components will be continued. The proteins will be cloned, expressed and reconstituted into liposomes. The interaction of synthetic peptides corresponding to those from human, beef, and rat liver aldehyde dehydrogenase will be studied when bound to the liposomes, reconstituted translocator/receptor components and processing peptidase. The structure and dynamics of the peptides, when bound, will be deduced from NMR spectroscopic data using transferred NOE experiments and relaxation rates. The effect of ethanol on mitochondrial import will be reinvestigated to determine if it inhibits import of proteins other than aldehyde dehydrogenase and how it inhibits the import of aldehyde dehydrogenase. A complete understanding of ethanol and acetaldehyde oxidation required that all aspects of the enzymology be understood, including gene regulation, reaction mechanisms and targeting to the proper organelle.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA010795-04
Application #
2894112
Study Section
Special Emphasis Panel (ZRG4-ALTX-1)
Program Officer
Isaki, Leslie
Project Start
1996-07-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Purdue University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Mukhopadhyay, Abhijit; Wei, Baoxian; Weiner, Henry (2013) Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol. Biochim Biophys Acta 1830:3391-8
Mukhopadhyay, Abhijit; Weiner, Henry (2007) Delivery of drugs and macromolecules to mitochondria. Adv Drug Deliv Rev 59:729-38
Mukhopadhyay, Abhijit; Yang, Chun-song; Wei, Baoxian et al. (2007) Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase. J Biol Chem 282:37266-75
Mukhopadhyay, Abhijit; Yang, Chun-Song; Weiner, Henry (2006) Binding of mitochondrial leader sequences to Tom20 assessed using a bacterial two-hybrid system shows that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom20 can still be imported. Protein Sci 15:2739-48
Mukhopadhyay, Abhijit; Zullo, Steven J; Weiner, Henry (2006) Factors that might affect the allotopic replacement of a damaged mitochondrial DNA-encoded protein. Rejuvenation Res 9:182-90
Mukhopadhyay, A; Ni, L; Yang, C-S et al. (2005) Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence. Cell Mol Life Sci 62:1890-9
Mukhopadhyay, Abhijit; Ni, Li; Weiner, Henry (2004) A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria. Biochem J 382:385-92
Mukhopadhyay, Abhijit; Heard, Thomas S; Wen, Xiaohui et al. (2003) Location of the actual signal in the negatively charged leader sequence involved in the import into the mitochondrial matrix space. J Biol Chem 278:13712-8
Yang, Chun-Song; Weiner, Henry (2002) Yeast two-hybrid screening identifies binding partners of human Tom34 that have ATPase activity and form a complex with Tom34 in the cytosol. Arch Biochem Biophys 400:105-10
Mukhopadhyay, Abhijit; Hammen, Philip; Waltner-Law, Mary et al. (2002) Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP). Protein Sci 11:1026-35

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