Biochemistry Faculty

Scott Briggs

Steve Broyles

Clint Chapple

Harry Charbonneau

James Clemens

James Forney

Frederick S. Gimble

Barbara Golden

Mark Hall

Mark Hermodson

Ann Kirchmaier

Xiaoqi Liu

Joe Ogas

Sandra Rossie

W. Andy Tao

Henry Weiner

H. Lee Weith

Adjunct Faculty

Jon Lebowitz

Emeritus Professors

Karl Brandt

Bernard Axelrod

Klaus Herrmann

Ki-Han Kim

Gunter Kohlhaw

David Krogmann

Victor Rodwell

Ronald Somerville

Roy Whistler

Howard Zalkin

Henry Weiner Professor of Biochemistry 305 Biochemistry, (765) 494-1650 Ph.D. Purdue University, 1963 hweiner@purdue.edu Investigators: Staff: Kwok Ki Ho Abhijit Mukhopadhyay Postdoctoral Fellows: Yifeng Li Soma Mukhopadhyay Area: Protein trafficking and the enzymology of aldehyde dehydrogenasess

The laboratory is investigating two seemingly unrelated problems, which actually are related for each employ the tools of molecular and structural biology. The first is involved with the import of proteins into mitochondria. The second with structure and functional analysis of aldehyde dehydrogenase, a detoxifying enzyme found in all tissue and species.

For a protein to be imported into mitochondria it must possess an N-terminal signal sequence, composed of 17-25 amino acids. After import the signal is removed in most cases by the action of a protease. We are interested in learning how the signals which all have different primary sequences can all be imported by the same translocators and processed by the same protease. Two approaches are employed to investigate the problem. One is to make mutations in the signal region and study their affect on the processes. The second is to study the interaction of synthetic signal peptides with a variety of components, including liposomes and proteins. For the later, physical techniques including 2D-NMR, CD, and fluorescence are employed. We are cloning components of the translocator so they can be characterized and incorporated into liposomes to reconstitute the translocator and identify new ones using a yeast two-hydrid system. In addition, a new co-translational import model is being developed.

The enzymology of aldehyde dehydrogenase is being investigated using various tools of molecular biology to probe for the active site of the enzyme. In collaboration with an X-ray chrystallographer, we recently determined the three dimensional structure of the enzyme. Projects include deleting the genes coding for the yeast enzyme so that this yeast can be used to produce mutants of the enzyme. The enzyme is being cloned in HeLa cells so we can study properties of the enzyme in vivo. Lastly, chimeric forms of the cytosolic and mitochondrial isozymes are being constructed in an attempt to understand why they have different properties. Allosteric interactions have been engineered into the enzyme, and we are studying them and subunit interactions. The aim is to try to be able to design an enzyme with properties not found in either parent enzyme which will be used to transform cells so they can be protected against the action of cytotoxic agents used in chemo-therapy. Directed evolution is being performed to produce enzymes with altered specificity.

Selected Publications:

• Hink U., Daiber A., Kayhan N., Trischler J., Kraatz C., Oelze M., Mollnau H., Wenzel P., Vahl C., Ho K.K., Weiner H., and Munzel T. Oxidative Inhibition of the Mitochondrial Aldehyde Dehydrogenase Promotes Nitroglycerin Tolerance in Human Blood Vessels. J. Am. Coll. Cardiol. 50: 2226-2232 (2007).

   

• Mukhopadhyay A., Yang C.S., Wei B. and Weiner H. Precursor Protein is Readily Degraded in Mitochondrial Matrix Space if the Leader is not Processed by Mitochondrial Processing Peptidase. J. Biol. Chem. 282: 37266-37275 (2007).

   

• Wenzel P, Oelze M, Coldewey M, Hortmann M, Seeling A, Hink U, Mollnau H, Stalleicken D, Weiner H, Lehmann J, Li H, Forstermann U, Munzel T, Daiber A. Heme Oxygenase-1. A Novel Key Player in the Development of Tolerance in Response to Organic Nitrates. Arterioscler. Thromb. Vasc. Biol. 27: 1729-1735 (2007).

   

• Mukhopadhyay A. and Weiner H. Delivery of drugs and macromolecules to mitochondria. Adv. Drug Deliv. Rev. 59: 729-738 (2007).

   

• Wenzel P., U. Hink, M. Oelze, A. Seeling, T. Isse, K. Bruns, L. Steinhoff, M. Brandt, A.L. Kleschyov, E. Schulz, K. Lange, H. Weiner, J. Lehmann, K.J. Lackner, T. Kawamoto, T. Munzel and A. Daiber. Number of nitrate groups determines reactivity and potency of organic nitrates: A proof of concept study in ALDH-2(-/-) mice. Br. J. Pharmacol. 150: 526-533 (2007).

   

• Brichac, J., K. Ho, A. Honzatko, R. Wang, X. Lu, H. Weiner and M. Picklo. Enantioselective oxidation of trans-4-hydroxy-2-nonenal is aldehyde dehydrogenase isozyme and Mg 2+-dependent. Chem. Res. Toxicol.20: 887-895 (2007).

   

• Larson, H.N., J. Zhou, Z. Chen, J.S. Stamler, H. Weiner and T.D. Hurley. Structural and functional consequences of coenzyme binding to the inactive Asian variant of mitochondrial aldehyde dehydrogenase: Roles of residues 475 and 487. J. Biol. Chem. 282: 12940-12950 (2007).

   

• Ho, K.K., T.D. Hurley and H. Weiner. Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis. Biochemistry 45: 9445-9453 (2006).

   

• Mukhopadhyay, A., C.-S. Yang and H. Weiner. Binding of mitochondrial leader sequences to Tom 20 assessed using a bacterial two-hybrid system show that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom 20 can still be imported. Protein Sci. 15: 2739-2748 (2006).

   

• Wenzel, P., U. Hink, M. Oelze, S. Schuppan, K. Schaeuble, S. Schildknecht, K.K. Ho, H. Weiner, M. Bachschmid, T. Munzel and A. Daiber. Role of reduced lipoic acid in the redox regulation of mitochondrial aldehyde dehydrogenase (aldh-2) activity: Implications for mitochondrial oxidative stress and nitrate tolerance. J. Biol. Chem. 282: 792-799.

   

• Rodriguez-Zavala J.S., Allali-Hassani A. and Weiner H. Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases. Protein Science 15: 1387-1396 (2006).

   

• Mukhopadhyay A., Zullo S.J. and Weiner H. Factors that might affect the Allotopic Replacement of a Damaged Mitochondrial DNA-encoded Protein. Rejuvenation Research 9: 182-190 (2006).

   

• Mukhopadhyay A., Ni L., Yang C.-S. and Weiner H. Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence. Cell. Mol. Life Sci. 62: 1890-1899 (2005).

   

• Larson H.N., Weiner H. and Hurley T.D. Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase “Asian” variant. J. Biol. Chem. 280(34): 30550-30556 (2005).

   

• Ho K.K., Allali-Hassani A., Hurley T.D. and Weiner H. Differential effects of Mg2+ ions on the individual kinetic steps of human cytosolic and mitochondrial aldehyde dehydrogenases. Biochemistry 44(22): 8022-8029 (2005).

   

• Oyama T., Isse N., Kagawa N., Kinaga T., Kim Y.D., Morita M., Sugio K., Weiner H., Yasumoto K., Kawamoto T. Tissue-distribution of aldehyde dehydrogenase 2 and effects of the ALDH2 gene-disruption and the expression of enzymes involved in alcohol metabolism. Frontiers in Bioscience 10: 951-960 (2005).

   

• Zullo S.J., Parks W.T., Chloupkova M., Wei B., Weiner H., Fenton W.A., Eisenstadt J.M. and Merril C.R. Stable transformation of CHO cells and human NARP cybrids confers oligomycin resistance (oli) following transfer of mitochondrial DNA-encoded olir ATPase6 gene to the nuclear genome: A model system for mtDNA gene therapy. Rejuvenation Research Vol. 8(1) (2005).

   

• Ho K.K. and Weiner H.  Isolation and Characterization of an Aldehyde Dehydrogenase encoded by the AldB gene of E. coli. J. of Bacteriology 187: 1067-1073 (2005).

   

• Mukhopadhyah A., Ni L. and Weiner H. A Co-translational Model to explain the in vivo Import of Proteins into HeLa Cell Mitochondria. Biochemical Journal 382: 385-392 (2004).

   

• Mukhopadhyah A., Heard T.A., Wen X.W., Hammen P.K. and Weiner H.  Location of the actual Signal in the Negatively Charged Leader Sequence Involved in the Import into the Mitochondrial Matrix Space. J. Biol. Chem. 278: 13712-13718 (2003).

   

• Mukhopadhyay A., Wei B., Zullo S.J., Wood J., Lauren V. and Weiner H.  In Vitro Evidence of Inhibition of Mitochondrial Protease Processing by HIV-1 Protease Inhibitors in Yeast: A Possible Contribution to Lipodystrophy Syndrome. Mitochondrion 1:511-518 (2002).

  

• Wei B., Mays D.C., Lipsky J.J. and Weiner H. Chemical modifications to study mutations that affect the ability of the general base (E268) to function in human liver mitochondrial aldehyde dehydrogenase. Chemio-Biological Interactions 143-144: 85-91 (2002).

   

• Rodriguez J. and Weiner H. Structural Aspects of Aldehyde Dehydrogenase that Influences Dimer-Tetramer Formation. Biochemistry 41: 8229-8237 (2002).

   

• Hammen P. K., Allali-Hassani A., Hallenga K., Hurley T.D. and Weiner H.  Multiple Conformations of NAD and NADH when bound to Human Cytosolic and Mitochondrial Aldehyde Dehydrogenase. Biochemistry 41: 7156-7168 (2002).

   

• Mukhopadhyay A., Hammen P., Waltner-Law M. and Weiner H.  Timing and Structural Considerations for the Processing of Mitochondrial Matrix Space Proteins by the Mitochondrial Processing Peptidase (MPP).  Protein Science 11: 1026-1035 (2002).

   

• Yang C-S and Weiner H. 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: 105-110 (2002).

   

• Mukhopadhyay A., Avramova L.V. and Weiner H.  Tom34 unlike Tom20 Does Not Interact with the Leader Sequence of Mitochondrial Precursor Proteins. Arch. Biochem. Biophys. 400: 97-104 (2002).