Structural Analysis of NAT Acetylation, Substrate Specificity and Polymorphisms

Summary

Principal Investigator: Kylie Walters
Abstract: Arylamine N-acetyltransferases (NATs) detoxify arylamine bladder carcinogens that are ingested as cooking, pesticide, tobacco, or dye byproducts. They perform this function by transferring to the arylamine an acetyl group from acetyl-CoA. Bladder cancer patients exhibit elevated levels of arylamine-adducts compared to control groups, some of which have been demonstrated to be poor substrates for NATs. As part of this proposal, we define the determinants of NAT substrate specificity to reveal why certain arylamine carcinogens are unable to be detoxified. NATs are highly polymorphic proteins and a large percentage of the population harbor NAT variants with reduced catalytic activity in vivo. We, and others, have recently demonstrated such variants to be rapidly degraded through the ubiquitin-proteasome pathway. One of our long-term goals is to use NATs as a model system to determine how ubiquitylation targets are identified in cells and to elucidate the mechanism(s) that lead to their delivery to the proteasome. This research will provide fundamental information on quality control pathways that exist to recognize and eliminate aberrant proteins. The following three general areas are pursued. 1) Develop a general model for NAT substrate specificity. NMR and steady state kinetics experiments are used to define the determinants of NAT substrate specificity and this knowledge used to generate variants that can acetylate additional arylamine carcinogens. 2) Determine the mechanisms that lead to NAT constitutive ubiquitylation. In previous work, we demonstrated NAT constitutive ubiquitylation to be linked to its aggregation state and our preliminary data indicate such ubiquitylation to occur at the endoplasmic reticulum. In the proposed research, we determine how NATs are recognized as aberrant, the determinants of their ubiquitylation, and whether they are processed through a common pathway that applies to other aggregated or mis-folded proteins. 3) Define how acetylation affects NAT structure and surface properties. The successful outcome of this research could aid in the prevention of bladder cancer and lead to new therapeutical strategies for diseases associated with mis-folded or aggregated proteins, including neurodegenerative diseases and cancer. Relevance of this research to public health This research has therapeutical implications for NAT-associated carcinogenesis, especially bladder cancer, as well as diseases associated misfolded or aggregated proteins, including neurodegenerative diseases. NATs detoxify chemicals known as arylamines and population-based studies have connected reduced NAT activity and arylamines that evade NAT detoxification to bladder cancer. We determine how certain arylamines evade NAT detoxification and why the NAT proteins of some people are destroyed before they can perform their protein function.
Funding Period: ----------------2007 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. ncbi Arylamine N-acetyltransferases: from structure to function
    Edith Sim
    Department of Pharmacology, University of Oxford, Oxford, United Kingdom
    Drug Metab Rev 40:479-510. 2008
  2. ncbi Ubiquitin and its binding domains
    Leah Randles
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
    Front Biosci (Landmark Ed) 17:2140-57. 2012
  3. pmc Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis
    Naixia Zhang
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
    Biochem Biophys Res Commun 385:395-401. 2009
  4. ncbi Ubiquitin-binding domains - from structures to functions
    Ivan Dikic
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Germany
    Nat Rev Mol Cell Biol 10:659-71. 2009
  5. pmc Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190
    Xin Zhou
    Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
    FEBS J 276:6928-41. 2009
  6. pmc Multitasking with ubiquitin through multivalent interactions
    Fen Liu
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA
    Trends Biochem Sci 35:352-60. 2010
  7. ncbi Identifying and studying ubiquitin receptors by NMR
    Xiang Chen
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA
    Methods Mol Biol 832:279-303. 2012

Scientific Experts

  • I Dikic
  • Kylie J Walters
  • Naixia Zhang
  • Xiang Chen
  • Leah Randles
  • Fen Liu
  • Xin Zhou
  • Edith Sim
  • Patrick E Hanna
  • Li Liu
  • Carston R Wagner
  • Kylie Walters
  • Sotiria Boukouvala

Detail Information

Publications8

  1. ncbi Arylamine N-acetyltransferases: from structure to function
    Edith Sim
    Department of Pharmacology, University of Oxford, Oxford, United Kingdom
    Drug Metab Rev 40:479-510. 2008
    ..Structural comparisons of NATs from prokaryotes and eukaryotes, particularly in relation to CoA binding, provide a platform for understanding how the unique NAT protein fold may lend itself to a wide range of functions...
  2. ncbi Ubiquitin and its binding domains
    Leah Randles
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
    Front Biosci (Landmark Ed) 17:2140-57. 2012
    ..In this manuscript, we highlight several ubiquitin receptors from the multiple UBD folds with a focus on the structural characteristics of their interaction with ubiquitin...
  3. pmc Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis
    Naixia Zhang
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
    Biochem Biophys Res Commun 385:395-401. 2009
    ..Our NMR data on a catalytically active state of hamster NAT2 suggest that structural rearrangements caused by its acetylation might contribute to this protection...
  4. ncbi Ubiquitin-binding domains - from structures to functions
    Ivan Dikic
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Germany
    Nat Rev Mol Cell Biol 10:659-71. 2009
    ..These new structure-based insights provide strategies for controlling cellular processes by targeting ubiquitin-UBD interfaces...
  5. pmc Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190
    Xin Zhou
    Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
    FEBS J 276:6928-41. 2009
    ..These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of the active site...
  6. pmc Multitasking with ubiquitin through multivalent interactions
    Fen Liu
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA
    Trends Biochem Sci 35:352-60. 2010
    ..Multivalent interactions regulate each stage of ubiquitin signaling pathways, and appear within the ubiquitin signal, the ubiquitylated substrate, ubiquitin processing enzymes and ubiquitin recognition proteins...
  7. ncbi Identifying and studying ubiquitin receptors by NMR
    Xiang Chen
    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA
    Methods Mol Biol 832:279-303. 2012
    ....