Arylamine N-acetyltransferase Aggregation and Constitutive Ubiquitylation

Fen Liu, Naixia Zhang, Xin Zhou, Patrick E. Hanna, Carston R. Wagner, Deanna M. Koepp, Kylie J. Walters

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation. To identify the properties that lead to the reduced cellular activity of certain NAT variants, we introduced one such polymorphism into the human NAT1 ortholog hamster NAT2. The polymorphism chosen was human NAT1*17, which results in the replacement of R64 with a tryptophan residue, and we demonstrate this substitution to cause hamster NAT2 to be constitutively ubiquitylated. Biophysical characterization of the hamster NAT2 R64W variant revealed that its overall protein structure and thermostability are not compromised. In addition, we used steady-state kinetics experiments to demonstrate that the R64W mutation does not interfere with NAT catalysis in vitro. Hence, the constitutive ubiquitylation of this variant is not caused by its inability to be acetylated. Instead, we demonstrate this mutation to cause the hamster NAT2 protein to aggregate in vitro and in vivo. Importantly, we tested and confirmed that the R64W mutation also causes human NAT1 to aggregate in cultured cells. By using homology modeling, we demonstrate that R64 is located at a peripheral location, which provides an explanation for how the NAT protein structure is not significantly disturbed by its mutation to tryptophan. Altogether, we provide fundamental information on why humans harboring certain NAT variants exhibit reduced acetylation capabilities.

Original languageEnglish (US)
Pages (from-to)482-492
Number of pages11
JournalJournal of Molecular Biology
Volume361
Issue number3
DOIs
StatePublished - Aug 18 2006

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Arylamine N-Acetyltransferase
Ubiquitination
Cricetinae
Mutation
Tryptophan
Proteins
Acetylation
Catalysis
Urinary Bladder Neoplasms
Carcinogens
Cultured Cells
N-acetyltalosaminuronic acid
DNA
Genes
Neoplasms

Keywords

  • arylamine N-acetyltransferase
  • polymorphism
  • protein aggregation
  • protein degradation
  • ubiquitylation

Cite this

Arylamine N-acetyltransferase Aggregation and Constitutive Ubiquitylation. / Liu, Fen; Zhang, Naixia; Zhou, Xin; Hanna, Patrick E.; Wagner, Carston R.; Koepp, Deanna M.; Walters, Kylie J.

In: Journal of Molecular Biology, Vol. 361, No. 3, 18.08.2006, p. 482-492.

Research output: Contribution to journalArticle

Liu, Fen ; Zhang, Naixia ; Zhou, Xin ; Hanna, Patrick E. ; Wagner, Carston R. ; Koepp, Deanna M. ; Walters, Kylie J. / Arylamine N-acetyltransferase Aggregation and Constitutive Ubiquitylation. In: Journal of Molecular Biology. 2006 ; Vol. 361, No. 3. pp. 482-492.
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