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.