Filling and mining the reactive metabolite target protein database

Abstract

The post-translational modification of proteins is a well-known endogenous mechanism for regulating protein function and activity. Cellular proteins are also susceptible to post-translational modification by xenobiotic agents that possess, or whose metabolites possess, significant electrophilic character. Such non-physiological modifications to endogenous proteins are sometimes benign, but in other cases they are strongly associated with, and are presumed to cause, lethal cytotoxic consequences via necrosis and/or apoptosis. The Reactive Metabolite Target Protein Database (TPDB) is a searchable, freely web-accessible (http://tpdb.medchem.ku.edu:8080/protein_database/) resource that attempts to provide a comprehensive, up-to-date listing of known reactive metabolite target proteins. In this report we characterize the TPDB by reviewing briefly how the information it contains came to be known. We also compare its information to that provided by other types of "-omics" studies relevant to toxicology, and we illustrate how bioinformatic analysis of target proteins may help to elucidate mechanisms of cytotoxic responses to reactive metabolites.The post-translational modification of proteins is a well-known endogenous mechanism for regulating protein function and activity (1,2). Modifications such as disulfide bond formation, acylation, phosphorylation, glycosylation, metal ion binding and others can affect enzymatic activity, intracellular signaling via protein-protein interactions (PPIs), subcellular trafficking and localization, or even protein degradation. Cellular proteins are also susceptible to posttranslational modification by xenobiotic agents that possess, or whose metabolites possess, significant electrophilic character (e.g., epoxides, Michael acceptors, aldehydes, acylating agents, benzylic sulfate esters, etc.). Such non-physiological modifications to endogenous proteins are sometimes benign, but in other cases they are strongly associated with (and presumed to cause) lethal cytotoxic consequences via necrosis and/or apoptosis. Understanding the mechanisms by which protein covalent binding leads to cytotoxicity has been a major concern in Toxicology for nearly 40 years.During this time, as technology evolved, peak interest progressed from identifying the structures of reactive metabolites and the chemistry and enzymology of their formation, to elucidating the nature of the adducts formed with proteins and biological nucleophiles, to the identification of the target proteins modified by particular reactive metabolites. With information on the latter becoming more readily available, interest is now turning to the elucidation of mechanisms connecting protein covalent binding events to downstream cytotoxic events and cell death. Although our knowledge about reactive metabolite target Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo...

Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners

“…Despite extensive investigation, the mechanisms by which covalent binding events trigger cytotoxic outcomes remain largely unclear [ 6 , 7 ]. A major reason for this gap is that only recently has it become technically feasible to identify numbers of individual proteins targeted by xenobiotic reactive metabolites.…”

“…Early target protein identifications were based on isolating individual adduct-bearing proteins, one at a time, using traditional protein separation methods. By 1997, only 28 proteins targeted by xenobiotic reactive metabolites had been isolated and identified, largely by N-terminal sequencing [ 6 ]. In 1998, however, the coupling of 2D gel electrophoresis with mass spectrometric methods of protein identification literally revolutionized the field [ 8 ].…”

“…Since then both the number of known target proteins and the number of small-molecule adduct-forming xenobiotics studied have increased nearly ten-fold. To help keep track of this information and to facilitate its analysis, we recently built the Reactive Metabolite Target Protein Database (TPDB) [ 6 , 9 , 10 ]. All proteins listed in this database came from studies using live animals or intact living cells.…”

“…As the number of known target proteins grew, so did the hope of elucidating mechanistic pathways connecting covalent adduction events to the observed cytotoxic outcomes. For example, we and others [ 7 , 11 - 14 ] have attempted to make sense of lists of target proteins by arbitrarily grouping them into categories according to function, but this approach has done little to reveal to a unifying mechanism of toxicity caused by a variety of reactive metabolites [ 6 , 7 ]. Another way to analyze target proteins is to sort them into Gene Ontology (GO) categories [ 15 ] and determine whether any show an over-abundance of target proteins relative to statistical expectations.…”

Liver Protein Targets of Hepatotoxic 4-Bromophenol Metabolites

“…18 In an attempt to gain insight into cellular mechanisms of reactive metabolite toxicity, our laboratory has identified numerous hepatocellular proteins that become covalently labeled by metabolites of [ 14 C]-BB. 19–21 It was thus of interest to identify proteins targeted by reactive metabolites of 4BP since these adduction events are not associated with hepatotoxic consequences.…”