Proteomic analysis of acrylamide-protein adduct formation in rat brain synaptosomes.
Evidence suggests that the neurological defects (gait abnormalities, foot splay, and skeletal muscle weakness) associated with acrylamide (ACR) intoxication are mediated by impaired neurotransmission at central and peripheral synapses. ACR can form adducts with nucleophilic residues on proteins and thereby alter corresponding structure and function. To evaluate protein adduction in nerve terminals as a possible mechanism of action, recombinant N-ethylmaleimide sensitive factor (NSF) was exposed in vitro to ACR (10 micromol) and mass spectrometry (MS) was used to identify adduct sites. MS analyses demonstrated that ACR formed adducts with sulfhydryl groups on cysteine residues (carbamoylethylcysteine, or CEC) of NSF. Ex vivo incubation of whole brain synaptosomes with ACR (0.001-1.0 M) produced concentration-dependent increases in CEC that were inversely correlated to reductions in neurotransmitter release that occurred over the same neurotoxicant concentration range. In synaptosomes isolated from rats intoxicated at a higher (50 mg/kg per day x 3, 5, 8, or 11 days) or a lower (21 mg/kg per day x 14, 21, or 28 day) ACR dose rate, CEC levels increased progressively up to a moderate level of neurotoxicity. To identify protein adducts, synaptosomal proteins labeled by ex vivo 14C-ACR exposure were separated by gel electrophoresis and probed by immunoblot analysis. Results showed that NSF and the SNARE protein, SNAP-25, were tentative ACR targets. Subsequent experiments indicated that ACR exposure increased synaptosomal levels of the 7S SNARE core complex, which is consistent with inhibition of NSF, SNAP-25 function, or both. These data suggest that adduction of cysteine residues on NSF and certain SNARE proteins might be causally involved in the nerve terminal dysfunction induced by ACR.
Barber
David S
DS
Center for Human and Environmental Toxicology, University of Florida, Gainesville, FL 32611, USA.
LoPachin
Richard M
RM
D000178:Acrylamides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D013058:Mass Spectrometry; D052066:N-Ethylmaleimide-Sensitive Proteins; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013574:Synaptosomes / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism / Q000494:pharmacology
D000178:Acrylamides; D011506:Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D003545:Cysteine