PMID- 12386159
OWN - NLM
STAT- MEDLINE
DCOM- 20030227
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 277
IP  - 52
DP  - 2002 Dec 27
TI  - Covalent modification of epithelial fatty acid-binding protein by 
      4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant 
      biology.
PG  - 50693-702
AB  - 4-Hydroxynonenal (4-HNE) is a cytotoxic alpha,beta-unsaturated acyl aldehyde that 
      is naturally produced from lipid peroxidation and cleavage in response to 
      oxidative stress and aging. Such reactive lipids covalently modify cellular 
      target proteins, thereby affecting biological structure and function. Herein we 
      report the identification of the epithelial fatty acid-binding protein (E-FABP) 
      as a molecular target for 4-HNE modification both in vitro and in vivo. 4-HNE 
      covalently modified (t(12) < 60 s) E-FABP in vitro, as revealed by a combination 
      of matrix-assisted laser desorption ionization-time of flight mass spectrometry 
      and immunochemical reactivity using antibodies directed to 4-HNE-protein 
      conjugates. Identification of Cys-120 as the major site of modification was 
      determined through tandem mass spectral sequencing of tryptic peptides, as well 
      as analysis of E-FABP mutants C120A, C127A, and C120A/C127A. The in vitro 
      modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and 
      temperature (4-37 degrees C) but was markedly potentiated by noncovalently bound 
      fatty acids. 4-HNE-modified E-FABP was more stable than unmodified E-FABP to 
      chemical denaturation by guanidine hydrochloride, as assessed by changes in 
      intrinsic tryptophan fluorescence. Analysis of soluble protein extracts from rat 
      retina with antibodies directed to 4-HNE-protein conjugates revealed 
      immunoreactivity with a 15-kDa protein that was identified by electrospray 
      ionization and matrix-assisted laser desorption ionization-time of flight mass 
      spectrometry as E-FABP. Evaluation of retinal pigment epithelial cell extracts 
      derived from E-FABP null mice by two-dimensional gel electrophoresis using 
      anti-4-HNE antibodies revealed increased modification in the null cells relative 
      to those from wild type cells. These results indicate that E-FABP is a molecular 
      target for 4-HNE modification and the hypothesis that E-FABP functions as an 
      antioxidant protein by scavenging reactive lipids through covalent modification 
      of Cys-120.
FAU - Bennaars-Eiden, Assumpta
AU  - Bennaars-Eiden A
AD  - Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 
      55455, USA.
FAU - Higgins, LeeAnn
AU  - Higgins L
FAU - Hertzel, Ann V
AU  - Hertzel AV
FAU - Kapphahn, Rebecca J
AU  - Kapphahn RJ
FAU - Ferrington, Deborah A
AU  - Ferrington DA
FAU - Bernlohr, David A
AU  - Bernlohr DA
LA  - eng
GR  - R03 AG19024/AG/NIA NIH HHS/United States
GR  - T32HL07741/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
DEP - 20021016
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Aldehydes)
RN  - 0 (Antioxidants)
RN  - 0 (Carrier Proteins)
RN  - 0 (Fabp5 protein, mouse)
RN  - 0 (Fabp7 protein, mouse)
RN  - 0 (Fabp7 protein, rat)
RN  - 0 (Fatty Acid-Binding Protein 7)
RN  - 0 (Fatty Acid-Binding Proteins)
RN  - 0 (Neoplasm Proteins)
RN  - 0 (Nerve Tissue Proteins)
RN  - 0 (Peptide Fragments)
RN  - 0 (Recombinant Proteins)
RN  - JU58VJ6Y3B (Guanidine)
RN  - K1CVM13F96 (4-hydroxy-2-nonenal)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Aldehydes/*pharmacology
MH  - Amino Acid Sequence
MH  - Amino Acid Substitution
MH  - Animals
MH  - Antioxidants/*pharmacology
MH  - Carrier Proteins/chemistry/drug effects/*metabolism
MH  - *Cysteine
MH  - Epithelial Cells/enzymology
MH  - Escherichia coli/metabolism
MH  - Fatty Acid-Binding Protein 7
MH  - Fatty Acid-Binding Proteins
MH  - Guanidine
MH  - Hydrogen-Ion Concentration
MH  - Kinetics
MH  - Mice
MH  - Mice, Knockout
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - *Neoplasm Proteins
MH  - *Nerve Tissue Proteins
MH  - Oxidative Stress/physiology
MH  - Peptide Fragments/chemistry
MH  - Protein Denaturation
MH  - Recombinant Proteins/chemistry/drug effects/metabolism
MH  - Thermodynamics
EDAT- 2002/10/19 04:00
MHDA- 2003/02/28 04:00
CRDT- 2002/10/19 04:00
PHST- 2002/10/19 04:00 [pubmed]
PHST- 2003/02/28 04:00 [medline]
PHST- 2002/10/19 04:00 [entrez]
AID - S0021-9258(19)31387-0 [pii]
AID - 10.1074/jbc.M209493200 [doi]
PST - ppublish
SO  - J Biol Chem. 2002 Dec 27;277(52):50693-702. doi: 10.1074/jbc.M209493200. Epub 
      2002 Oct 16.