Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.
The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.
Kapphahn
Rebecca J
RJ
Department of Ophthalmology, University of Minnesota, 380 Lions Research Building, 2001 6th Street SE, Minneapolis, MN 55455, USA.
Giwa
Babatomiwa M
BM
Berg
Kristin M
KM
Roehrich
Heidi
H
Feng
Xiao
X
Olsen
Timothy W
TW
Ferrington
Deborah A
DA
D000375:Aging / Q000378:metabolism; D000447:Aldehydes / Q000032:analysis / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000032:analysis; D004847:Epithelial Cells / Q000378:metabolism; D005136:Eye Proteins / Q000032:analysis*; D050883:HSC70 Heat-Shock Proteins / Q000032:analysis; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis; D010084:Oxidation-Reduction; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism; D040901:Proteomics; D051381:Rats; D011916:Rats, Inbred F344; D012160:Retina / Q000187:drug effects / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014305:Triose-Phosphate Isomerase / Q000032:analysis; D038206:beta-Crystallin B Chain / Q000032:analysis
D000447:Aldehydes; D003432:Cross-Linking Reagents; D005136:Eye Proteins; D050883:HSC70 Heat-Shock Proteins; D008565:Membrane Proteins; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2; D010751:Phosphopyruvate Hydratase; D014305:Triose-Phosphate Isomerase; C027576:4-hydroxy-2-nonenal