Carbonylation of ER chaperone proteins in aged mouse liver

doi:10.1016/S0006-291X(03)00826-X

Biochemical and Biophysical Research Communications

Volume 305, Issue 3, 6 June 2003, Pages 566-572

https://doi.org/10.1016/S0006-291X(03)00826-X Get rights and content

Abstract

Progressive accumulation of oxidative damage to macromolecules in aged tissues is thought to contribute to the decline in tissue function characteristic of the aged phenotype. Mitochondria are a major intracellular source of reactive oxygen species (ROS); however, other organelles are also endogenous sources of oxyradicals and oxidants, which can damage macromolecules. We, therefore, sought to examine the relationship between aging and oxidative damage to ER resident proteins, which exist in a strongly oxidizing environment necessary for disulfide bond formation. In these studies, we have fractionated young and aged liver homogenates, resolved the proteins by 2D gel electrophoresis, assayed for oxidative damage as indicated by protein carbonylation, and identified BiP/Grp78, protein disulfide isomerase (PDI), and calreticulin as exhibiting an age-associated increase in oxidative damage. Increased carbonylation of these key proteins in aged liver suggests an age-associated impairment in protein folding, disulfide crosslinking, and glycosylation in the aged mouse liver.

Section snippets

Materials and methods

Isolation of tissue. Young (3 months) and aged (24 months) male C57BL/6 mice from the National Institute on Aging colonies (Bethesda, MD) were obtained through the Charles River Laboratories (Wilmington, MA), maintained with a 12 h light/dark cycle, and fed ad libitum on a standard chow diet for at least 1 week before use. The animals were sacrificed by decapitation, and the livers were harvested immediately, rinsed in ice-cold PBS, and then prepared for subcellular fractionation.

Subcellular

Characterization of ER/mitochondrial fraction

Solubilized 8000g pellets were resolved by single dimension, gradient SDS–PAGE, and immunoblotting was used to detect organelle-specific (ER/mitochondrial) protein markers to assess the relative enrichment of these subcellular compartments. The relative immunoreactivity of calnexin (ER) and cytochrome C (mitochondria) demonstrates the enrichment of these marker proteins in the 8000g pellet, with very little cytoplasmic contamination (p38 MAPK). Additional fractionation of the ER/mitochondrial

Discussion

We have shown by protein carbonyl assay that oxidative damage to critical ER proteins increases in the aged mouse liver. Carbonylated proteins have been reported to be indicators of oxidative damage, to increase with age, and to significantly impair the function of such oxidized proteins [7], [8], [17], [18], [19]. For example, the selective carbonylation of aconitase and ANT in aged muscle mitochondria affects their enzymatic and functional activities, and thereby may contribute to the

Acknowledgments

This project was supported by U.S.P.H.S. Grant PO2 AG10514 awarded by the National Institute on Aging, and by grants from the Sealy Center on Aging and the Claude D. Pepper Older Americans Independence Centers. We thank the University of Texas Medical Branch Protein Core for Sequencing and MALDI/TOF analysis, and we thank Diane Strain for clerical assistance.

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Carbonylation of ER chaperone proteins in aged mouse liver

Abstract

Section snippets

Materials and methods

Characterization of ER/mitochondrial fraction

Discussion

Acknowledgments

Mech. Aging Dev.

Mutat. Res.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Mol. Cell

J. Biol. Chem.

Exp. Gerontol.

Cell

Cell

J. Nutr.

Oxidants, oxidative stress, and biology of aging

Nature

Does oxidative damage to DNA increase with age?

Proc. Natl. Acad. Sci. USA

Protein oxidation and aging

Science

Protein oxidation in aging and age-related diseases

Ann. N. Y. Acad. Sci.

Mitochondrial free radical generation; site of production in states 4 and 3, organ specificity and relationship with aging rate

J. Bioenerg. Biomembr.

Oxidative damage during aging targets mitochondrial aconitase

Proc. Natl. Acad. Sci. USA