doi:10.1016/j.mad.2008.04.007 
Copyright © 2008 Elsevier Ireland Ltd All rights reserved.
Aging and dietary restriction effects on ubiquitination, sumoylation, and the proteasome in the heart
Feng Lia, Le Zhangb, Jeffrey Craddocka, Annadora J. Bruce-Kellerb, Kalavathi Dasurib, AnhThao Nguyenb and Jeffrey N. Kellerb, 
, 
aSanders-Brown Center on Aging, University of Kentucky, Lexington, KY, USA
bPennington Biomedical Research Center/Louisiana State University System, 6400 Perkins Road, Baton Rouge, LA 70808-4124, USA
Received 28 January 2008;
revised 15 April 2008;
accepted 22 April 2008.
Available online 30 April 2008.
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Abstract
Dietary restriction (DR), in the absence of malnutrition, is the only intervention known to reliably increase average and maximal lifespan in a variety of organisms including mammals. Because the effects of DR on the heart are poorly understood, in the present study we examined the effects of DR on the ubiquitin-proteasome pathway (UPP) in the heart. In these studies we observed that DR significantly reduced age-related impairments in proteasome-mediated protein degradation, and reduced age-related increases in ubiquitinated, oxidized, and sumoylated protein in the heart. Interestingly, DR did not significantly increase the expression of 20S proteasome subunits or the proteasome maturation factor (POMP-1). These data demonstrate for the first time the effects of aging and DR on proteasome biogenesis and sumoylation in the heart. Cumulatively, our data indicate that DR has many beneficial effects towards the UPP in the heart, and suggests that a preservation of the UPP may be a potential mechanism by which DR mediates beneficial effects on the cardiovascular system.
Keywords: Aging; Heart; Oxidative stress; Proteasome; Sumo; Ubiquitin
Abbreviations: AL, ad libitum; DR, dietary restriction; NIA, National Institute of Aging; POMP, proteasome maturation protein; ROS, reactive oxygen species; Sumo, small ubiquitin-like modifier; Ub, ubiquitin
Fig. 1. Effects of aging and dietary restriction on proteasome-mediated protein degradation in the heart. The levels of proteasome-mediated protein degradation were determined by measuring the amount of oxidized bovine serum albumin (oxBSA) in heart lysates from 3-month-old AL, 25-month-old AL, and 25-month-old DR rodents. The graphs represent the mean and S.E.M. of the degradation of rhodamine labeled oxBSA from six animals in each experimental group. *p < 0.05 compared to 3-month-old AL animals; **p < 0.05 compared to 25-month-old AL rodents.
Fig. 2. Analysis of heart lysate reveals an age-related impairment in 20S, and age-related increase in 26S, proteasome activity. Analysis of 26S proteasome activity in heart lysates (ATP-stimulated) revealed a significant elevation in chymotrypsin-like activity in both aged and DR heart lysates. Analysis of 20S proteasome activity (SDS stimulated) revealed an age-related impairment in chymotrypsin-like activity in 25-month-old tissues relative to heart lysates from 3-month-old animals. The specificity for proteasome activity in heart lysate studies was determined by subtracting the background protease activity (chymotrypsin-like activity not inhibited by lysate pretreatment with 10 μM MG132). *p < 0.05 compared to 3-month-old AL animals.
Fig. 3. Oxidative modification to the proteasome in the heart does not appear to be modulated by aging or DR. Immunoprecipitation of the proteasome was conducted in heart lysates from 3-month-old AL, 25-month-old AL, and 25-month-old DR rodents. The resulting immunoprecipitate was analyzed for protein carbonyls by derivatization and subsequent detection of the derivatized products using an anti-DNP antibody based detection system. Results are representative of results from six separate experiments.
Fig. 4. Effects of aging and dietary restriction on the levels of 20S alpha and 20S beta subunits in the heart. The level of 20S beta subunits (A) and 20S alpha subunits (B) were analyzed in the hearts of 3-month-old AL, 25-month-old AL, and 25-month-old DR rodents. A representative blot for the analyses is provided, with the molecular weight markers provided on left hand margin of the representative blot. The graph represent the mean and S.E.M. of the optical density of the immunoreactivity from six animals in each experimental group. *p < 0.05 compared to 3-month-old AL animals. Equal loading was confirmed by Commassie staining, data not shown.
Fig. 5. Effects of aging and dietary restriction on the levels of POMP-1 in the heart. The level of POMP-1 were analyzed in the hearts of 3-month-old AL, 25-month-old AL, and 25-month-old DR rodents. A representative blot for the analyses is provided, with the molecular weight markers provided on left hand margin of the representative blot. The graph represent the mean and S.E.M. of the optical density of the immunoreactivity from six animals in each experimental group. Equal loading was confirmed by Commassie staining, data not shown.
Fig. 6. Effects of aging and dietary restriction on the levels of ubiquitinated protein in the heart. The level of ubiquitinated protein was analyzed in the hearts of 3-month-old AL, 25-month-old AL, and 25-month-old DR rodents. A representative blot for the analyses is provided. The graph represent the mean and S.E.M. of the optical density of the immunoreactivity from six animals in each experimental group. *p < 0.05 compared to 3-month-old AL animals; **p < 0.05 compared to 25-month-old AL animals. Equal loading was confirmed by Commassie staining, data not shown.
Fig. 7. Effects of aging and dietary restriction on the levels of sumoylated protein in the heart. The level of sumoylated protein was analyzed in the hearts of 3-month-old AL, 25-month-old AL, and 25-month-old DR rodents. A representative blot for the analyses is provided. The graphs represent the mean and S.E.M. of the optical density of the immunoreactivity from six animals in each experimental group. *p < 0.05 compared to 3-month-old AL animals; **p < 0.05 compared to 25-month-old AL animals. Equal loading was confirmed by Commassie staining, data not shown.
Fig. 8. Effects of aging and dietary restriction on the levels of oxidized protein in the heart. The level of oxidized protein was determined by analyzing the amount of HNE-modified protein and protein carbonyls present in the hearts lysates from 3-month-old AL, 25-month-old AL, and 25-month-old DR rodents. A representative blot for each analyses is provided, which represents the data obtained from six separate experiments.
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