In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes.
The ubiquitin-proteasome system (UPS) is responsible for turnover of most cellular proteins in eukaryotes. Protein degradation by the UPS serves quality control and regulatory functions. Proteasome inhibition showed great promise in effectively treating cancer and restenosis. UPS dysfunction in cardiac hypertrophy and failure has recently been suspected but remains to be investigated. A system capable of monitoring dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ would no doubt benefit significantly efforts to decipher the pathogenic significance of UPS dysfunction in the heart and to evaluate the effect of proteasome inhibition on cardiac myocytes. We successfully established such a system in cultured cardiac myocytes by delivering and expressing a modified green fluorescence protein (GFPu) gene using recombinant adenoviruses. GFPu contains a ubiquitination signal sequence fused to the COOH terminus. Fluorescence microscopy and Western blots revealed that protein abundance of modified green fluorescent protein (GFPu), but not wild-type green fluorescent protein, in cultured cardiac myocytes was incrementally increased when function of the proteasomes was inhibited in various degrees by specific inhibitors. The increase in GFPu protein levels and fluorescence intensity is paralleled by a decrease in the in vitro peptidase activity of the proteasomes. Our results demonstrate that GFPu can be used as a surrogate marker to monitor dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ. Application of this novel system reveals that moderate levels of H2O2, a reactive oxygen species generator, impair proteolytic function of the UPS in cultured cardiac myocytes.
Dong
Xin
X
Cardiovascular Research Institute, University of South Dakota, Sioux Falls, South Dakota 57105, USA.
Liu
Jinbao
J
Zheng
Hanqiao
H
Glasford
Joseph W
JW
Huang
Wei
W
Chen
Quan Hai
QH
Harden
Niels R
NR
Li
Faqian
F
Gerdes
A Martin
AM
Wang
Xuejun
X
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D002470:Cell Survival; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007202:Indicators and Reagents; D007976:Leupeptins / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D008991:Monitoring, Physiologic / Q000379:methods*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000502:physiology; D016877:Oxidants / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000494:pharmacology; D025801:Ubiquitin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D007202:Indicators and Reagents; D007976:Leupeptins; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D016877:Oxidants; D011506:Proteins; D017382:Reactive Oxygen Species; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde