In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes

Xin Dong, Jinbao Liu, Hanqiao Zheng, Joseph W. Glasford, Wei Huang, Quan Hai Chen, Niels R. Harden, Faqian Li, A. Martin Gerdes, Xuejun Wang

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)H1417-H1425
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume287
Issue number3 56-3
DOIs
StatePublished - Sep 1 2004

Fingerprint

Proteasome Endopeptidase Complex
Ubiquitin
Cardiac Myocytes
Green Fluorescent Proteins
Proteins
Fluorescence
Ubiquitination
Cardiomegaly
Protein Sorting Signals
Eukaryota
Fluorescence Microscopy
Adenoviridae
Quality Control
Proteolysis
Reactive Oxygen Species
Peptide Hydrolases
Heart Failure
Biomarkers
Western Blotting

Keywords

  • Adenoviruses
  • Proteasome inhibition
  • Protein degradation
  • Rat
  • Tissue culture

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes. / Dong, Xin; Liu, Jinbao; Zheng, Hanqiao; Glasford, Joseph W.; Huang, Wei; Chen, Quan Hai; Harden, Niels R.; Li, Faqian; Gerdes, A. Martin; Wang, Xuejun.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 287, No. 3 56-3, 01.09.2004, p. H1417-H1425.

Research output: Contribution to journalArticle

Dong, Xin ; Liu, Jinbao ; Zheng, Hanqiao ; Glasford, Joseph W. ; Huang, Wei ; Chen, Quan Hai ; Harden, Niels R. ; Li, Faqian ; Gerdes, A. Martin ; Wang, Xuejun. / In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes. In: American Journal of Physiology - Heart and Circulatory Physiology. 2004 ; Vol. 287, No. 3 56-3. pp. H1417-H1425.
@article{1a829b2c14834c41a1e57c693c032de4,
title = "In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes",
abstract = "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.",
keywords = "Adenoviruses, Proteasome inhibition, Protein degradation, Rat, Tissue culture",
author = "Xin Dong and Jinbao Liu and Hanqiao Zheng and Glasford, {Joseph W.} and Wei Huang and Chen, {Quan Hai} and Harden, {Niels R.} and Faqian Li and Gerdes, {A. Martin} and Xuejun Wang",
year = "2004",
month = "9",
day = "1",
doi = "10.1152/ajpheart.01233.2003",
language = "English (US)",
volume = "287",
pages = "H1417--H1425",
journal = "American Journal of Physiology - Renal Physiology",
issn = "0363-6127",
publisher = "American Physiological Society",
number = "3 56-3",

}

TY - JOUR

T1 - In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes

AU - Dong, Xin

AU - Liu, Jinbao

AU - Zheng, Hanqiao

AU - Glasford, Joseph W.

AU - Huang, Wei

AU - Chen, Quan Hai

AU - Harden, Niels R.

AU - Li, Faqian

AU - Gerdes, A. Martin

AU - Wang, Xuejun

PY - 2004/9/1

Y1 - 2004/9/1

N2 - 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.

AB - 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.

KW - Adenoviruses

KW - Proteasome inhibition

KW - Protein degradation

KW - Rat

KW - Tissue culture

UR - http://www.scopus.com/inward/record.url?scp=4143114519&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=4143114519&partnerID=8YFLogxK

U2 - 10.1152/ajpheart.01233.2003

DO - 10.1152/ajpheart.01233.2003

M3 - Article

C2 - 15105173

AN - SCOPUS:4143114519

VL - 287

SP - H1417-H1425

JO - American Journal of Physiology - Renal Physiology

JF - American Journal of Physiology - Renal Physiology

SN - 0363-6127

IS - 3 56-3

ER -