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Genetic Inhibition of 20S Proteasomes in the Heart
Author(s) -
zheng hanqiao,
Horak Kathleen,
Robbins Jeffrey,
Wang Xuejun
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb12-b
Subject(s) - proteasome , microbiology and biotechnology , biology , transgene , endogeny , endocrinology , biochemistry , gene
The ubiquitin‐proteasome system (UPS) degrades most cellular proteins, thereby playing critical roles in virtually all aspects of cell function. It is becoming increasingly clear that proteasomal malfunction is a common and potentially very important pathological process in the heart. To investigate the role(s) of the proteasome in the heart, a model of cardiomyocyte‐restricted (CR) genetic inhibition of the proteasome is needed for both in vitro and in vivo studies. Method PSMB5 encodes the beta5 subunit of 20S proteasomes. Recombinant adenoviruses capable of delivering a peptidase‐dead mutant (T60A) mouse PSMB5 into cultured cardiomyocytes were engineered and tested. Stable transgenic (tg) mouse lines that express various levels of T60A‐PSMB5 in a CR manner were created using an attenuated, inducible Mhc6 promoter. Results When delivered by the adenoviruses, T60A‐PSMB5 replaces the endogenous PSMB5 protein, inhibits proteasomal chymotrypsin‐like peptidase activities (PCLPA), and accumulates a UPS surrogate substrate (GFPu) dose‐dependently in cultured cardiomyocytes. As expected, tg T60A‐PSMB5 replaces endogenous PSMB5 dose‐dependently and can inhibit myocardial PCLPA by ~50%. Cross‐breeding with the GFPdgn reporter mice showed a significant accumulation of GFPdgn in the heart by T60A‐PSMB5 at 1 and 4 months, indicating a successful CR inhibition of the proteasome in vivo. Conclusion These new reagents will considerably benefit the research into the pathogenic role of the UPS in cardiac remodeling and failure. Supports: AHA Postdoc Fellowship #0620032Z; NIH HL072166.