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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 21, 2005 as doi:10.1096/fj.05-4869fje. |
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,1
* Cardiovascular Research Institute, South Dakota Health Research Foundation, University of South Dakota School of Medicine and Sioux Valley Hospitals and Health System, Sioux Falls, South Dakota, USA;
Department of Pathophysiology, Guangzhou Medical College, Guangzhou, Guangdong, China;
Cardiovascular Research Institute, Nanjing Medical University, Nanjing, Jiangsu, China; and
The Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois, USA
2 Correspondence: Cardiovascular Research Institute, University of South Dakota School of Medicine, 1100 E. 21st St., Suite 700, Sioux Falls, South Dakota 57105, USA. E-mail: xwang{at}usd.edu
SPECIFIC AIMS
Protein misfolding and aberrant aggregation are associated with many severe disorders, such as neural degenerative diseases, desmin-related myopathy (DRM), and congestive heart failure. Intrasarcoplasmic amyloidosis and increased ubiquitinated proteins are observed in human failing hearts. The pathogenic roles of these derangements in the heart remain unknown. The ubiquitin-proteasome system (UPS) plays a central role in intracellular proteolysis and regulates critical cellular processes. In cultured cells, aberrant aggregation by a mutant (MT) or misfolded protein impairs the UPS. However, this has not been demonstrated in intact animals and it is unclear how the UPS is impaired. This study was aimed to: 1) test whether abnormal desmin aggregation impairs UPS proteolytic function in intact animals by introducing a recently validated UPS function reporter into a well established DRM mouse model; 2) decipher at which step of UPS-mediated proteolysis the defect resides, if the UPS is significantly impaired in the DRM hearts; and 3) test whether expression of a MT-desmin (MT-des) is sufficient to impair the UPS in a non-muscle cell culture system.
PRINCIPAL FINDINGS
1. The UPS is impaired in DRC mouse hearts
Caused by mutations in the desmin gene, desminopathy is a subgroup of DRM which is characterized by the presence of abnormal desmin aggregates in muscle cells. DRM often affects the heart and resultant cardiomyopathy is known as desmin-related cardiomyopathy (DRC). It has been previously shown by cardiac specific transgenesis that moderate overexpression of a MT-des (R172 through E178 deletion) but not wild-type- (WT) des causes intrasarcoplasmic aberrant protein aggregation and cardiac hypertrophy and malfunction in mice. To probe UPS function in MT-des Tg, WT-des Tg, and non-Tg (Ntg) hearts, we measured ubiquitinated proteins in ventricular myocardium. Indicated by myocardial levels of desmin transcripts, overexpression of MT-des (6 folds over Ntg littermates) is significantly less than that of WT-des Tg controls (16 folds) at 9 wk. Compared with Ntg hearts, ubiquitinated proteins were significantly increased in both the soluble fractions and the total protein extracts of MT-des Tg hearts but not in WT-des Tg hearts, suggesting that the 26S proteasome is likely impaired in MT-des hearts.
To test whether the proteolytic function of the UPS is indeed impaired in the MT-des hearts, we then cross-bred MT-des Tg or WT-des Tg mice with the GFPdgn Tg mice. GFPdgn protein, a surrogate substrate of the UPS, was markedly increased in MT-des/GFPdgn double Tg hearts, compared with either WT-des/GFPdgn double Tg, or GFPdgn single Tg hearts of littermates (Fig. 1
A). Consistently, direct fluorescence confocal microscopic examinations of myocardial sections showed increased green fluorescence intensities in the cardiomyocytes of MT-des/GFPdgn double Tg hearts (Fig. 1C
). This increase in GFPdgn protein in the MT-des/GFPdgn double Tg hearts is due to slower degradation rather than increased synthesis because the steady-state GFPdgn transcript level in MT-des/GFPdgn double Tg hearts was not different from that in GFPdgn single Tg hearts (Fig. 1B
). These data clearly prove that the proteolytic function of the UPS in the heart is significantly inhibited by expression of a MT-des.
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2. The impairment of the UPS is primarily due to diminished entry of ubiquitinated proteins into the 20S proteasome
Theoretically, defects in either ubiquitination or the 26S proteasomes can impair UPS-mediated proteolysis. The abundance of ubiquitin remained unchanged; and more importantly, the ubiquitinated proteins in both the soluble and insoluble fractions of MT-des Tg hearts were significantly greater than in the WT-des Tg or Ntg hearts. These indicate that the UPS malfunction is unlikely caused by a deficiency in ubiquitination. Therefore, the primary impairment resides in the 26S proteasome which is composed of the 19S and the 20S proteasomes. The 19S proteasome recognizes, binds, de-ubiquitinates, and unfolds poly-ubiquitinated proteins and feeds the unfolded protein into the 20S proteasome. The target protein is then cleaved in the chamber of the 20S proteasome. Therefore, defects in either the 19S or the 20S proteasomes can affect the function of the 26S proteasomes. The function of 20S proteasomes further depends upon the activity of its peptidase subunits: ß5, ß2, and ß1 subunits which are respectively responsible for chymotrypsin-like, trypsin-like, and caspase-like activities. Using synthetic fluorogenic substrates, we examined proteasomal peptidase activities in the heart. We found that the activities of all the three peptidase subunits were not decreased in MT-des Tg hearts. On the contrary, all the three peptidase activities were significantly increased in the MT-des Tg hearts, compared with both Ntg and WT-des Tg hearts (Fig. 2
). Consistently, the protein abundance of components of the 20S proteasomes such as 
6, ß5 precursor, and ß2 subunit were significantly increased in the MT-des Tg hearts. These findings strongly suggest that the primary defect is not in the proteolytic core of 20S proteasomes but rather in the entry of ubiquitinated proteins into the 20S proteasomes. Impairments of the 19S proteasome, the -rings of the 20S complex, or both are formerly possible causes. Our further examination revealed that key 19S proteasome components such as Rpt3 and Rpt5, were significantly down-regulated in the MT-des expressing hearts but not in the WT-des overexpressing hearts, suggesting that depletion of the 19S proteasomes is at least partially responsible for the proteasomal malfunction caused by MT-des.
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3. MT-des impairs the UPS in cultured non-muscle cells
Expression of MT-des can directly compromise the desmin filament network that is unique to myocytes. To rule out the possibility that UPS impairment by MT-des is due to disruption of the desmin filament network, we further tested the effect of MT-des on the UPS in human embryonic kidney (HEK) cells. We established clonal cell lines stably express GFPu, a surrogate protein substrate for the UPS, as previously reported by Bence et al. and designated as GFPu-2 cells. As expected, GFPu protein accumulates in GFPu-2 cells and the cells become fluorescent upon proteasomal inhibition. Transient transfection of MT-des but not WT-des led to formation of aberrant desmin aggregates and significantly increased GFPu protein levels in GFPu-2 cells as evidenced by both Western blot and fluorescence microscopic analyses, indicating that UPS impairment by MT-des can occur in a cell type which does not normally express desmin. This demonstrates that MT-des impairing the UPS is not through disrupting the desmin filaments and that proteasomal malfunction observed in MT-des Tg hearts is not necessarily secondary to cardiac malfunction.
CONCLUSIONS AND SIGNIFICANCE
In this study, we have proven for the first time in intact animals that abnormal desmin aggregation impairs UPS proteolytic function in the heart. Our further analyses suggest that the defect resides in the entry of protein substrates into the proteolytic core of the 20S proteasome and depletion of key components of the 19S proteasome is likely responsible.
Abnormal protein aggregation such as intrasarcoplasmic amyloidosis, has recently observed in the majority of human hearts with end-stage congestive heart failure, a common but highly lethal and disabling disorder. The pathophysiological importance of this discovery remains unclear. Our study suggests that aberrant protein aggregation may represent a major pathogenic factor in congestive heart failure by impairing UPS proteolytic function in the heart. Notably, findings presented here are not only very important to cardiac pathogenesis but also of potentially major significance for many other areas of research, such as pathogenesis of neural degenerative diseases.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4869fje;
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