Hyperubiquitination of proteins in dilated cardiomyopathy

Abstract

Protein degradation by the ubiquitin-proteasome pathway plays an important role in a variety of fundamental cellular processes, including cell cycle regulation, transcription, antigen processing and muscle remodelling. Research into disorders associated with the ubiquitin-proteasome system has been mainly in the field of neurodegenerative diseases. It is however becoming increasingly apparent that defects in the system are responsible for a number of non-neurological pathologies. Based on initial observations made as part of a proteomic analysis of an animal model of dilated cardiomyopathy (DCM) which indicated increased activity of the ubiquitin-proteasome system, we sought to determine whether this system was perturbed in hearts of human DCM patients. We studied explanted hearts from 12 DCM, 9 ischaemic (IHD) and 12 unused donor hearts. Protein expression was examined using two-dimensional polyacrylamide gel electrophoresis, Western blotting and immunohistochemistry. Expression of mRNA was examined using real-time quantitative polymerase chain reaction. Ubiquitinated proteins were affinity purified using a ubiquitin-binding column and identified using peptide mass fingerprinting. All DCM hearts showed significantly higher expression of certain key enzymes of the ubiquitin-proteasome pathway. mRNA expression of ubiquitin carboxyl-terminal hydrolase (UCH) was significantly higher (5.4-fold) in DCM hearts than in control hearts. Myocytes in sections from DCM hearts stained positively for UCH, whereas control hearts were negative. Overall protein ubiquitination was increased two-fold in DCM relative to IHD hearts and five-fold relative to donor hearts. The ubiquitination of a number of distinct proteins was greatly enhanced in DCM hearts as revealed by anti-ubiquitin Western blots. A number of these proteins were identified using affinity purification and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

“…Unlike similar methods, which have been reported in other organisms, our method does not require predeleting the native Ub genes (21) or high expression levels of a recombinant Ub gene (21)(22)(23). Two methods for identifying Ub proteins from human cells were published recently (24,25). In common with our approach, these methods do not require any genetic manipulation of the cells used for protein extraction.…”

Angiotensin II Type 2 Receptor–Mediated Gene Expression Profiling in Human Coronary Artery Endothelial Cells

“…This is especially interesting because Weekes et al have previously reported hyperubiquitination in both dilated cardiomyopathy and ischemia of the heart. 17 Another interesting gene that was differentially regulated by the AT 2 receptor was RGS7. Although there is no direct evidence, it can be speculated that the AT 2 receptor may decrease AT 1 receptormediated increases in calcium mobilization through the regulation of this gene.…”

The ubiquitin proteasome system in human cardiomyopathies and heart failure

“…A consistent finding among all of the studies is the observation that polyubiquitinated proteins are increased in DCM/failing hearts compared with control hearts (Table 1). This has been measured by quantitative Western blot analysis using antibodies that recognize ubiquitin with densitometric analysis of high molecular weight proteins (9,38,71,96,101). Immunolabeling for ubiquitin in fixed tissue sections has also revealed a greater number and size of ubiquitin-positive deposits in failing myocardium compared with controls (25, 29,38,60,88,101).…”

“…A functional UPS is critical for normal protein turnover and removes damaged or misfolded proteins in a multistep process (24,39,66,93). Dysfunction of the UPS is rapidly gaining recognition as a potentially important mechanism involved in the pathogenesis of a number of cardiac diseases, including heart failure (38,71,88,93,96,101), cardiomyopathies (12,44,71,78), hypertrophy (18,29,54,72), atrophy (2,72), ischemia-reperfusion (41,67), and atherosclerosis (30), providing a strong rationale for further study of specific mechanisms of proteasome impairment and identification of specific targets for therapy. In vitro and animal models have proven highly valuable in assessing effects of various stressors on the UPS and, in some cases, suggesting a causal link between defective protein clearance and disease phenotypes (14,41,46).…”