Hyperubiquitination of proteins in dilated cardiomyopathy.

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.
Mesh Terms:
Blotting, Western, Cardiomyopathy, Dilated, Cells, Cultured, Electrophoresis, Gel, Two-Dimensional, Glutathione Transferase, Humans, Immunohistochemistry, Myocardial Ischemia, Myocardium, Phenotype, RNA, Messenger, Reverse Transcriptase Polymerase Chain Reaction, Thiolester Hydrolases, Ubiquitin, Ubiquitin Thiolesterase
Proteomics
Date: Feb. 01, 2003
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