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.
Weekes
John
J
Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine, Heart Science Centre, Harefield, UK.
Morrison
Karen
K
Mullen
Anthony
A
Wait
Robin
R
Barton
Paul
P
Dunn
Michael J
MJ
D015153:Blotting, Western; D002311:Cardiomyopathy, Dilated / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017202:Myocardial Ischemia / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013869:Thiolester Hydrolases / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase
D012333:RNA, Messenger; D025801:Ubiquitin; D005982:Glutathione Transferase; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase