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Detection of Protein Ubiquitination
Published on: August 19, 2009
Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in
S U Sixt1, H P Jennissen, M Winterhalter
1Klinik für Anästhesiologie, Universitätsklinikum Düsseldorf, Germany. StephanUrs.Sixt@med.uni-duesseldorf.de
Abstract:
The selective degradation of many proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved protein [1]. Ubiquitylated proteins were degraded by the 26S proteasome in an ATP-depended manner. The degradation of ubiquitylated proteins were controlled by isopeptidase cleavage. A well characterised system of ubiquitylation and deubiquitylation is the calmodulin system in vitro [2]. Detection of ubiquityl-calmodulin conjugtates in vivo have not been shown so far. In this article we discuss the detection of ubiquitin calmodulin conjugates in vivo by incubation with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues. Proteins with a molecular weight of ubiquityl-calmodulin conjugates could be detected in all organs tested. Incubation with ubiquitylprotein-isopeptidase showed clearly a decrease of ubiquitin calmodulin conjugates in vivo with an origination of unbounded ubiquitin. These results suggest that only few ubiquitin calmodulin conjugates exist in rabbit tissues.
Insights
Researchers detected ubiquitin-calmodulin conjugates in rabbit tissues using a novel isopeptidase. This study provides the first in vivo evidence of these conjugates, suggesting they exist in low levels across various organs.
Area of Science:
- Cellular Biology
- Biochemistry
- Protein Degradation
Background:
- The ubiquitin system mediates selective protein degradation in eukaryotic cells.
- Ubiquitylation targets proteins for degradation by the 26S proteasome.
- Ubiquitin-calmodulin conjugation and deubiquitylation are well-characterized in vitro.
Purpose of the Study:
- To detect ubiquitin-calmodulin conjugates in vivo.
- To investigate the presence of these conjugates in rabbit tissues.
Main Methods:
- Incubation of rabbit tissues with a novel high-molecular weight ubiquitylprotein-isopeptidase.
- Detection of proteins with the molecular weight of ubiquitin-calmodulin conjugates.
Main Results:
- Ubiquitin-calmodulin conjugates were detected in all tested rabbit organs.
- Incubation with ubiquitylprotein-isopeptidase reduced ubiquitin-calmodulin conjugates, releasing free ubiquitin.
- These findings indicate low levels of ubiquitin-calmodulin conjugates in vivo.
Conclusions:
- The study provides the first in vivo evidence for ubiquitin-calmodulin conjugates.
- Ubiquitin-calmodulin conjugates are present at low levels in various rabbit tissues.
- The developed method allows for the detection of these conjugates in vivo.
