Linked Life Data

15963938

Source:http://linkedlifedata.com/resource/pubmed/id/15963938

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2005-8-1
pubmed:abstractText
Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
343
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
244-55
pubmed:dateRevised
2005-11-29
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2.
pubmed:affiliation
Discovery Technologies, Novartis Institutes for Biomedical Research, CH-4058 Basel, Switzerland.
pubmed:publicationType
Journal Article