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Homogeneous Assays for Cellular Protein Degradation Usingβ-Galactosidase Complementation: NF-κB/IκB Pathway Signaling

Authors :
Yali Shu
Inna Vainshtein
Xue Mei Wang
Richard M. Eglen
Xiaoning Zhao
Peter Fung
Joseph L. Horecka
Robert Gellibolian
Hyna Dotimas
Betty Lou Bosano
Source :
ASSAY and Drug Development Technologies. 1:823-833
Publication Year :
2003
Publisher :
Mary Ann Liebert Inc, 2003.

Abstract

Activation of cells by the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor-kappaB (NF-kappaB) via proteasomal degradation of an associated IkappaB molecule. To monitor cellular IkappaB, the protein was recombinantly expressed as a fusion protein with a novel enzymatic tag, ProLabel (PL). ProLabel is a small 5.5-kDa sequence from the amino-terminal amino acids of beta-galactosidase, possesses a simple ribbon structure, and can be fused to many proteins via the amino or carboxyl terminus. Expression of this construct allows quantitative detection of the recombinant protein in crude lysates by using a method based on beta-galactosidase enzyme fragment complementation (EFC). Transient transfection of IkappaB-PL in HeLa cells generated an EFC signal that was highly correlated with a western analysis of the protein construct. ProLabel expressed alone in the cells did not show any EFC activity, due to rapid proteolytic degradation, indicating a very low background signal from the protein tag. TNF-alpha and IL-1 treatment induced a concentration-dependent degradation of IkappaB-PL, with potency values similar to those reported using other methods. IkappaBM-PL (mutant of IkappaB-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF-alpha or IL-1, demonstrating that degradation of IkappaB-PL was specific to the NF-kappaB pathway activation. TNF-alpha and IL-1 induced maximal IkappaB-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF-alpha antibodies and the proteasome inhibitor, MG-132. The assay was amenable to HTS systems, with good precision and reproducibility. Z' values and coefficients of variance for IkappaB-PL degradation were 0.6 and9%, respectively.

Details

ISSN :
15578127 and 1540658X
Volume :
1
Database :
OpenAIRE
Journal :
ASSAY and Drug Development Technologies
Accession number :
edsair.doi.dedup.....bab332fe067b2089adf0f014ce46902f
Full Text :
https://doi.org/10.1089/154065803772613453