Homogeneous Assays for Cellular Protein Degradation Using β -Galactosidase Complementation: NF- κ B/I κ B Pathway Signaling
AbstractActivation of cells by the tumor necrosis factor- α (TNF- α ) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor- κ B (NF- κ B) via proteasomal degradation of an associated I κ B molecule. To monitor cellular I κ B, 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 β -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 β -galactosidase enzyme fragment complementation (EFC). Transient transfection of I κ B-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- α and IL-1 treatment induced a concentration-dependent degradation of I κ B-PL, with potency values similar to those reported using other methods. I κ BM-PL (mutant of I κ B-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF- α or IL-1, demonstrating that degradation of I κ B-PL was specific to the NF- κ B pathway activation. TNF- α and IL-1 induced maximal I κ B-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF- α 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 I κ B-PL degradation were 0.6 and <9%, respectively.