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Direct observation of a transient ternary complex during I?B?-mediated dissociation of NF-?B from DNA.

ABSTRACT: We previously demonstrated that I?B? markedly increases the dissociation rate of DNA from NF-?B. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and I?B? binding sites on NF-?B are overlapping. The kinetics of interaction of I?B? with NF-?B and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in I?B? were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-?B association with DNA is extremely rapid with a rate constant of 1.5 × 10(8) M(-1)?s(-1). The NF-?B-DNA complex dissociates with a rate constant of 0.41 s(-1), yielding a KD of 2.8 nM. When I?B? is added to the NF-?B-DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this I?B?-mediated dissociation is nearly equal to the rate constant of association of I?B? with the NF-?B-DNA complex, showing that I?B? is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant I?B? were also measured. This mutant associates with NF-?B more rapidly than wild-type I?B?, but it associates with the NF-?B-DNA complex more slowly and also is less efficient at mediating dissociation of the NF-?B-DNA complex.

SUBMITTER: Alverdi V

PROVIDER: S-EPMC3890772 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA.

Alverdi Vera V Hetrick Byron B Joseph Simpson S Komives Elizabeth A EA

Proceedings of the National Academy of Sciences of the United States of America 20131223 1

We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate consta ...[more]

PMID: 24367071

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Project description:BackgroundRegulator of cullins 1 (ROC1) is an important catalytic subunit of cullin-RING E3 ligase. Nuclear factor-kappa B (NF-κB) signaling is closely related to tumor invasion and metastasis. Earlier, we reported that ROC1 was associated with a poor prognosis in patients with bladder cancer (BCa). However, it is unclear whether ROC1 is involved in the NF-κB signaling associated with malignant BCa progression.MethodsThe expression of ROC1 and p65 in bladder cancer and paracancerous tissues were detected by immunohistochemistry (IHC). Pearson correlation was used to assess correlation between ROC1 and p65 protein expressions. The wound-healing and transwell assays were used to monitor cell invasion and migration. The effect of ROC1 on the expression of key proteins in the NF-κB signaling was determined by immunofluorescence and western blot (WB). Cycloheximide (CHX), MG132 and immunoprecipitation assays were used to evaluate the effect of ROC1 on the ubiquitination of phosphorylated inhibitor of kappa B alpha (p-IκBα). A lung metastasis mouse model was generated to detect the role of ROC1 in tumor metastasis.ResultsWe found that ROC1 was up-regulated in BCa tissues and cell lines, and high ROC1 levels were positively correlated with higher tumour grade, lymph node metastasis, distant metastasis and poor prognosis. Linear-regression analysis showed significant a Pearson correlation between ROC1 and nuclear p65 expression in BCa tissue microarray (TMA) samples. Functional studies demonstrated that ROC1 promoted BCa cell invasion and migration. In vitro and in vivo experiments showed that ROC1 activated NF-κB signaling by enhancing the ubiquitination of p-IκBα, which caused p65 nuclear translocation and promoted the transcription of some metastasis-related target genes, such as urokinase-type plasminogen activator receptor (uPAR), intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and matrix metalloproteinase 9 (MMP9), resulting in promoting BCa metastasis.ConclusionROC1 plays an important role in the progression of BCa and serves as a potential diagnostic and therapeutic target for patients with BCa.

Dataset Information

Direct observation of a transient ternary complex during I?B?-mediated dissociation of NF-?B from DNA.

Publications

Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA.

Similar Datasets

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