Atteinte ganglionnaire sentinelle et cancer de la prostate : quel impact sur la survie sans recidive ? À propos de 200 patients

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Heterologous SUMO-2/3-Ubiquitin Chains Optimize IκBα Degradation and NF-κB Activity

The NF-κB pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule IκBα, the IKK subunit γ/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of IκBα and activation of NF-κB mediated by TNFα. We found that under conditions of deficient SUMOylation, an important delay in both TNFα-mediated proteolysis of IκBα and NF-κB dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs), revealed the formation of chains on IκBα containing SUMO-2/3 and ubiquitin after TNFα stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on IκBα after activation of the TNFα signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of IκBα by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of IκBα modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified IκBα to the action of 26S proteasome, contributing to the optimal control of NF-κB activity after TNFα-stimulation

Between pathological prostate cancer lymph node and sentinel lymph node status : which one is the most informative?

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SUMOylation contributes to the optimal TNFα-mediated NF-κB activation and degradation of IκBα.

<p>(A) HeLa cells were transfected 72 h with control or Ubc9 siRNA (100 nM). Cells were co-transfected with a NF-κB-luciferase reporter plasmid (3EnhancerConA) and β-galactosidase reporter. Twenty-four hours later cells were stimulated with TNFα (15 ng/ml) as indicated and luciferase and β-galactosidase activities measured as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051672#pone.0051672-Rodriguez2" target="_blank">[50]</a>. The graph corresponds to the mean of three independent experiments. (B) HeLa cells were transfected during 72 h with control or Ubc9 siRNA (100 nM) and stimulated with TNFα (15 ng/ml) as indicated. Western-blot analyses were performed with the indicated antibodies.</p

SUMO-2 and Ubiquitin promote efficient chain extension on IκBα.

<p>(A) Strategy used to make the different fusions proteins. (B) HEK293 cells were co-transfected with His6-ubiquitin and IκBα fusion-proteins as indicated. Cells were pre-treated with MG132 and stimulated 20 min with TNFα. His₆-ubiquitylated proteins were purified using denaturing conditions and Ni²⁺ chromatography. EV: Empty Vector. (C) HEK293 cells were co-transfected with His6-SUMO-2 and IκBα fusions protein as indicated. Cells were pre-treated with MG132 and stimulated 20 min with TNFα as in A. His₆-sumoylated proteins were purified using Ni²⁺ chromatography procedure. (D) <i>In vitro</i> SUMOylation assay using IκBα WT or fusion proteins as substrates. (E) HEK293 cells were transfected as indicated, pre-treated with MG132 and stimulated 20 min with TNFα. Cells were lysed in a buffer containing 3.5 µM of TUBE hHR23A. TUBE-captured material was eluted and submitted to IκBα immunoprecipitation. EV: Empty Vector.</p

SUMO-2/3-Ubiquitin chains drive an efficient IκBα degradation by the 26S proteasome.

<p>(A) (B) <i>In vitro</i> ubiquitylation, SUMOylation or mixed assays using IκBα WT (A) or S³⁵ IκBα WT (B) as substrates. Suboptimal conditions of conjugation were used in this assay (see materials and methods). (A) Western blot detection with the indicated antibodies. (B) Detection of radio-labelled material. (C) <i>In vitro</i> ubiquitylation, SUMOylation or mixed assays using S³⁵ IκBα WT as substrate in the presence (+) or absence (-) of 26S proteasome. Saturating conditions of conjugation were used in this assay (see materials and methods). Different Ubiquitin: SUMO-2/SUMO-3 molar ratios were tested as follows: lane 1 = 4∶0/0, lane 2 = 3∶0.5/0.5, lane 3 = 2∶1/1, lane 4 = 1∶1.5/1.5, lane 5∶0:2/2. Detection of radio labelled material. (D) <i>In vitro</i> ubiquitylation, SUMOylation or mixed assays using S³⁵ IκBα WT as substrate in the presence (+) or absence (−) of 26S proteasome. Replicated reactions using saturating conditions and the following ubiquitin: SUMO-2/SUMO-3 ratios: 4∶0/0 for lanes 1 and 2, 0∶2/2 for lanes 3 and 4 and 2∶1/1 for lanes 5 and 6. Phosphorimager quantification of modified forms of S³⁵ IκBα WT in the presence or absence of 26S (n = 5). Standard deviation is indicated in the histograms. (E). Seventy-two hours after transfection with control or Ubc9 siRNA (100 nM), HeLa cells were pre-treated with MG132, stimulated with TNFα and lysed in a buffer containing TUBE-hHR23A. TUBE-captured material was submitted to IκBα immunoprecipitation. After IκBα-IP, extracts were eluted with glycine 200 mM pH2.5, equilibrated at pH 7.5 and submitted to an <i>in vitro</i> proteasome-mediated degradation assay at the indicated times.</p