Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1

Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition

Técnicas cirúrgicas para correção de aneurisma de Aorta : Surgical techniques for aortic aneurysm repair

O aneurisma de aorta abdominal é definido como uma dilatação segmentar maior que 50% do diâmetro normal, podendo variar, entre vários fatores, principalmente com a idade, sexo e hábitos de vida, necessitando de acompanhamento ambulatorial e, em casos selcionados, de intervenção cirúrgica. A grande maioria dos pacientes são assintomáticos, podendo em poucos casos apresentar sintomas inespecíficos como dor abdominal ou até mesmo massa abdominal pulsátil. Foram apresentados os fatores de risco, epidemiologia, etiologia, fisiopatologia, rastreamento, diagnóstico e possibilidades de tratamento cirúrgico e endovascular, bem como prognóstico. O Objetivo do artigo foi avaliar o melhor método intervencionista para tratamento de aneurisma de aorta abdominal de acordo com os diferentes tipos de pacientes que envolvem suas determinadas comorbidades, propiciando adequado tratamento sendo este endovascular ou cirúrgico convencional. Foram analisadas as taxas de complicações, necessidade de reintervenções, morbimortalidade e tempo de internação pós procedimento, além do prognóstico

Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10

Adenosine diphosphate-ribosylation is a post-translational modification mediated by intracellular and membrane-associated extracellular enzymes and many bacterial toxins. The intracellular enzymes modify their substrates either by poly-ADP-ribosylation, exemplified by ARTD1/PARP1, or by mono-ADP-ribosylation. The latter has been discovered only recently, and little is known about its physiological relevance. The founding member of mono-AD-Pribosyltransferases is ARTD10/PARP10. It possesses two ubiquitin-interaction motifs, a unique feature among ARTD/PARP enzymes. Here, we find that the ARTD10 ubiquitin-interaction motifs bind to K63-linked poly-ubiquitin, a modification that is essential for NF-kappa B signalling. We therefore studied the role of ARTD10 in this pathway. ARTD10 inhibits the activation of NF-kB and downstream target genes in response to interleukin-1 beta and tumour necrosis factor-a, dependent on catalytic activity and poly-ubiquitin binding of ARTD10. Mechanistically ARTD10 interferes with poly-ubiquitination of NEMO, which interacts with and is a substrate of ARTD10. Our findings identify a novel regulator of NF-kB signalling and provide evidence for cross-talk between K63-linked poly-ubiquitination and mono-ADP-ribosylation

Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle

Biotechnologie

Erioflorin inhibits AP-1- and NF-κB-trancriptional activities.

<p>(A) HEK293 cells were co-transfected with an AP-1 <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TPA (10 nM) with or without erioflorin (2.5 and 5 µM). Relative AP-1 activity was normalized to <i>renilla</i> luciferase and presented relative to TPA-only treated cells. (B) HEK293 cells were co-transfected with a NF-κB <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TNFα (20 mg mL^–1) with or without erioflorin (2.5 and 5 µM). Relative NF-κB activity was normalized to <i>renilla</i> luciferase and presented relative to TNFα-only treated cells. All data are given as means ± SEM (n≥3, **p<0.01).</p

Erioflorin specifically inhibits E3-ligase β-TrCP1 activity.

<p>(A) HEK293 cells were treated with TNFα (20 ng mL^–1) for the indicated times with or without erioflorin (10 µM). (B) HEK293 cells were maintained under full medium conditions (10% serum) or serum deprived for 24 h following treatment with erioflorin (5 µM) for 8 h. (C) HEK293 cells were treated for 8 h with erioflorin (1.25–10 µM) or the prolyl-hydroxylase inhibitor dimethyloxallylglycine (DMOG, 1 µM). (D) HeLa cells were serum deprived for 48 h prior to treatment with erioflorin (10 µM) or the proteasome inhibitor MG132 (10 µM) for 8 h. Whole cell extracts were subjected to western analysis and probed with the indicated antibodies. Blots are representative for at least three independent experiments.</p

Erioflorin stabilizes Pdcd4_(39–91)luc from TPA-induced degradation.

<p>(A) Structure of erioflorin (Mwt = 348.4). (B) Stably Pdcd4_(39–91)luc expressing HEK293 cells were treated for 8 h with TPA (10 nM) with increasing concentrations of erioflorin (0.0625–10 µM). Pdcd4 stabilizing activity was determined relative to Δ(RLU_control–RLU_TPA-only). (C) Stably Pdcd4_(mut39–91)luc expressing HEK293 cells were treated as in (B). Luciferase activity is given relative to TPA-treated controls. All data are presented as means ± SEM (n≥3, *p<0.05, **p<0.01).</p