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

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 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