No abstract

Nuclear factor-jB (NF-jB) and p53 critically determine cancer development and progression. Defining the cross talk between these transcription factors can expand our knowledge on molecular mechanisms of tumorigenesis. Here, we show that induction of replicational stress activates NF-jB p65 and triggers its interaction with p53 in the nucleus. Experiments with knockout cells show that p65 and p53 are both required for enhanced NF-jB activity during S-phase checkpoint activation involving ataxiatelangiectasia mutated and checkpoint kinase-1. Accordingly, the pro-inflammatory cytokine tumor necrosis factora (TNF-a) also triggers formation of a transcriptionally active complex containing nuclear p65 and p53 on jB response elements. Gene expression analyses revealed that, independent of NF-jB activation in the cytosol, TNFinduced NF-jB-directed gene expression relies on p53. Hence, p53 is unexpectedly necessary for NF-jB-mediated gene expression induced by atypical and classical stimuli. Remarkably, data from gain-and loss-of function approaches argue that anti-apoptotic NF-jB p65 activity is constitutively evoked by a p53 hot-spot mutant frequently found in tumors. Our observations suggest explanations for the outstanding question why p53 mutations rather than p53 deletions arise in tumors of various origins.

No abstract

Histone deacetylase 2 (HDAC2) is relevant for homeostasis and plays a critical role in gastrointestinal cancers. Here, we report that post-translational modification of endogenous HDAC2 with small ubiquitin-related modifier 1 (SUMO1) is a new regulatory switch for the tumor suppressor p53. Sumoylation of HDAC2 at lysine 462 allows binding of HDAC2 to p53. Moreover, sumoylated HDAC2 is a previously not recognized biologically relevant site-specific deacetylase for p53. Deacetylation of p53 at lysine 320 by sumoylated HDAC2 blocks recruitment of p53 into promoter-associated complexes and p53-dependent expression of genes for cell cycle control and apoptosis. Thereby, catalytically active sumoylated HDAC2 restricts p53 functions and attenuates DNA damage-induced apoptosis. Genotoxic stress evokes desumoylation of HDAC2, enabling p53-dependent gene expression. Our data show a new molecular mechanism involving a dynamically controlled HDAC2-sumoylation/p53-acetylation switch that regulates cell fate decisions following genotoxic stress.

Potassium channels allow for the passive movement of potassium ions across the cell membrane and are instrumental in controlling the membrane potential in all cell types. Quaternary ammonium (QA) compounds block potassium channels and have long been used to study the functional and structural properties of these channels. Here we describe the interaction between three symmetrical hydrophobic QAs and the prokaryotic potassium channel KcsA. The structures demonstrate the presence of a hydrophobic pocket between the inner helices of KcsA and provide insight into the binding site and blocking mechanism of hydrophobic QAs. The structures also reveal a structurally hidden pathway between the central cavity and the outside membrane environment reminiscent of the lateral fenestration observed in sodium channels that can be accessed through small conformational changes in the pore wall. We propose that the hydrophobic binding pocket stabilizes the alkyl chains of long-chain QA molecules and may play a key role in hydrophobic drug binding in general.

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.

No abstract

No abstract