Small molecules intercept Notch signaling and the early secretory pathway.

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.

NOD2-C2 - a novel NOD2 isoform activating NF-kappaB in a muramyl dipeptide-independent manner.

BACKGROUND: The innate immune system employs several receptor families that form the basis of sensing pathogen-associated molecular patterns. NOD (nucleotide-binding and oligomerization domain) like receptors (NLRs) comprise a group of cytosolic proteins that trigger protective responses upon recognition of intracellular danger signals. NOD2 displays a tandem caspase recruitment domain (CARD) architecture, which is unique within the NLR family. FINDINGS: Here, we report a novel alternative transcript of the NOD2 gene, which codes for a truncated tandem CARD only protein, called NOD2-C2. The transcript isoform is highest expressed in leucocytes, a natural barrier against pathogen invasion, and is strictly linked to promoter usage as well as predominantly to one allele of the single nucleotide polymorphism rs2067085. Contrary to a previously identified truncated single CARD NOD2 isoform, NOD2-S, NOD2-C2 is able to activate NF-kappaB in a dose dependent manner independently of muramyl dipeptide (MDP). On the other hand NOD2-C2 competes with MDPs ability to activate the NOD2-driven NF-kappaB signaling cascade. CONCLUSION: NOD2 transcripts having included an alternative exon downstream of exon 3 (exon 3a) are the endogenous equivalents of a previously described in vitro construct with the putative protein composed of only the two N-terminal CARDs. This protein form (NOD2-C2) activates NF-kappaB independent of an MDP stimulus and is a potential regulator of NOD2 signaling.

Klotho is a substrate for alpha-, beta- and gamma-secretase.

Klotho is an anti-aging protein with different functions of the full-length membrane protein and the secreted hormone-like form. Using overexpression and knock-down approaches as well as embryonic fibroblasts of knock-out mice we present evidence that Klotho is shedded by the alpha-secretases ADAM10 and 17 as well as by the beta-secretase beta-APP cleaving enzyme 1. The remaining membrane-bound fragment is a substrate for regulated intramembrane proteolysis by gamma-secretase. Our data suggest that therapeutic approaches targeting these proteases should be carefully analyzed for potential side effects on Klotho-mediated physiological processes.