Identifizierung und Charakterisierung neuer tumorrelevanter Gene, die unter Kontrolle des Wnt/β-Catenin-Signalwegs stehen

Mittels DNA-Microarray-Analyse wurden die Expressionsmuster von C57MG-Wnt-1- und C57MG-pLNCx-Zellen verglichen. 140 differentiell exprimierte Gene wurden identifiziert. Neun Wnt-1-induzierte Gene wurden näher analysiert. Die Gene $\it 24p3$, $\it JE$ und $\it Pedf$ werden entgegengesetzt durch Wnt-1 und durch LiCl reguliert. Untersuchungen des $\it Pedf$-Promotors weisen auf die Repression von $\it Pedf$ durch den Wnt-Signalweg hin. Das $\it 24p3$-Gen unterliegt einer Inhibition durch LiCl und Wnt-3a, während langfristige Wnt-1-Einwirkung zur Induktion des Gens in C57MG-Zellen führt, wie Expressions- und Promotoruntersuchungen ergaben. In den verwendeten Zellen kann $\it 24p3$ seine Expression autokrin verstärken und kolokalisiert mit dem PML-Protein. In Brusttumoren der Maus lokalisiert das Protein in Bereichen mit TUNEL-positiven Zellen. NGAL, das menschliche Homolog von $\it 24p3$, wurde in Darmzelllinien, Tumor- und Normalgeweben untersucht. Die erhaltenen Daten deuten auf komplexe Regulationsmechanismen der Wnt-Zielgene hin.The expression patterns of C57MG-Wnt-1 und C57MG-pLNCx cells were compared by using the DNA microarray technique. 140 differentially expressed genes were identified. Nine Wnt-1 induced genes were further analysed. For $\it 24p3$, $\it JE$ and $\it Pedf$ an opposite regulation by Wnt-1 and by LiCl in the C57MG cells was detected. Studies with the $\it Pedf$ promoter are indicative of the down regulation of the $\it Pedf$ gene by an active Wnt pathway. The $\it 24p3$ gene is subjected to a negative regulation by LiCl and Wnt-3a, whereas long-term activation of the Wnt pathway results in the induction of $\it 24p3$, as detected by expression and promoter analysis. In the used cells, $\it 24p3$ enhances its own expression in an autokrine manner. The protein localizes together with PML and is detected in TUNEL-positive regions in mouse breast tumours. NGAL, the human homologue of $\it 24p3$, was analysed in colon cell lines, normal and tumour tissues. The obtained data suggest complex mechanisms of regulation of the Wnt target genes

Identifizierung und Charakterisierung neuer tumorrelevanter Gene, die unter Kontrolle des Wnt/β\beta-Catenin-Signalwegs stehen

Mittels DNA-Microarray-Analyse wurden die Expressionsmuster von C57MG-Wnt-1- und C57MG-pLNCx-Zellen verglichen. 140 differentiell exprimierte Gene wurden identifiziert. Neun Wnt-1-induzierte Gene wurden näher analysiert. Die Gene $\it 24p3$, $\it JE$ und $\it Pedf$ werden entgegengesetzt durch Wnt-1 und durch LiCl reguliert. Untersuchungen des $\it Pedf$-Promotors weisen auf die Repression von $\it Pedf$ durch den Wnt-Signalweg hin. Das $\it 24p3$-Gen unterliegt einer Inhibition durch LiCl und Wnt-3a, während langfristige Wnt-1-Einwirkung zur Induktion des Gens in C57MG-Zellen führt, wie Expressions- und Promotoruntersuchungen ergaben. In den verwendeten Zellen kann $\it 24p3$ seine Expression autokrin verstärken und kolokalisiert mit dem PML-Protein. In Brusttumoren der Maus lokalisiert das Protein in Bereichen mit TUNEL-positiven Zellen. NGAL, das menschliche Homolog von $\it 24p3$, wurde in Darmzelllinien, Tumor- und Normalgeweben untersucht. Die erhaltenen Daten deuten auf komplexe Regulationsmechanismen der Wnt-Zielgene hin.The expression patterns of C57MG-Wnt-1 und C57MG-pLNCx cells were compared by using the DNA microarray technique. 140 differentially expressed genes were identified. Nine Wnt-1 induced genes were further analysed. For $\it 24p3$, $\it JE$ and $\it Pedf$ an opposite regulation by Wnt-1 and by LiCl in the C57MG cells was detected. Studies with the $\it Pedf$ promoter are indicative of the down regulation of the $\it Pedf$ gene by an active Wnt pathway. The $\it 24p3$ gene is subjected to a negative regulation by LiCl and Wnt-3a, whereas long-term activation of the Wnt pathway results in the induction of $\it 24p3$, as detected by expression and promoter analysis. In the used cells, $\it 24p3$ enhances its own expression in an autokrine manner. The protein localizes together with PML and is detected in TUNEL-positive regions in mouse breast tumours. NGAL, the human homologue of $\it 24p3$, was analysed in colon cell lines, normal and tumour tissues. The obtained data suggest complex mechanisms of regulation of the Wnt target genes

Total Synthesis of (−)-Doliculide, Structure–Activity Relationship Studies and Its Binding to F-Actin

Actin, an abundant protein in most eukaryotic cells, is one of the targets in cancer research. Recently, a great deal of attention has been paid to the synthesis and function of actin-targeting compounds and their use as effective molecular probes in chemical biology. In this study, we have developed an efficient synthesis of (−)-doliculide, a very potent actin binder with a higher cell-membrane permeability than phalloidin. Actin polymerization assays with (−)-doliculide and two analogues on HeLa and BSC-1 cells, together with a prediction of their binding mode to F-actin by unbiased computational docking, show that doliculide stabilizes F-actin in a similar way to jasplakinolide and chondramide C.

Inducin Triggers LC3‐Lipidation and ESCRT‐Mediated Lysosomal Membrane Repair

: Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death

A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics

Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy