Katja Hübel

A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the α13 subunits of G proteins (Gα13). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Gα13 both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Gα13 might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.

Natural product–inspired cascade synthesis yields modulators of centrosome integrity

In biology-oriented synthesis, the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is, in particular, met by the scaffolds of natural products selected in evolution. The synthesis of natural product-inspired compound collections calls for efficient reaction sequences that preferably combine multiple individual transformations in one operation. Here we report the development of a one-pot, twelve-step cascade reaction sequence that includes nine different reactions and two opposing kinds of organocatalysis. The cascade sequence proceeds within 10-30 min and transforms readily available substrates into complex indoloquinolizines that resemble the core tetracyclic scaffold of numerous polycyclic indole alkaloids. Biological investigation of a corresponding focused compound collection revealed modulators of centrosome integrity, termed centrocountins, which caused fragmented and supernumerary centrosomes, chromosome congression defects, multipolar mitotic spindles, acentrosomal spindle poles and multipolar cell division by targeting the centrosome-associated proteins nucleophosmin and Crm1.

Prieurianin/endosidin 1 is an actin‐stabilizing small molecule identified from a chemical genetic screen for circadian clock effectors in Arabidopsis thaliana

Chemical modulators are powerful tools to investigate biological processes. To identify circadian clock effectors, we screened a natural product library in the model plant Arabidopsis thaliana. Two compounds, prieurianin (Pri) and prieurianin acetate, were identified as causing a shorter circadian period. Recently, Pri was independently identified as a vesicle trafficking inhibitor and re-named endosidin 1 (ES1). Here we show that Pri primarily affects actin filament flexibility in vivo, later resulting in reduced severing and filament depolymerization. This stabilization of the actin cytoskeleton subsequently causes changes in vesicle trafficking. Pri also affected microfilaments in mammalian cells, indicating that its target is highly conserved; however, it did not alter actin dynamics in vitro, suggesting that its activity requires the presence of actin-associated proteins. Furthermore, well-characterized actin inhibitors shortened the period length of the Arabidopsis clock in a similar way to Pri, supporting the idea that Pri affects rhythms by altering the actin network. We conclude that actin-associated processes influence the circadian system in a light-dependent manner, but their disruption does not abolish rhythmicity. In summary, we propose that the primary effect of Pri is to stabilize the actin cytoskeleton system, thereby affecting endosome trafficking. Pri appears to stabilize actin filaments by a different mechanism from previously described inhibitors, and will be a useful tool to study actin-related cellular processes.

ATP competitive inhibitors of D-alanine-D-alanine ligase based on protein kinase inhibitor scaffolds.

D-Alanine-D-alanine ligase (DDl) is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. Here, we describe a new approach to identify new inhibitor scaffolds for DDl based on similarity in the ATP binding region of different kinases and DDl. After an initial screening of several protein kinase inhibitors, we found that the Brutonss tyrosine kinase inhibitor LFM-A13, an analog of the Leflunomide metabolite A771726, inhibits DDl with a K(i) of 185 microM. A series of malononitrilamide and salicylamide derivatives of LFM-A13 has been synthesized to confirm the validity of this scaffold as an inhibitor of DDl.

The Ras Pathway Modulator Melophlin A Targets Dynamins

The Ras/mitogen-activated protein (MAP) kinase signal transduction pathway regulates numerous biological programs including cell growth and differentiation, and harbors several important anticancer-drug targets. Recent research, in particular inspired by systems biology approaches, revealed the importance of dynamic spatiotemporal regulation of and interplay between the Ras network members and their interaction with other signaling modules for fully functional Ras signaling. Because of their rapid, conditional, and reversible mode of action, small-molecule modulators of protein function are particularly suitable tools for the conditional analysis of such dynamic biological processes, and hold great promise for the study of biological systems. Therefore, the identification of novel small-molecule modulators of signaling through the Ras network and the identification of their molecular targets are of major interest. 6] The naturally occurring tetramic acids melophlin A and B (1 and 2, Scheme 1A) reverse the morphology of HRas-transformed NIH3T3 fibroblasts at a concentration of 5 mgmL 1 (that is, IC50= 14 mm). [7] However, the biological targets of the melophlins and their link to the Ras network have not been identified. Herein, we report the synthesis of a melophlin-inspired compound collection and a subsequent chemical proteomics investigation, which revealed that melo-