Search

The anti-malarial artemisinins have also been implicated in the regulation of various other cellular pathways. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin-B6 (also known as pyridoxal 5’-phosphate, PLP), in complex with artesunate at 2.4-Å resolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

Glioblastomas (GBMs) are the most frequent malignant brain tumors with very limited treatment options and nearly all GBM patients dying within 1 year. Pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) is a secreted growth factor that shows mitogenic, chemotactic and transforming activity. While PTN expression is tightly regulated during embryogenesis and very limited in normal adult tissues, a marked PTN upregulation is seen in tumors including glioblastomas. Targeting of the PTN receptors, ALK and RPTP-zeta, indicates a contribution of PTN-activated signaling pathways in glioblastomas. However, the relevance of PTN expression itself is unknown especially since, besides PTN, at least one more growth factor, midkine (MK), signals through ALK and is expressed in glioblastoma. Here we demonstrate the biologic relevance of PTN in 2 glioblastoma cell lines in vitro and in vivo. We show that stable ribozyme-targeting leads to a robust reduction of PTN mRNA and protein levels. This results in decreased cell proliferation, cell migration and soft agar colony formation in vitro. Comparing clonal ribozyme-transfected cells with different residual PTN levels, we establish a PTN gene-dose effect of glioblastoma cell proliferation. In a subcutaneous tumor xenograft mouse model, in vivo growth is markedly reduced upon PTN depletion, which is paralleled by decreased PTN serum levels. Furthermore, the immunohistochemical analysis of the tumors shows reduced angiogenesis in PTN-depleted tumors. We conclude that PTN is a rate-limiting growth factor in glioblastoma. Since PTN is overexpressed in glioblastomas but rarely found in normal tissue, PTN may represent an attractive therapeutic target.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.