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.

Trypanosomal and leishmanial infections claim tens of thousands of lives eachy ear.T he metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasitesh ydroperoxide-clearance cascade.I nvitro and in vivo functional assays show that as mall, selective inhibitor efficiently inhibits Tpx. With X-rayc rystallography,S AXS,a nalytical SEC,S EC-MALS,M Ds imulations,I TC,a nd NMR spectroscopy, we show howc ovalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra-and intermolecular inhibitor-inhibitor,p rotein-protein, and inhibitor-protein interactions stabilizet he dimer.T he behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with as mall, monovalent ligand that can be exploited for future drug design.

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. ' 2005 Wiley-Liss, Inc.Key words: pleiotrophin; PTN; glioblastoma; ribozyme-targeting; tumor growth; tumor angiogenesis Gliomas are the most common primary CNS tumors in humans, which are subdivided according to the WHO into low-grade astrocytomas (grade 2), anaplastic astrocytomas (grade 3) and glioblastomas (GBMs; grade 4). Glioblastomas, which can arise de novo or progress from lower-grade gliomas, are the most frequent and most malignant brain tumors, with nearly all GBM patients dying within 1 year despite aggressive treatment.1 Clearly, an insight into the molecular basis of glioblastoma growth may provide new therapeutic strategies.Pleiotrophin (PTN), also referred to as heparin-binding growthassociated molecule (HB-GAM), heparin-binding growth factor 8, heparin-binding neurotrophic factor (HBNF), or osteoblast-specific protein-1 (OSF-1), is a 15.3 kDa secreted protein that was originally purified from human breast cancer cells, 2 bovine uterus, 3 as well as neonatal rat brain. 4,5 Based on its time-and tissuespecific expression pattern during rodent development, PTN represents a developmentally regulated cytokine whose expression gets increasingly restricted with progressing organ differentiation and drops after the perinatal phase. In the CNS, PTN expression is high in neuroepithelial progenitor cells as well as in glial cells and neurons du...

Highlights d Human TRPML2 extracytosolic/lumenal domain (ELD) structures at pH 4.5 and 6.5 d Comparison of the three human TRPML channel ELDs at acidic and near-neutral pH d Ca 2+ interacts with the TRPML2 acidic pre-pore loop in a pHdependent manner d Ca 2+ and pH do not influence ELD structure but play a role in oligomer integrity

Targeting multiprotein receptor complexes, rather than receptors directly, is a promising concept in drug discovery. This is particularly relevant to the GABAB receptor complex, which plays a prominent role in many brain functions and diseases. Here, we provide the first studies targeting a key protein–protein interaction of the GABAB receptor complexthe interaction with KCTD proteins. By employing the μSPOT technology, we first defined the GABAB receptor-binding epitope mediating the KCTD interaction. Subsequently, we developed a highly potent peptide-based inhibitor that interferes with the KCTD/GABAB receptor complex and efficiently isolates endogenous KCTD proteins from mouse brain lysates. X-ray crystallography and SEC-MALS revealed inhibitor induced oligomerization of KCTD16 into a distinct hexameric structure. Thus, we provide a template for modulating the GABAB receptor complex, revealing a fundamentally novel approach for targeting GABAB receptor-associated neuropsychiatric disorders.