Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and Recycling.

SummaryClathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact μ-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling

Benzothiazole derivatives as human DNA topoisomerase IIα inhibitors

Benzothiazole derivatives resembling the structure of DNA purine bases were tested to determine their topoisomerase inhibition activities. Based on DNA topoisomerase I and II relaxation assay results, all 12 derivatives acted as human topoisomerase IIα inhibitors, whereas only two compounds inhibited Calf thymus topoisomerase I. 3-amino-2-(2-bromobenzyl)-1,3-benzothiazol-3-ium 4-methylbenzensulfonate (BM3) was observed to be the most effective human topoisomerase IIα inhibitor with the lowest IC50 value of 39 nM. The mechanistic studies suggested that BM3 was neither a DNA intercalator nor a topoisomerase poison, it was only a DNA minor groove-binding agent. BM3 initially bound to the DNA topoisomerase IIα enzyme, then to DNA. As a result, the tested benzothiazole derivatives were obtained as strong topoisomerase IIα inhibitors. The benzothiazole tosylated salt form BM3 was found as the most effective topoisomerase IIα inhibitor. BM3's mechanisms of action might be its direct interaction with the enzyme. BM3's minor groove-binding property might also contribute to this action. Hence, BM3 could be a good candidate as a new anticancer agent. © 2013 Springer Science+Business Media New York

Molecular profiling and combinatorial activity of CCT068127: a potent CDK2 and CDK9 inhibitor

Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimised from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2-family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer

Mutagenic potentials of dental cements as detected by the Salmonella/microsome test

The potential mutagenicity of a zinc phosphate (Poscal(R)), a polycarboxylate (Aqualox(R)) and glass ionomer cements with (Argion(R)) and without (Meron(R)) silver reinforcement were characterized by employing the Ames Salmonella/microsome test. The materials were eluted in dimethyl sulphoxide or physiologic saline and the aliquots were used either immediately or after an incubation period of 24 h at 37degreesC. Mutagenic effects of the materials were tested on Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535 using the standard plate incorporation assay, and in the presence or absence of S9 fraction from rat liver. Poscal(R) and Aqualox(R) elicited mutagenic effects on S. typhimurium TA 98 and TA 1535, whereas Meron(R) exhibited mutagenic effects on S. typhimurium TA 98. No mutagenic effects were detected for Argion(R). The type of solvent, dose of the material and incubation as well as the interactions between these factors exhibited varying degrees of influences on the mutagenic activities of the cements (P<0.05 and P<0.1). We conclude that zinc phosphate, polycarboxylate, and glass ionomer cements may have possible mutagenic activities. (C) 2003 Elsevier Ltd. All rights reserved

Functional dissection of the interactions of stonin 2 with the adaptor complex AP-2 and synaptotagmin

Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. Here, we demonstrate that the endocytotic protein stonin 2 localizes to axonal vesicle clusters through its mu-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to mu2. Mutation of the tyrosine motif-binding pocket of mu2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. We hypothesize that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling

Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P2]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domain-containing accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the “side sites” of the AP2 α- and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrin-mediated SV recycling in synapses