Vesicular Glutamate Transporter Expression Ensures High-Fidelity Synaptic Transmission at the Calyx of Held Synapses.

Recycling of synaptic vesicles (SVs) at presynaptic terminals is required for sustained neurotransmitter release. Although SV endocytosis is a rate-limiting step for synaptic transmission, it is unclear whether the rate of the subsequent SV refilling with neurotransmitter also influences synaptic transmission. By analyzing vesicular glutamate transporter 1 (VGLUT1)-deficient calyx of Held synapses, in which both VGLUT1 and VGLUT2 are co-expressed in wild-type situation, we found that VGLUT1 loss causes a drastic reduction in SV refilling rate down to ∼25% of wild-type values, with only subtle changes in basic synaptic parameters. Strikingly, VGLUT1-deficient synapses exhibited abnormal synaptic failures within a few seconds during high-frequency repetitive firing, which was recapitulated by manipulating presynaptic Cl- concentrations to retard SV refilling. Our data show that the speed of SV refilling can be rate limiting for synaptic transmission under certain conditions that entail reduced VGLUT levels during development as well as various neuropathological processes.

Current status of the development of Ras inhibitors.

Despite the importance of ras as driver genes in many cancers, clinically effective anti-cancer drugs targeting their products, Ras, have been unavailable so far, which was in part ascribable to the apparently 'undruggable' nature of their tertiary structures. Nonetheless, recent studies in academia and industry have identified novel surface pockets accepting small-molecule ligands in both their active GTP-bound and inactive GDP-bound forms (Ras•GTP and Ras•GDP, respectively), which has led to a surge of investigations into the discovery of Ras-specific inhibitors particularly by utilizing their structural information for structure-based drug design (SBDD). We have been developing Ras inhibitors by SBDD targeting a novel conformation of Ras•GTP called state 1, possessing 'druggable' surface pockets, which emerges from the conformational dynamics. In this article, we will survey Ras functions from the structural biological point of view and summarize the current status of the development of Ras inhibitors including our own.

High-throughput screening system to identify small molecules that induce internalization and degradation of HER2.

Overexpression of growth factor receptors in cancers, e.g., human epidermal growth factor receptor 2 (HER2) in ovarian and breast cancers, is associated with aggressiveness. A possible strategy to treat cancers that overexpress those receptors is blockade of receptor signaling by inducing receptor internalization and degradation. In this study, we developed a cell-based high-throughput screening (HTS) system to identify small molecules that induce HER2 internalization by employing our recently developed acidic-pH-activatable probe in combination with protein labeling technology. Our HTS system enabled facile and reliable quantification of HER2 internalization with a Z' factor of 0.66 and a signal-to-noise ratio of 44.6. As proof of concept, we used the system to screen a ∼155,000 small-molecule library and identified three hits that induced HER2 internalization and degradation via at least two distinct mechanisms. This HTS platform should be adaptable to other disease-related receptors in addition to HER2.

Artificial control of subtype-specific platelet-derived growth factor-receptor signaling.

Platelet-derived growth factor (PDGF) signaling controls various physiological functions via two receptor subtypes: PDGF receptor (PDGFR) alpha and PDGFRbeta. Nevertheless, our understanding of their roles is limited because of a lack of pharmacological tools to discriminate subtype-specific signaling. We developed a chimeric receptor by combining ligand-binding-domain truncated PDGFRbeta with anti-fluorescein single chain antibody, expecting the control of PDGFRbeta-specific signaling by oligomerized fluorescein as an artificial agonist. Results show that calcium mobilization, Cdc42 activation, and cell migration were elicited specifically by the artificial ligand in cells expressing the chimeric receptor. Our method is expected to be useful to understand the subtype-specific roles of PDGFRs in various cellular functions.