👤 Taeko Kakizawa

Peptide library screening is used to detect optimal sequences for enzymatic cleavage; moreover, the data obtained through this screening are useful for the establishment of a fast screening system and designing of substrate-based enzyme inhibitors. In this study, peptide libraries were prepared and digested with the beta-site amyloid precursor protein cleaving enzyme (BACE1). BACE1 has been used as a target enzyme for drug development against Alzheimer's disease (AD). The library sequences were derived from our previous screening study based on amyloid-beta precursor protein (APP) substrates. Then, newly selected non-natural amino acids were incorporated into several positions on these sequences. After digestion with BACE1, the reaction mixtures were analyzed with high-performance liquid chromatography followed by mass spectrometry to identify the peptides undergoing efficient cleavage. The data obtained from this study can be used for designing drugs against AD in the future. Show less

Membrane-associated guanylate kinases (MAGUKs) are abundant postsynaptic density (PSD)-95/discs large/zona occludens-1 (PDZ)-containing proteins that can assemble receptors and associated signaling enzymes at sites of cell-cell contact, including synapses. PSD-93, a postsynaptic neuronal MAGUK, has three PDZ domains that can bind to specific ion channels, including NMDA delta2 type glutamate receptors, as well as Shaker and inward rectifier type K(+) channels, and can mediate clustering of these channels in heterologous cells. Genetic analyses of Drosophila show that MAGUKs play critical roles in synaptic development because mutations of discs large disrupt the subsynaptic reticulum and block postsynaptic clustering of Shaker K(+) channels. It is uncertain whether MAGUKs play an essential role in the development of central synapses. There are four neuronal MAGUKs with overlapping expression patterns in the mammalian brain; however, we find PSD-93 is the only MAGUK expressed in cerebellar Purkinje neurons. Therefore, we targeted disruption of PSD-93 in mouse. Despite the absence of MAGUK immunoreactivity in Purkinje neurons from the knock-outs, these mice have no structural or functional abnormality in cerebellum. Both the dendritic architecture and the postsynaptic localization of PSD-93 interacting proteins remain intact at light and electron microscopic levels in the knock-outs. Postsynaptic Purkinje cell responses, monosynaptic climbing fiber innervation, and cerebellar-dependent behaviors are also normal. Our data demonstrate that MAGUK proteins of the PSD-93/95 family are not essential for development of certain central synapses but may instead participate in specialized aspects of synaptic signaling and plasticity. Show less