Cast: a novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and munc13-1.

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of approximately 120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca2+-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein-protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody-coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

Nectin: an adhesion molecule involved in formation of synapses.

The nectin-afadin system is a novel cell-cell adhesion system that organizes adherens junctions cooperatively with the cadherin-catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin-afadin system colocalizes with the cadherin-catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin-afadin system in the formation of synapses.

Mouse homologue of skin-specific retroviral-like aspartic protease involved in wrinkle formation.

Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.

Identification of activity-regulated proteins in the postsynaptic density fraction.

BACKGROUND: The postsynaptic density (PSD) at synapses is a specialized submembranous structure where neurotransmitter receptors are linked to cytoskeleton and signalling molecules. Activity-dependent dynamic change in the components of the PSD is a mechanism of synaptic plasticity. Identification of the PSD proteins and examination of their modulations dependent on synaptic activity will be valuable for an understanding of the molecular basis of learning and memory. RESULT: We attempted here to identify proteins in the PSD fraction by two-dimensional (2D) gel electrophoresis and mass spectrometry. About 1.7 x 103 protein spots were detected on 2D gels. A total of 90 spots were identified, containing 47 different protein species. In addition to previously identified PSD proteins such as PSD-95/SAP90, several new proteins were identified in the PSD fraction. They included stomatin-like protein 2 and NIPSNAP1. We also examined activity-dependent modulations of PSD proteins by 2D gel electrophoresis. The spot concentration of G protein beta subunit 5 and NIPSNAP1 increased 2 h after kainate treatment that caused generalized seizures. CONCLUSION: These results indicate that the combination of 2D gel electrophoresis and mass spectrometry is an excellent tool for the identification of activity-regulated PSD proteins.