PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K⁺-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.     

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells.     

Nitric oxide synthase in the glossopharyngeal and vagal afferent pathway of a teleost, Takifugu niphobles

To examine the presence of nitric oxide synthase (NOS) in the sensory system of the glossopharyngeal and vagus nerves of teleosts, nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity and immunoreactivity for NOS were examined in the puffer fish Takifugu niphobles. The nitrergic sensory neurons were located in the ganglia of both the glossopharyngeal and the vagal nerves. In the vagal ganglion, positive neurons were found in the subpopulations for the branchial rami and the coelomic visceral ramus, but not for the posterior ramus or the lateral line ramus. In the medulla, nitrergic afferent terminals were found in the glossopharyngeal lobe, the vagal lobe, and the commissural nucleus. In the gill structure, the nitrergic nerve fibers were seen in the nerve bundles running along the efferent branchial artery of all three gill arches. These fibers appeared to terminate in the proximal portion of the efferent filament arteries of three gill arches. On the other hand, autonomic neurons innervating the gill arches were unstained. These results suggest that nitrergic sensory neurons in the glossopharyngeal and vagal ganglia project their peripheral processes through the branchial rami to a specific portion of the branchial arteries, and they might play a role in baroreception of this fish. A possible role for nitric oxide (NO) in baroreception is also discussed.     

Auxin-inducible protein depletion system in fission yeast

Background  

Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe.     

The multiple promoter methylation profile of PR gene and ERalpha gene in tumor cell lines

The changes of methylation status of various gene promoters are a common feature of malignant cells and these changes can occur early in the progression process. Therefore, abnormal methylation can be used as cancer marker. Such studies will first require the development of a panel of methylated markers that are methylated in cancer tissues but unmethylated in normal tissues or methylated status is different between cancer tissues and normal tissues. By using methylation-specific PCR (MSP) assay method, we observed alterations in DNA methylation at the double promoter regions of the progesterone receptor (PR) gene and estrogen receptor (ERalpha) gene in various tumor cell lines. Compared with normal white blood cell, the methylation status of PRA promoter in various cancer cell lines changed from unmethylation pattern to methylation pattern. That of PRB promoter changed from both unmethylated and methylated alleles to only methylated allele. The methylation status of ERalpha-A and ERalpha-B promoter in various cancer cell lines are cell -specific. This study indicates that PR promoter methylation may be a molecular marker in various cancer detections. And the methylation status of ERalpha-A and ERalpha-B is cell-specific.