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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic peptide with anti-inflammatory and anti-oxidative effects. In order to increase the efficiency of traversing biological barriers, a novel fusion peptide PACAP-TAT was produced by tagging PACAP at its C-terminus with 11-amino acid TAT protein transduction domain. The results of characteristic assays showed that PACAP-TAT activated PACAP specific receptor PAC1 with the same potency as PACAP and PACAP-TAT crossed blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) with the efficiency about 2.5-fold higher than that of PACAP. Both PACAP-TAT and PACAP were used treat the mice with lung injury induced by repeated smoke inhalation. It was shown that both PACAP-TAT and PACAP decreased the mortality, increased the body weight and inhibited the edema and vascular permeability in the lungs of the mice received repeated smoke inhalation, while PACAP-TAT displayed more marked effects than PACAP. PACAP-TAT decreased myeloperoxidase (MPO) activity, increased catalase (CAT) activity and down-regulated interleukin 6 (IL-6) and malondialdehyde (MDA) levels in the lungs with a significantly higher efficiency than PACAP. The histopathological analysis also showed that PACAP-TAT attenuated the cell filtration and bronchi epithelial hyperplasia more significantly than PACAP. Moreover the leukocyte count in blood and the serum superoxide dismutase (SOD) activity in the mice treated with PACAP-TAT were significantly different from that in mice treated with PACAP (p0.05). All these data indicated that PACAP-TAT with increased traversing ability was more effective than PACAP in protecting the mice from the lung injury induced by repeated smoke inhalation.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with cytoprotective ability mediated by its specific receptor PAC1. In this research, firstly the thymus index and the expression of PAC1 in the normal and degenerative thymus with different gender were assayed; secondly PACAP in different dose was used to treat the female mice with cyclophosphamide (CPS) and the changes in thymus index, the expression of PAC1, histopathology, apoptosis, oxidative status and the caspase 3 activity in thymus were determined and compared. It was found that in the mice of age from 1 to 9 weeks in the stage of sex development, the thymus index was significantly higher in female mice than in male mice. And it was found for the first time that the PAC1 expression level in thymus of female mice was significantly higher than that of male mice and the expression of the PAC1 and PACAP increased significantly in the degenerative thymus induced by CPS. After PACAP was co-injected with CPS to the female mice, it was shown that only low dose (1 nmol/kg) of PACAP promoted the thymus index, inhibited the cell apoptosis, ameliorated the oxidative status and decreased the caspase activity significantly, while high dose (10 nmol/kg) of PACAP had no significant protective effects against CPS-induced thymus atrophy. It was concluded that the expression of PAC1 in the thymus changes in reverse ratio with thymus index and in direct ratio with cell apoptosis and only low dose of PACAP had positive effects against the CPS-induced thymus atrophy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood-brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p

PAC1 is PACAP (pituitary adenylate cyclase-activating polypeptide) preferring receptor belonging to class B G protein coupled receptor (GPCR) mediating the most effects of PACAP. The important role of G protein coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. The bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) assay were used in this research to confirm the dimerization of PAC1 for the first time. The structure-activity relationship focused on the N-terminal HSDCIF motif, which locates behind the signal sequence and has high homology with PACAP (1-6), was assayed using a receptor mutant with the deletion of the HSDCIF motif. The fluorescence confocal microscope observation showed that the deletion of the HSDCIF motif impaired the cell delivery of PAC1. The results of BiFC, BRET and westernblot indicated that the deletion of HSDCIF motif and the replacement of the Cys residue with Ala in HSDCIF motif resulted in the disruption of receptor dimerization. And the exogenous chemically synthesized oligopeptide HSDCIF (100 nmol/L) not only down-regulated the dimerization of PAC1, induced the internalization of PAC1, but also inhibited the proliferation of CHO cells expressing PAC1 stably and decreased the activity of PACAP on the cell viability. All these data suggested that the N-terminal HSDCIF motif played key role in the trafficking and the dimerization of PAC1, and the exogenous oligopeptide HSDCIF had effects on the cell signaling, trafficking and the dimerization of PAC1.