Journal: Acta Biochimica et Biophysica Sinica

Article Title: Positive allosteric regulation of PAC1-R up-regulatesPAC1-R and its specific ligand PACAP

doi: 10.3724/abbs.2022041

Figure Lengend Snippet: Fluorescence confocal microscopy and immunofluorescence analysis on the nuclear translocation of PAC1-R induced by PACAP(28-38) and TAT in PAC1-CHO and RGC-5 cells PACAP(28-38) and TAT induce nuclear translocation of PAC1-R in PAC1-CHO cells, and PACAP(28-38) and TAT promote PAC1-R expression, which is positively correlated with the nuclear translocation of PAC1-R in RGC-5 cells. (A) Fluorescence microscope imaging (upper) and corresponding statistical analysis (lower) showed that PACAP(28-38) and TAT induce significant trafficking and aggregation of PAC1R-eGFP fluorescence signal (green) into the nucleus of PAC1-CHO cells. *P<0.01 vs peptides-. (B) PACAP(28-38) and TAT can not induce significant trafficking or aggregation of M-PAC1R(Cys25/Ala25)-eGFP fluorescence signal (green) into the nucleus of M-PAC1-CHO cells. *P<0.01 vs peptides-. (C,D) The results of immunofluorescence confocal imaging (upper) and the coresponding immunofluorescence signal data analysis (lower) showed that both PACAP(28-38) and TAT can significantly induce the nuclear translocation of PAC1-R and up-regulate the expression level of PAC1-R, and both are significantly inhibited by 2-bromopalmitate. Data are presented as the mean±SEM of three experiments. *P<0.01 vs TAT- or P(28-38)-. #P<0.01 vs TAT+. Scale bar=5 μm. P(28-38): PACAP(28-38).

Article Snippet: Oligo-peptides PACAP(28-38) and TAT with 95% purity were synthesized by GL Biochem (Shanghai, China).

Techniques: Fluorescence, Confocal Microscopy, Immunofluorescence, Translocation Assay, Expressing, Microscopy, Imaging

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Positive allosteric regulation of PAC1-R up-regulatesPAC1-R and its specific ligand PACAP

doi: 10.3724/abbs.2022041

Figure Lengend Snippet: PACAP(28-38) and TAT increase both the promoter activities and expression levels of PAC1-R and PACAP (A) The ChIP-PCR results obtained by using nuclear extracts of RGC-5 cells with the primers of F: 5′-TGCCAATGTCCCATGTTTCA-3′ and R: 5′-TAAGAGTGGTCAGGACCCCC-3′ targeting PAC1-R promoter sequence (–2019 bp to –1739 bp). The positive 280bp band illustrates the binding of the C-terminus of PAC1-R with the PAC1-R promoter sequence in some way. (B) The dual-luciferases reporter assay results showed that the promoter activity of PAC1-R is significantly enhanced by PACAP(28-38) (0.1–100 μM) and TAT (0.1–1 μM). (C) Western blots of PAC1-R in whole RGC-5 cells incubated with PACAP(28-38) or TAT in concentrations from 0.1 μM to100 μM (upper) and the corresponding statistical analysis (lower). (D) The ChIP-PCR results obtained by using nuclear extracts of RGC-5 cells with the primers of F: 5′-TAGCAGCATCTTCAGACGCA-3′ and R: 5′-GGAGACTTGTTTGCCGAGG-3′ targeting PACAP promoter sequence (–858 bp to –536 bp). The positive 320 bp band illustrated the binding of the C-terminus of PAC1-R with the PACAP promoter sequence in some way. (E) The dual-luciferases reporter assay result showed that the promoter activity of PACAP is significantly enhanced by PACAP(28-38) (0.1 μM–10 μM) and TAT (0.01 μM–10 μM). *P<0.01 vs 0 μM. (F) The ELISA result showed that PACAP(28-38) (0.01 μM–10 μM) and TAT (0.1 μM–10 μM) significantly increase the expression level of PACAP. Data are presented as the mean±SEM of three experiments. *P<0.01 vs 0 μM. (A,D) ChIP-PCRs were conducted with templates including: 1, the nuclear extraction of RGC-5 cells before precipitation by the antibody targeting the C-terminus of PAC1-R; 2, ChIP product from RGC-5 cells treated with PACAP(28-38) (1 μM) with PAC1-R antibody; 3, ChIP product from RGC-5 cells treated with TAT (1 μM) with PAC1-R antibody; 4, ChIP product from RGC-5 cells treated with PACAP(28-38) (1 μM) with antibody targeting VPAC2-R; 5, ChIP product from RGC-5 cells treated with TAT (1 μM) with antibody targeting VPAC2-R; M, DNA marker. P(28-38): PACAP(28-38).

Article Snippet: Oligo-peptides PACAP(28-38) and TAT with 95% purity were synthesized by GL Biochem (Shanghai, China).

Techniques: Expressing, Sequencing, Binding Assay, Reporter Assay, Activity Assay, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Extraction, Marker

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Positive allosteric regulation of PAC1-R up-regulatesPAC1-R and its specific ligand PACAP

doi: 10.3724/abbs.2022041

Figure Lengend Snippet: Palmitoylation inhibitor 2-bromopalmitateinterferes with the promoter activities and expression levels of PAC1-R and PACAP induced by PACAP(28-38) and TAT (A) The dual-luciferases reporter assay result showed that PAC1-R promoter activity induced by PACAP(28-38) and TAT is significantly inhibited by 2-bromopalmitate. (B) The dual-luciferases reporter assay result showed that PACAP promoter activity induced by PACAP(28-38) and TAT is significantly inhibited by 2-bromopalmitate. (C) Western blots (upper) and the subsequent quantitative analysis (lower) showed that the PAC1-R expression level induced by PACAP(28-38) and TAT treatment is significantly inhibited by 2-bromopalmitate. (D) The ELISA result showed that the PACAP expression level induced by PACAP(28-38) and TAT treatment is significantly inhibited by 2-bromopalmitate. Data are presented as the mean±SEM of three experiments. *P<0.01 vs peptides-, P(28-38)-, or TAT-. #P<0.01 vs peptides+, P(28-38)+, or TAT+. 2-BP: 2-bromopalmitate; P(28-38): PACAP(28-38).

Article Snippet: Oligo-peptides PACAP(28-38) and TAT with 95% purity were synthesized by GL Biochem (Shanghai, China).

Techniques: Expressing, Reporter Assay, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Positive allosteric regulation of PAC1-R up-regulatesPAC1-R and its specific ligand PACAP

doi: 10.3724/abbs.2022041

Figure Lengend Snippet: Effects of PACAP(28-38) and TAT on the cell viability in RGC-5 cell PD model induced by MPP (A) MTT assay showed that both PACAP(28-38) and TAT in concentrations from 0.1 to 100 μM significantly increase the cell viability decreased by MPP treatment. *P<0.01 vs 0 μM. (B) MTT assay showed that PACAP(28-38) and TAT have significant cytoprotective effects which are significantly inhibited by PACAP(6-38). *P<0.01 vs peptides-. #P<0.01 vs peptides+. (C) PACAP(28-38) and TAT have significant cytoprotective effects which are significantly inhibited by 2-bromopalmitate. *P<0.01 vs peptides-. #P<0.01 vs peptides+. (D) Effects of PACAP(28-38) and TAT with or without 2-bromopalmitate on apoptosis as determined by TUNEL assay in RGC-5 cells induced by MPP. Microscopic images of cells treated with DMEM without MPP as control (CON) or with 8 mM MPP, PACAP(28-38)+MPP, TAT+MPP, PACAP(28-38)+MPP+2-BP and TAT+MPP+2-BP. Yellow arrows indicate non-apoptotic cells and red arrows indicate apoptotic cells (Scale bar=10 μm). (E) Quantitative analysis of the TUNEL images showed that PACAP(28-38) and TAT have significant cytoprotective effects which are significantly inhibited by 2-bromopalmitate. Data are presented as the mean±SEM of three experiments. *P<0.01 vs control. #P<0.01 vs peptides-. &,ΔP<0.01. 2-BP: 2-bromopalmitate; P(28-38): PACAP(28-38).

Article Snippet: Oligo-peptides PACAP(28-38) and TAT with 95% purity were synthesized by GL Biochem (Shanghai, China).

Techniques: MTT Assay, TUNEL Assay, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Positive allosteric regulation of PAC1-R up-regulatesPAC1-R and its specific ligand PACAP

doi: 10.3724/abbs.2022041

Figure Lengend Snippet: The working mechanism of PAC1-R nuclear translocation and the signalling pathway mediated by nuclear PAC1-R The binding of the allosteric modulators at the N-terminal extracellular domain of PAC1-R such as PACAP(28-38) and TAT trigger the significant endocytosis and nuclear translocation of PAC1-R; then the nuclear PAC1-R promotes the transcriptions of itself and its specific ligand PACAP in two ways. (A) The C-terminus of nuclear PAC1-R recruits TFs such as SP1, Zic2, GATA1, REST, YY1, or directly binding PAC1-R/PACAP promoter regions to enhance the promoters activities of PAC1-R/PACAP. (B) The C-terminus of nuclear PAC1-R recruits TFs to regulat some key protein expression such as Htt and reduces the silencer activity related to PACAP by regulating the function of NRSE-NRSF system.

Article Snippet: Oligo-peptides PACAP(28-38) and TAT with 95% purity were synthesized by GL Biochem (Shanghai, China).

Techniques: Translocation Assay, Binding Assay, Expressing, Activity Assay