A New Peptide With Membrane-permeable Function Derived From Human

OBJECTIVETo study the translocation ability of a new peptide (Circadian locomotor output cycles kaput protein' s DNA-binding peptide, hCLOCK' s DNA_BIND, hClock-(35-47)) from human circadian proteins through the membrane and the blood-brain barrier, we also observed the inhibiting effect of the presence of heparan sulfate (HS) on the internalization of hClock-(35-47). Moreover, we study the circadian of internalization of hClock-(35-47) into NIH3T3 cells treated by serum shock. Furthermore, we studied the effect of internalization of hClock-(35-47) into NIH3T3 cells by serum shock on the transcription of cellular circadian gene. METHODShClock-(35-47) , which was chemically synthesized and labeled with FITC at N-terminal, was incubated with the vascular endothelial cell (ECV-304) and the primary cultured neuroglial cells in the culture medium, the distribution of it was observedon a fluorescence microscope. The fluorescence intensity of every cell is measured using the Image-Pro Plus (Version 4. 5 for WindowsTM) system. The cytotoxicity was determined by the MTT assay. Moreover, the FITC-labeled hClock-(35-47) was injected into the SD rat through the common carotid artery. Then brain tissues were dissected and cryostat sections were prepared. The brain sections were observed under a fluorescence microscopy. ECV-304 cells were pretreated with HS, and then FITC-labeled hClock-(35-47) was added to the medium. The inhibiting effect of HS on the internalization of hClock-(35-47) were analyzed by the different degrees of internalization. In the study of the circadian of internalization of hClock-(35-47) into NIH3T3 cells treated by serum shock, the relative quantification of cellular uptake of hClock-(35-47) was measured by flow cytometry analysis. Moreover, the mRNA expression levels ofthe circadian gene perl is measured by RT-PCR assays.RESULTShClock-(35-47) has the internalization ability into the ECV-304 and the neuroglial cells. The internalization of hClock-(35-47) seemed to have no selectivity to cells. The concentration of the hClock-(35-47) in the cells kept increasing as the incubation time increased during the observed time period. And the amount of the internalized peptideincreased as the applied peptide concentration increased. The internalized tendency in the time course and dose-dependent experiments for the hClock-(35-47) was basically similar with the results obtained by Tomoki Suzuki et al for the Tat-(48-60), Rev-(34-50), and R8 peptides. Moreover, hClock-(35-47) did not induce any toxicity in all conditions .even at 100 μM for 24h. The strong fluorescein signal in the brain sections shows that the peptide had the ability to translocate through the blood-brain barriar. The fluorescence intensity in the cells preincubated with HS was lower than that of the control. These results confirmed that HS inhibit the internalization effect of hClock-(35-47). It is possible that the electrostatic interaction between the MPPs and cell surface sulfated polysaccharides should contribute to the internalization of the peptide. Furthemore, we found that the internalization of hClock-(35-47) into NIH3T3 cells treaded by serum shock has the circadian rhythm variation. In addition, we found that the mRNA expression levels of the circadian gene perl were depressed by the uptake of hClock-(35-47) into NIH3T3 cells treaded by serum shock. The possible reason is that hClock-(35-47) has the competitive inhibition effect on the combination between the hClock -BMAL1 dimer and the DNA certain region.CONCLUSIONIt is promising to provide a new safe carrier for the intracellular and encephalic treament because of these characteristics of hClock-(35-47). Moreover, it is possible that hClock- (35-47) should become a new tool to intervene the cellular circadian rhythm clock.