Novel Cell Permeable Peptide Based on Multi Basic Furin-dependent Cleavage Site of Respiratory Syncytial Virus Fusion Protein

Cell permeable peptide (CPP) is able to transport itself or conjugated molecules such as nucleotides, peptides, and proteins into cells. Since short peptide of human immunodeficiency virus-1 Tat has been discovered as CPP, it has been continuously studied for their ability to transport heterologous cargoes into cells. In this study, we have focused on the fusion protein of respiratory syncytial virus (RSV), which has six basic amino acids in multi basic furin-dependent cleavage site (MBFCS) required to be cationic CPP. To develop more efficient CPP, the sequence, which linked two MBFCS, was synthesized (called RS-CPP). To assess cell permeable efficiency of RS-CPP or MBFCS, the peptides was conjugated with fluorescein isothiocyanate, and cell permeable efficiency was measured by fluorescence-activated cell sorting. Cell permeability of RS-CPP or MBFCS was increased in a dose-dependent manner, but RS-CPP showed more efficient cell permeability than MBFCS in MDCK, HeLa, Vero E6, and A549 cells. To evaluate whether RS-CPP can transport its conjugated functional peptide (VIVIT) in CD8+ T cell, it was confirmed that IL-2 and β-galactosidase expression were significantly inhibited through selective block of nuclear factor activated T-cell. To investigate endocytic pathways, Cre-mediated DNA recombination (loxP-STOP-loxP-LacZ reporter system) was investigated with divergent endocytosis inhibitors in TE671 cells, and RS-CPP endocytosis is occurred via binding cell surface glycosaminoglycan and clathrin-mediated endocytosis, or macropinocytosis. These results indicated that RS-CPP could be a novel cationic CPP, and it would help understanding for delivery of biologically functional molecules based on viral basic amino acids.

Importance of the putative furin recognition site 742RNRR745 for antiangiogenic Sema3C activity in vitro

Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.

Effect of Furin Inhibitor on Growth of Breast Cancer M CF￣7 Cell / 医药导报

Objective To investigate the role of Furin in breast cancer cell proliferation and provide a theoretical basis for in￣depth study of breast cancer. Methods Different concentrations of Furin inhibitor were added in MCF￣7 cell culture to test MCF￣7 cell proliferation by MTT essay.Hochest 33342 staining was used to detect the morphological change of apoptosic cells.Western blot analysis was applied to measure the level of cell apoptosis associated proteins,such as Caspase￣3,Caspase￣8 andCaspase￣9.The enzyme￣linked immunosorbent assay was used for detection the CAT and SOD levels in cell culture. ResultsMCF￣7 cell growth was inhibited by Furin inhibitor in a time and dose dependent manner.The results of Western blot and Hochest33342 staining indicated that MCF￣7 cells were apoptosis after Furin inhibitor treatment. The level of CAT was increasedsignificantly,associated with the level of SOD. Conclusion Furin inhibitor could induce MCF cell apoptosis, thereby inhibitcell proliferation by modulating MCF￣7 cell redox state.

Effects of furin inhibitor on metastasis of human breast cancer MCF-7 cells / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the mechanism underlying breast cancer metastasis and to provide theoretical da-ta for studying the pathogenesis of breast cancer onset and development.METHODS: Human breast cancer MCF-7 cells were treated with different concentrations of furin inhibitorα1-PDX for 48 h.Wound healing assay and Transwell assay were applied to detect the migration and invasion abilities of the MCF-7 cells.The expression of cell migration-associated proteins, including membrane-type 1 matrix metalloproteinase ( MT1-MMP) , vascular endothelial growth factor ( VEGF)-C and VEGF-D, was determined by Western blotting.The protein levels of MMP2 and MMP9 in the supernatant were measured by ELISA. RESULTS:Compared with control group, 200 nmol/L of furin inhibitor exerted significant inhibitory effects on the cell mi-gration (P<0.05).The expression of cell migration-associated proteins MT1-MMP, VEGF-C and VEGF-D was significantly inhibited after treated withα1-PDX ( P<0.05 ) .Significant inhibitory effects of α1-PDX on the expression of MMP9 and MMP2 (P<0.05) in the supernatant were observed.CONCLUSION:Furin inhibitor suppresses the metastasis of MCF-7 cells via down-regulating the expression of MMPs and VEGFs.

Relationship between single nucleotide polymorphism-229C/T in P1 promoter of furin gene and functions of hepatocytes in patients with liver cirrhosis / 中国病理生理杂志

AIM: To study the influences of P1 promoter activity of furin gene on the functions of hepatocytes in patients with liver cirrhosis.METHODS: The patients with liver cirrhosis of 180 cases were recruited.The single nucleotide polymorphism(SNP-229 C/T) in P1 promoter of furin gene was genotyped using competitively differentiated polymerase chain reaction.The relationships between the promoter activity based on genotyping and the serum levels of liver enzymes,total bilirubin,albumin and prothrombin were observed.RESULTS: The distribution frequencies of allele C and T were 75.3%(271/360) and 24.7%(89/360).Those of genotypes CC,CT and TT were 62.2%(112/180),26.1%(47/180) and 11.7%(21/180),respectively.The distribution frequencies of the genotypes were not related to the serum levels of major liver enzymes,albumin,total bilirubin and prothrombin,except for alkaline phosphatase and ?-glutamyl transferase.CONCLUSION: The activity of furin promoter exerts no effects on the main functions of hepatocytes,suggesting that furin may be a new therapeutic target for HBV infection.

Inhibition of proprotein convertases by mung bean trypsin inhibitor / 第二军医大学学报

Objective:To extract the mung bean trypsin inhibitor(MBTI) from mung bean and to study the inhibitory activity of MBTI against proprotein convertases(PCs).Methods: MBTI was purified to homogeneity by ammonium sulfate precipitation, sequential chromatography of gel filtration,ion exchange,affinity chromatography and HPLC.The high expression strains of 2 PCs,Kexin and Furin,were selected.Kexin and Furin were purified by ammonium sulfate precipitation and gel filtration.The inhibitory activity of MBTI against Kexin and Furin was assayed and the inhibitory constants(Ki) of MBTI against the 2 PCs were calculated by Dixon's plot.Results:The purified MBTI showed a single peak on HPLC and a single band on SDS-PAGE.The inhibitory activity of MBTI against Kexin(Ki= 3.9?10~(-9)mol/L) was stronger than that against Furin.Conclusion: MBTI can inhibit both Kexin and Furin,especially Kexin,and also can be an ideal inhibitor against PCs after further modification.