Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and...Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and trefoil factors(TFFs). As a toad af-PFP, Bm ALP1 can be reversibly regulated between active and inactive forms, with its paralog Bm ALP3 acting as a negative regulator. Bm ALP1 interacts with Bm TFF3 to form a cellular active complex called βγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of βγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence,we hereby propose a secretory endolysosome channel(SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with βγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.展开更多
Functionalized carbon nanotubes have already demonstrated great biocompatibility and potential for drug delivery.We have synthesized acid oxidized and non-covalently PEGlyated single-walled carbon nanotubes(SWNTs),whi...Functionalized carbon nanotubes have already demonstrated great biocompatibility and potential for drug delivery.We have synthesized acid oxidized and non-covalently PEGlyated single-walled carbon nanotubes(SWNTs),which were previously prepared for drug delivery purposes,and explored their potential for detoxification in the bloodstream.Our investigations of the binding of SWNTs to a pore-forming toxin pyolysin show that SWNTs prevented toxin-induced pore formation in the cell membrane of human red blood cells.Quantitative hemolysis assay and scanning electron microscopy were used to evaluate the inhibition of hemolytic activity of pyolysin.According to Raman spectroscopy data,human red blood cells,unlike HeLa cells,did not internalize oxidized SWNTs.Molecular modeling and circular dichroism measurements were used to predict the 3-D structure of pyolysin(domain 4)and its interaction with SWNTs.The tryptophan-rich hydrophobic motif in the membrane-binding domain of pyolysin,a common construct in a large family of cholesterol-dependent cytolysins,shows high affinity for SWNTs.展开更多
Objective： To achieve the co-expression of GrB and PFP in Hep-2 cells and analyze the growth inhibiting effects on Hep-2 cells. Methods： Lymphocytes were separated from human laryngeal carcinoma tissue, complete Exo...Objective： To achieve the co-expression of GrB and PFP in Hep-2 cells and analyze the growth inhibiting effects on Hep-2 cells. Methods： Lymphocytes were separated from human laryngeal carcinoma tissue, complete Exon fragments of GrB and PFP were amplified by RT-PCR via extracting lymphocytes total RNA, and they were recombined to the downstream of T7 promoter in the vector pVAX1. The recombinant plasmid pVAX1-PIG was transfected into Hep-2 cells with Lipofectamine 2000. The expression of proteins was identified by RT-PCR, MTT and western blot assay. Results： The gene sequence of the RT-PCR products of GrB and PFP were consistent with the data of GenBank by DNA sequencing analysis. The GrB and PFP cDNA fragment were cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB and PFP were maintained. The target proteins were detected in the transfected Hep-2 cells, and the inhibitive effect of PFP and GrB on Hep-2 cells growth were studied by thiazolyl blue （MTT） test. Conclusion： The pVAX1-PFP-IRES-GrB plasmid was successfully constructed and expressed, and the expression of PFP and GrB could inhibit the growth of Hep-2 cells.展开更多
基金supported by the National Natural Science Foundation of China (31572268, U1602225, 31872226)Yunling Scholar Program to Y.Z。
文摘Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and trefoil factors(TFFs). As a toad af-PFP, Bm ALP1 can be reversibly regulated between active and inactive forms, with its paralog Bm ALP3 acting as a negative regulator. Bm ALP1 interacts with Bm TFF3 to form a cellular active complex called βγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of βγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence,we hereby propose a secretory endolysosome channel(SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with βγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.
基金by Dr.Tang’s startup fund from University of Waterloo and by grants from the Natural Science and Engineering Research Council(NSERC)of Canada.
文摘Functionalized carbon nanotubes have already demonstrated great biocompatibility and potential for drug delivery.We have synthesized acid oxidized and non-covalently PEGlyated single-walled carbon nanotubes(SWNTs),which were previously prepared for drug delivery purposes,and explored their potential for detoxification in the bloodstream.Our investigations of the binding of SWNTs to a pore-forming toxin pyolysin show that SWNTs prevented toxin-induced pore formation in the cell membrane of human red blood cells.Quantitative hemolysis assay and scanning electron microscopy were used to evaluate the inhibition of hemolytic activity of pyolysin.According to Raman spectroscopy data,human red blood cells,unlike HeLa cells,did not internalize oxidized SWNTs.Molecular modeling and circular dichroism measurements were used to predict the 3-D structure of pyolysin(domain 4)and its interaction with SWNTs.The tryptophan-rich hydrophobic motif in the membrane-binding domain of pyolysin,a common construct in a large family of cholesterol-dependent cytolysins,shows high affinity for SWNTs.
基金supported in part by the grants from the National Natural Science Foundation of China (No.30670102 & 30770111 & 30770820)the National High Technology Research and Development Program of China (2006AA02Z176)Shanghai Natural Science Foundation (06ZR14056)
基金the Foundation of Doctoral Subject in Jinan University(51205069)the Natural Science Foundation of Guangdong,China(5300804)
文摘Objective： To achieve the co-expression of GrB and PFP in Hep-2 cells and analyze the growth inhibiting effects on Hep-2 cells. Methods： Lymphocytes were separated from human laryngeal carcinoma tissue, complete Exon fragments of GrB and PFP were amplified by RT-PCR via extracting lymphocytes total RNA, and they were recombined to the downstream of T7 promoter in the vector pVAX1. The recombinant plasmid pVAX1-PIG was transfected into Hep-2 cells with Lipofectamine 2000. The expression of proteins was identified by RT-PCR, MTT and western blot assay. Results： The gene sequence of the RT-PCR products of GrB and PFP were consistent with the data of GenBank by DNA sequencing analysis. The GrB and PFP cDNA fragment were cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB and PFP were maintained. The target proteins were detected in the transfected Hep-2 cells, and the inhibitive effect of PFP and GrB on Hep-2 cells growth were studied by thiazolyl blue （MTT） test. Conclusion： The pVAX1-PFP-IRES-GrB plasmid was successfully constructed and expressed, and the expression of PFP and GrB could inhibit the growth of Hep-2 cells.
