Severe acute respiratory syndrome coronavirus(SARS-Co V) encodes eight accessory proteins, the functions of which are not yet fully understood. SARS-Co V protein 6(P6) is one of the previously studied accessory protei...Severe acute respiratory syndrome coronavirus(SARS-Co V) encodes eight accessory proteins, the functions of which are not yet fully understood. SARS-Co V protein 6(P6) is one of the previously studied accessory proteins that have been documented to enhance viral replication and suppress host interferon(IFN) signaling pathways. Through yeast two-hybrid screening, we identified eight potential cellular P6-interacting proteins from a human spleen c DNA library. For further investigation, we targeted the IFN signaling pathway-mediating protein, N-Myc(and STAT) interactor(Nmi). Its interaction with P6 was confirmed within cells. The results showed that P6 can promote the ubiquitin-dependent proteosomal degradation of Nmi. This study revealed a new mechanism of SARS-Co V P6 in limiting the IFN signaling to promote SARS-Co V survival in host cells.展开更多
OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） s...OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS： Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma （NSG） mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS： We observed that treatment with pristimerin, at as low as 1 IJmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co- administration with pristimerin at 4 mg/（kg.d） for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION： These studies suggest the potential beneficial use of pristimerin and pristimerincontaining herb extract in future liver-targeted gene therapy with rAAV vectors.展开更多
AIM: To determine if proteasome inhibition induces apoptosis in human cholangiocarcinoma cells, and if so, to elucidate the cellular mechanisms. METHODS: Studies were performed in the human KMCH, KMBC, and Mz-ChA-1 ch...AIM: To determine if proteasome inhibition induces apoptosis in human cholangiocarcinoma cells, and if so, to elucidate the cellular mechanisms. METHODS: Studies were performed in the human KMCH, KMBC, and Mz-ChA-1 cholangiocarcinoma, and normal rat cell lines. MG132, a peptide aldehyde, which inhibits the chymotrypsin-like activity of the proteaosome was employed for this study. Apoptosis was assessed morphologically by 4'-6-Diamidino-2-phenylindole (DAPI) nuclear staining and fluorescence microscopy. Mitochondrial membrane potential was examined using a fluorescent unquenching assay. Ultrastructural changes during cell death were examined using transmission electron microscopy (TEM). Caspase 3/7 activity was assessed using an enzymatic-based fluorescent assay. Cytosolic-free calcium concentrations were measured using Fura-2 and digitized fluorescent microscopy. RESULTS: MG132, a proteasome inhibitor, induced apoptosis in all the cholangiocarcinoma cell lines examined. In contrast, minimal cytotoxicity was observed in normal rat cholangiocytes. Apoptosis was time-and -concentration-dependent. There was no change in the mitochondrial membrane potential between treated and untreated cells. Ultrastructural examination by transmission electron microscopy displayed the classic features of apoptosis, but in addition, there was also dramatic vacuolization of the endoplasmic reticulum (ER). Unexpectedly, no increase in caspase 3/7 activity was observed in MG132 treated cells, nor did the pancaspase inhibitor, Q-VD-OPh prevent cell death. The protein synthesis inhibitor, cycloheximide, blocked apoptosis induced by proteosome inhibitor indicating that ER dysfunction was dependent upon the formation of new proteins. CONCLUSION: Proteosome inhibition induces ERdysfunction and caspase-independent cell death selectively in human cholangiocarcinoma cells. Proteasome inhibitors warrant evaluation as anticancer agents for the treatment of human cholangiocarcinoma.展开更多
基金supported by China NSFC grants (#31170152 and 81130083)
文摘Severe acute respiratory syndrome coronavirus(SARS-Co V) encodes eight accessory proteins, the functions of which are not yet fully understood. SARS-Co V protein 6(P6) is one of the previously studied accessory proteins that have been documented to enhance viral replication and suppress host interferon(IFN) signaling pathways. Through yeast two-hybrid screening, we identified eight potential cellular P6-interacting proteins from a human spleen c DNA library. For further investigation, we targeted the IFN signaling pathway-mediating protein, N-Myc(and STAT) interactor(Nmi). Its interaction with P6 was confirmed within cells. The results showed that P6 can promote the ubiquitin-dependent proteosomal degradation of Nmi. This study revealed a new mechanism of SARS-Co V P6 in limiting the IFN signaling to promote SARS-Co V survival in host cells.
基金supported in part by the Alex's Lemonade Foundation,and Bankhead-Coley Cancer Research Program,Florida Department of Health(to CL), Public Health Service grants R01 HL-097088 and R21 EB-015684 from the National Institutes of Health,and an institutional grant from the Children's Miracle Network (to AS,CL and GA)supported in part by the state-sponsored program for Graduate Students from China Scholarship Council,Government of China, and the National Natural Science Foundation of China (No.30730114)
文摘OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS： Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma （NSG） mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS： We observed that treatment with pristimerin, at as low as 1 IJmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co- administration with pristimerin at 4 mg/（kg.d） for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION： These studies suggest the potential beneficial use of pristimerin and pristimerincontaining herb extract in future liver-targeted gene therapy with rAAV vectors.
基金NIH grants DK63947 (to GJG)the Mayo and Palumbo Foundations
文摘AIM: To determine if proteasome inhibition induces apoptosis in human cholangiocarcinoma cells, and if so, to elucidate the cellular mechanisms. METHODS: Studies were performed in the human KMCH, KMBC, and Mz-ChA-1 cholangiocarcinoma, and normal rat cell lines. MG132, a peptide aldehyde, which inhibits the chymotrypsin-like activity of the proteaosome was employed for this study. Apoptosis was assessed morphologically by 4'-6-Diamidino-2-phenylindole (DAPI) nuclear staining and fluorescence microscopy. Mitochondrial membrane potential was examined using a fluorescent unquenching assay. Ultrastructural changes during cell death were examined using transmission electron microscopy (TEM). Caspase 3/7 activity was assessed using an enzymatic-based fluorescent assay. Cytosolic-free calcium concentrations were measured using Fura-2 and digitized fluorescent microscopy. RESULTS: MG132, a proteasome inhibitor, induced apoptosis in all the cholangiocarcinoma cell lines examined. In contrast, minimal cytotoxicity was observed in normal rat cholangiocytes. Apoptosis was time-and -concentration-dependent. There was no change in the mitochondrial membrane potential between treated and untreated cells. Ultrastructural examination by transmission electron microscopy displayed the classic features of apoptosis, but in addition, there was also dramatic vacuolization of the endoplasmic reticulum (ER). Unexpectedly, no increase in caspase 3/7 activity was observed in MG132 treated cells, nor did the pancaspase inhibitor, Q-VD-OPh prevent cell death. The protein synthesis inhibitor, cycloheximide, blocked apoptosis induced by proteosome inhibitor indicating that ER dysfunction was dependent upon the formation of new proteins. CONCLUSION: Proteosome inhibition induces ERdysfunction and caspase-independent cell death selectively in human cholangiocarcinoma cells. Proteasome inhibitors warrant evaluation as anticancer agents for the treatment of human cholangiocarcinoma.
