Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymph...Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid malignancies.Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials.In this study,we prepared an engineered fusion protein,scFv-LDP,consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination.After purification and refolding,scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays.The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP.MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,respectively.An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized.Drugs were given intravenously on day 14 and 21 after tumor transplantation.In terms of maximal tolerated doses,scFv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (P<0.05).Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.展开更多
N^(6)-methyladenosine (m^(6)A) is a prevalent internal post-transcriptional modification in eukaryotic RNAs executed by m^(6)A-binding proteins known as “readers.” Our previous research demonstrated that the Arabido...N^(6)-methyladenosine (m^(6)A) is a prevalent internal post-transcriptional modification in eukaryotic RNAs executed by m^(6)A-binding proteins known as “readers.” Our previous research demonstrated that the Arabidopsis m^(6)A reader ECT2 positively regulates transcript levels of the proteasome regulator PTRE1 and several 20S proteasome subunits, thereby enhancing 26S proteasome activity. However, mechanism underlying the selective recognition of m^(6)A targets by readers, such as ECT2, remains elusive. In this study, we further demonstrate that ECT2 physically interacts with PTRE1 and several 20S proteasome subunits. This interaction, which occurs on the ribosome, involves the N terminus of PTRE1, suggesting that ECT2 might bind to the nascent PTRE1 polypeptide. Deleting ECT2’s protein interaction domain impairs its mRNA-binding ability, whereas mutations in the m^(6)A-RNA-binding site do not affect protein-protein interactions. Moreover, introducing a novel protein-binding domain into ECT2 increases transcript levels of proteins interacting with this domain. Our findings indicate that interaction with the PTRE1 protein enhances ECT2’s binding to PTRE1 m^(6)A mRNAs during translation, thereby regulating PTRE1 mRNA levels.展开更多
In the present study, we investigate effect of amylin on the insulin sensitivity of rat skeletal muscle extensor digitorum longus (EDL) using in vitro intact muscle incubation in combination with metabolic radioactive...In the present study, we investigate effect of amylin on the insulin sensitivity of rat skeletal muscle extensor digitorum longus (EDL) using in vitro intact muscle incubation in combination with metabolic radioactive labeling. The molecular basis of the amylin action was further examined using proteomic analysis. In particular, proteins of interest were characterized using an integrated microcharacterization procedure that involved in-gel trypsin digestion, organic solvent extraction, high performance liquid chromatography separation, microsequencing and microsequence analysis. We found that amylin significantly decreased the insulin-stimulated glucose incorporation into glycogen (p < 0.01) and produced a protein spot of approximately 20 ku in size. This amylin responsive protein (hereby designated as amylin responsive protein 1, APR1) was identified to be protein p20. Moreover, ARP1 spots on gels were found to consistently produce a corresponding radioactive spot on X-ray films in 32Pi but not in 35S-methionine labeling experiments. In conclusion, our results showed that in vitro amylin concomitantly evoked the production of ARP1 and caused insulin resistance in EDL muscle. It is suggested that protein p20 may be involved in amylin signal transduction and the appearance of ARP1 may be a step in a molecular pathway leading to the development of insulin resistance. ARP1 might therefore be a useful molecular marker for amylin action, insulin resistance and Type 2 diabetes.展开更多
It is well established that different sites within a protein evolve at different rates according to their role within the protein; identification of these correlated mutations can aid in tasks such as ab initio protei...It is well established that different sites within a protein evolve at different rates according to their role within the protein; identification of these correlated mutations can aid in tasks such as ab initio protein structure, structure function analysis or sequence alignment. Mutual Information is a standard measure for coevolution between two sites but its application is limited by signal to noise ratio. In this work we report a preliminary study to investigate whether larger sequence sets could circumvent this problem by calculating mutual information arrays for two sets of drug naive sequences from the HIV gpl20 protein for the B and C subtypes. Our results suggest that while the larger sequences sets can improve the signal to noise ratio, the gain is offset by the high mutation rate of the HIV virus which makes it more difficult to achieve consistent alignments. Nevertheless, we were able to predict a number of coevolving sites that were supported by previous experimental studies as well as a region close to the C terminal of the protein that was highly variable in the C subtype but highly conserved in the B subtype.展开更多
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02A255)the National Natural Science Foundation of China(Grant No. 30701029)the National Science and Technology Major Projects(Grant Nos.2009ZX09103-720 and 2010ZX09401-407)
文摘Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid malignancies.Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials.In this study,we prepared an engineered fusion protein,scFv-LDP,consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination.After purification and refolding,scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays.The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP.MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,respectively.An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized.Drugs were given intravenously on day 14 and 21 after tumor transplantation.In terms of maximal tolerated doses,scFv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (P<0.05).Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.
基金Double first-class discipline promotion project(2021B10564001)Laboratory of Lingnan Modern Agriculture Project(NT2021001 and NG2021004).
文摘N^(6)-methyladenosine (m^(6)A) is a prevalent internal post-transcriptional modification in eukaryotic RNAs executed by m^(6)A-binding proteins known as “readers.” Our previous research demonstrated that the Arabidopsis m^(6)A reader ECT2 positively regulates transcript levels of the proteasome regulator PTRE1 and several 20S proteasome subunits, thereby enhancing 26S proteasome activity. However, mechanism underlying the selective recognition of m^(6)A targets by readers, such as ECT2, remains elusive. In this study, we further demonstrate that ECT2 physically interacts with PTRE1 and several 20S proteasome subunits. This interaction, which occurs on the ribosome, involves the N terminus of PTRE1, suggesting that ECT2 might bind to the nascent PTRE1 polypeptide. Deleting ECT2’s protein interaction domain impairs its mRNA-binding ability, whereas mutations in the m^(6)A-RNA-binding site do not affect protein-protein interactions. Moreover, introducing a novel protein-binding domain into ECT2 increases transcript levels of proteins interacting with this domain. Our findings indicate that interaction with the PTRE1 protein enhances ECT2’s binding to PTRE1 m^(6)A mRNAs during translation, thereby regulating PTRE1 mRNA levels.
文摘In the present study, we investigate effect of amylin on the insulin sensitivity of rat skeletal muscle extensor digitorum longus (EDL) using in vitro intact muscle incubation in combination with metabolic radioactive labeling. The molecular basis of the amylin action was further examined using proteomic analysis. In particular, proteins of interest were characterized using an integrated microcharacterization procedure that involved in-gel trypsin digestion, organic solvent extraction, high performance liquid chromatography separation, microsequencing and microsequence analysis. We found that amylin significantly decreased the insulin-stimulated glucose incorporation into glycogen (p < 0.01) and produced a protein spot of approximately 20 ku in size. This amylin responsive protein (hereby designated as amylin responsive protein 1, APR1) was identified to be protein p20. Moreover, ARP1 spots on gels were found to consistently produce a corresponding radioactive spot on X-ray films in 32Pi but not in 35S-methionine labeling experiments. In conclusion, our results showed that in vitro amylin concomitantly evoked the production of ARP1 and caused insulin resistance in EDL muscle. It is suggested that protein p20 may be involved in amylin signal transduction and the appearance of ARP1 may be a step in a molecular pathway leading to the development of insulin resistance. ARP1 might therefore be a useful molecular marker for amylin action, insulin resistance and Type 2 diabetes.
文摘It is well established that different sites within a protein evolve at different rates according to their role within the protein; identification of these correlated mutations can aid in tasks such as ab initio protein structure, structure function analysis or sequence alignment. Mutual Information is a standard measure for coevolution between two sites but its application is limited by signal to noise ratio. In this work we report a preliminary study to investigate whether larger sequence sets could circumvent this problem by calculating mutual information arrays for two sets of drug naive sequences from the HIV gpl20 protein for the B and C subtypes. Our results suggest that while the larger sequences sets can improve the signal to noise ratio, the gain is offset by the high mutation rate of the HIV virus which makes it more difficult to achieve consistent alignments. Nevertheless, we were able to predict a number of coevolving sites that were supported by previous experimental studies as well as a region close to the C terminal of the protein that was highly variable in the C subtype but highly conserved in the B subtype.
