Introduction: As a chaperone, heat shock protein acts as central integrators of protein homeostasis in cell. The form of these functions is to help setting up a complex protein molecular fold (folded protein) in many ...Introduction: As a chaperone, heat shock protein acts as central integrators of protein homeostasis in cell. The form of these functions is to help setting up a complex protein molecular fold (folded protein) in many important settings, such as growth, differentiation, and the ability to live. It has become clear that the control system plays an important role if the folding process fails or an error occurs, causing folding abnormalities and targeted functionality to accumulate. The accumulation of faulty protein folding would harm cells and can result in death. Apparently, there is a correlation between protein folding error with various diseases, such as diabetes mellitus and cancer. Method: We examined protein levels in all samples using Dotblott with monoclonal antibody anti-Hsp40 and anti-Hsp70. Levels of the protein content was read using a densitometer. Modification of Dot Blot was as follows: treatment was conducted with 3 × SSC, added with 20 mL blocking solution, add with total protein samples of 10 mg/ml on nitrocellulose paper, prehybridized, incubated at 70° for 30 seconds, incubated at 70° for 30 seconds with primary antibody anti-Hsp40 or Hsp70 protein and then added with second antibody HRP anti-Hsp40 or Hsp70 protein, treated with 3 × SSC and visualized with TSA HRP, and then administered with streptavidin, biothynil tyramide, and, finally, added with chromogen (DAB) in a confined space. Result: From the analysis of the data using Manova test with Wilk’s Lambda, there were significant differences in the levels of Hsp40 between Benign Oral Lesion (mean 688.31 area) and OSCC (mean 1354.59 area) patients (p 0.070), there was also a highly significant difference in Hsp70 levels between patients who experienced Benign Oral Lesion (mean 529.82 area) and OSCC (mean 1346.32 area) patients (p 0.006). Conclusion: OSCC patients have increased Hsp70 levels, so it is possible that something is going wrong in protein folding. Errors in protein folding result in a new homeostasis or inhibition of apoptosis and increasing cell proliferation that triggers carcinogenesis. Hsp40 acts as co-chaperones.展开更多
Influenza virus contains three integral membrane proteins:haemagglutinin,neuraminidase,and matrix protein(M1 and M2).Among them,M2 protein functions as an ion channel,important for virus uncoating in endosomes of viru...Influenza virus contains three integral membrane proteins:haemagglutinin,neuraminidase,and matrix protein(M1 and M2).Among them,M2 protein functions as an ion channel,important for virus uncoating in endosomes of virus-infected cells and essential for virus replication.In an effort to explore potential new functions of M2 in the virus life cycle,we used yeast two-hybrid system to search for M2-associated cellular proteins.One of the positive clones was identified as human Hsp40/Hdj1,a DnaJ/Hsp40 family protein.Here,we report that both BM2(M2 of influenza B virus)and A/M2(M2 of influenza A virus)interacted with Hsp40 in vitro and in vivo.The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40.Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^(IPK) that is a cellular inhibitor of PKR.PKR is a crucial component of the host defense response against virus infection.We therefore attempted to understand the relationship among M2,Hsp40 and p58^(IPK) by further experimentation.The results demonstrated that both A/M2 and BM2 are able to bind to p58^(IPK)in vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^(IPK),and consequently induce cell death.These results suggest that influenza virus M2 protein is involved in p58^(IPK)mediated PKR regulation during influenza virus infection,therefore affecting infected-cell life cycle and virus replication.展开更多
文摘Introduction: As a chaperone, heat shock protein acts as central integrators of protein homeostasis in cell. The form of these functions is to help setting up a complex protein molecular fold (folded protein) in many important settings, such as growth, differentiation, and the ability to live. It has become clear that the control system plays an important role if the folding process fails or an error occurs, causing folding abnormalities and targeted functionality to accumulate. The accumulation of faulty protein folding would harm cells and can result in death. Apparently, there is a correlation between protein folding error with various diseases, such as diabetes mellitus and cancer. Method: We examined protein levels in all samples using Dotblott with monoclonal antibody anti-Hsp40 and anti-Hsp70. Levels of the protein content was read using a densitometer. Modification of Dot Blot was as follows: treatment was conducted with 3 × SSC, added with 20 mL blocking solution, add with total protein samples of 10 mg/ml on nitrocellulose paper, prehybridized, incubated at 70° for 30 seconds, incubated at 70° for 30 seconds with primary antibody anti-Hsp40 or Hsp70 protein and then added with second antibody HRP anti-Hsp40 or Hsp70 protein, treated with 3 × SSC and visualized with TSA HRP, and then administered with streptavidin, biothynil tyramide, and, finally, added with chromogen (DAB) in a confined space. Result: From the analysis of the data using Manova test with Wilk’s Lambda, there were significant differences in the levels of Hsp40 between Benign Oral Lesion (mean 688.31 area) and OSCC (mean 1354.59 area) patients (p 0.070), there was also a highly significant difference in Hsp70 levels between patients who experienced Benign Oral Lesion (mean 529.82 area) and OSCC (mean 1346.32 area) patients (p 0.006). Conclusion: OSCC patients have increased Hsp70 levels, so it is possible that something is going wrong in protein folding. Errors in protein folding result in a new homeostasis or inhibition of apoptosis and increasing cell proliferation that triggers carcinogenesis. Hsp40 acts as co-chaperones.
基金supported by National Natural Sciences Foundation of China(NSFC)(Grant Nos.30670091 and 30599434)National Basic Research Program(Project 973)of China Ministry of Science and Technology(Grant No.2011CB504703)+1 种基金National Key Technologies R&D Program(Grant No.2006BAD06A01)GFG is a leading principal investigator of the NSFC Innovative Research Group(Grant No.81021003).
文摘Influenza virus contains three integral membrane proteins:haemagglutinin,neuraminidase,and matrix protein(M1 and M2).Among them,M2 protein functions as an ion channel,important for virus uncoating in endosomes of virus-infected cells and essential for virus replication.In an effort to explore potential new functions of M2 in the virus life cycle,we used yeast two-hybrid system to search for M2-associated cellular proteins.One of the positive clones was identified as human Hsp40/Hdj1,a DnaJ/Hsp40 family protein.Here,we report that both BM2(M2 of influenza B virus)and A/M2(M2 of influenza A virus)interacted with Hsp40 in vitro and in vivo.The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40.Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^(IPK) that is a cellular inhibitor of PKR.PKR is a crucial component of the host defense response against virus infection.We therefore attempted to understand the relationship among M2,Hsp40 and p58^(IPK) by further experimentation.The results demonstrated that both A/M2 and BM2 are able to bind to p58^(IPK)in vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^(IPK),and consequently induce cell death.These results suggest that influenza virus M2 protein is involved in p58^(IPK)mediated PKR regulation during influenza virus infection,therefore affecting infected-cell life cycle and virus replication.
