AIM To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195(?60HDAg), ...AIM To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195(?60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization.METHODS Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ?60HDAg. Nucleic acid binding properties were investigated by gel retardation assays. RESULTS Results showed that the truncated ?60HDAg protein is intrinsically disordered but compact, whereas the RNA binding domain, comprising residues 94-146, adopts a dynamic helical conformation. We also found that ?60HDAg fails to multimerize but still contains nucleic acid binding activity, indicating that multimerization is not essential for nucleic acid binding. Moreover, in agreement with what has been previously reported for full-length protein, no apparent specificity was found for the truncated protein regarding nucleic acid binding.CONCLUSION Taken together these results allowed concluding that ?60HDAg is intrinsically disordered but compact; ?60HDAg is not a multimer but is still capable of nucleic acid binding albeit without apparent specificity.展开更多
Primary biliary cirrhosis(PBC),primary sclerosing cholangitis(PSC)and autoimmune hepatitis(AIH)are the major forms of autoimmune liver diseases each characterized by the destruction of a specific liver cell type and t...Primary biliary cirrhosis(PBC),primary sclerosing cholangitis(PSC)and autoimmune hepatitis(AIH)are the major forms of autoimmune liver diseases each characterized by the destruction of a specific liver cell type and the presence of differing auto-antibodies.We took a proteomic approach utilizing in situ matrix-assisted laser desorption/ionization mass spectrometry(MALDI MS)to obtain profiles directly from liver samples of patients with PBC,PSC,AIH and controls.The ability to precisely localize the region for acquisition of MALDI MS allowed us to obtain profiles from bile ducts,inflammatory infiltrates and hepatocytes from each biopsy sample.Analysis tools developed to identify peaks and compare peaks across diseases and cell types were used to develop models to classify the samples.Using an initial set of testing samples from PBC patients and controls,we identified unique peaks present in bile ducts,inflammatory infiltrates and hepatocytes that could classify samples in a validation cohort with 88–91%accuracy.Interestingly,profiles of PSC and AIH did not differ significantly from PBC.Identification of proteins in these peaks may represent novel autoantigens or effector molecules.These findings illustrate the potential of a proteomic approach to autoimmune diseases with in situ MALDI MS.展开更多
基金Supported by Fundação para a Ciência e Tecnologia,FCT,to GHTM-UID/Multi/04413/2013Carolina Alves and Ana Casaca were recipients of FCT PhD grantsJoão Paulo Tavanez is a recipient of a FCT post-doctoral fellowship SFRH/BPD/87494/2012.
文摘AIM To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195(?60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization.METHODS Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ?60HDAg. Nucleic acid binding properties were investigated by gel retardation assays. RESULTS Results showed that the truncated ?60HDAg protein is intrinsically disordered but compact, whereas the RNA binding domain, comprising residues 94-146, adopts a dynamic helical conformation. We also found that ?60HDAg fails to multimerize but still contains nucleic acid binding activity, indicating that multimerization is not essential for nucleic acid binding. Moreover, in agreement with what has been previously reported for full-length protein, no apparent specificity was found for the truncated protein regarding nucleic acid binding.CONCLUSION Taken together these results allowed concluding that ?60HDAg is intrinsically disordered but compact; ?60HDAg is not a multimer but is still capable of nucleic acid binding albeit without apparent specificity.
基金This work was supported by NIH/NIGMS 5R01 GM58008Vanderbilt Ingram Cancer Center Core Support Grant P30 CA068485,NIH DK39588National Foundation for Cancer Research:Vanderbilt Center for Proteomics and Drug Action.
文摘Primary biliary cirrhosis(PBC),primary sclerosing cholangitis(PSC)and autoimmune hepatitis(AIH)are the major forms of autoimmune liver diseases each characterized by the destruction of a specific liver cell type and the presence of differing auto-antibodies.We took a proteomic approach utilizing in situ matrix-assisted laser desorption/ionization mass spectrometry(MALDI MS)to obtain profiles directly from liver samples of patients with PBC,PSC,AIH and controls.The ability to precisely localize the region for acquisition of MALDI MS allowed us to obtain profiles from bile ducts,inflammatory infiltrates and hepatocytes from each biopsy sample.Analysis tools developed to identify peaks and compare peaks across diseases and cell types were used to develop models to classify the samples.Using an initial set of testing samples from PBC patients and controls,we identified unique peaks present in bile ducts,inflammatory infiltrates and hepatocytes that could classify samples in a validation cohort with 88–91%accuracy.Interestingly,profiles of PSC and AIH did not differ significantly from PBC.Identification of proteins in these peaks may represent novel autoantigens or effector molecules.These findings illustrate the potential of a proteomic approach to autoimmune diseases with in situ MALDI MS.
