Evolutionary history and genetic diversity study of heat-shock protein 60 of Rhizophagus irregularis

Despite the ubiquitous occurrence of heat-shock protein 60 (Hsp60) and their role in maintenance of cell activity and integrity, this protein remains poorly characterized in many of the symbiotic soil mycorrhizal fungi such as Rhizophagus irregularis. Thus, in the current study, an attempt has been made to elucidate the evolutionary history, time of divergence followed by estimation of population genetic parameters of hsp60 using R. irregularis as a model organism. Sequence alignment reported here identified several close homologues for hsp60 (gene) and Hsp60 (protein) from diverse taxa, while the output from protein-based phylogenetic tree indicates that mitochondrial Hsp60 of R. irregularis shares close evolutionary relationship with classical α-proteobacteria. This is perhaps the first line of evidence elucidating the likelihood of hsp60 from fungal taxa sharing a close evolutionary relationship with classical α-proteobacteria as a common ancestor. Comprehensive analysis of mitochondrial hsp60 from selected fungal taxa from the evolutionary point of view explains the possibility of gene duplication and or horizontal gene transfer of this gene across various fungal species. Synteny relationships and population genetics credibly explain high genetic variability associated with fungal hsp60 presumably brought by random genetic recombination events. The results presented here also confirm a high level of genetic differentiation of hsp60 among all the three fungal populations analysed. In this context, the outcome of the current study, basedon computational approach, stands as a testimony for explaining the possibility of increased genetic differentiation experienced by hsp60 of R. irregularis.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Cpn60 deficiency may play a role in the intrapancreatic enzyme activation in acute pancreatitis / 中国病理生理杂志

AIM: To investigate the change of Cpn60 content,the alterations of pancreatic enzymes and lysosome,in order to better understand the mechanism of intrapancreatic enzyme activation in acute pancreatitis(AP). METHODS: The AP model was replicated by retrograde infusion of 4% sodium-deoxycholate in the choledocus of SD rats. The levels of amylase in plasma and TNF-? in pancreatic tissue were measured by biochemical technique at 5 h and 10 h after AP induction. The content of Cpn60 and pancreatic enzymes in different compartments of the acinar cells were tested by quantitative protein A-gold immunocytochemistry technique. The change of lysosome in the acinar cells was observed under the electronic microscope. RESULTS: After AP was induced,the levels of amylase in the plasma and TNF-? in the pancreatic tissue increased significantly. Lysosomes with different forms were found inside the acinar cells,and some of them located in the Golgi apparatus. Cpn60 content decreased,which was accompanied by an increase of lipase or chymotrypsinogen content in the pancreatic secretory pathway. CONCLUSION: In the pancreatic acinar cells of AP rats,Cpn60 content decreased,suggesting an insufficient chaperone capacity,and combining with the change of lysosome both in its amount and location,which may take part in the intrapancreatic enzyme activation and the development of AP.