| Prion-related diseases, also been defined as transmissible spongiform encephalopathies (TSE), affect many mammalian hosts including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and Kuru in humans, scrapie in sheep, chronic wasting disease in deer and elk. bovine spongiform encephalopathy in cattle and transmissible mink encephalopathy in minks. In90th of last century, Stanley, B, Prusiner had identified that the pathological factor of prion disease is PrPSc, which derived from the conformational isoform of PrPc protein which are widely expressed in various mammal cell (including neuron). More documents they proposed that after go-through the conformational conversion from PrPC to PrPSc (from alpha-helix rich status to beta-sheet rich status), the monomers of PrPSc molecular will aggregate, and finally produce the fibers of prion (which called as prion rod). As the physiological function of PrPC remains unclear, why the accumulation of PrPSc can prion disease phenomenon and the pathological mechanism of prion disease keep as hotspot in prion field? Summarily, the accumulation of PrPSc molecular cause aberrant of cellular regulation network, which will result in seriously apoptosis and loss of neuron in infected brain; alternatively, PrPSc can cause acute-or chronic injury on host cell, and interact with other proteins, misconduct the normal function of key-point of cellular physiology.Protein disulfide isomerase, PDI, is chaperon molecular retained on lumen of endoplasmic reticulum and widely expressed on eukaryote. PDI is member of thioredoxin superfamily, which contain enzymatic motif " CGHC " in site of53-56,379-400of PDI molecular organization. It is well documented that PDI act as protective factor in neurodegenerative disease. In Alzheimer’s disease and Parkinson’s disease, expression of PDI is up-regulated and the enzymatic activity of PDI is inhibited. However, there were no documents on PDI and prion disease.In the present study, we performed Western blot, immunofluorescence assay, biotin-switch-test, Caspase-3activity assay on scrapie263K infected hamster brain tissue and cell models expressed mutanted PrP protein. We proposed the role of PDI in the process of Prion disease, and revealed that PDI regulate the endoplasmic reticulum stress and mitochondrial dysfunction induced by mutanted prion protein.The main results of this study are as follows:1. The Scrapie263K infected brain tissue homogenate was subjected to immunoblot assay with specific PDI antibody to investigate the expression profile of PDI protein during prion disease. Results indicated that PDI protein and its homologue Grp58are up-regulated in the terminal stage of prion disease, and the expression of hallmark of ER stress (Grp78) and apoptosis (Caspase-3) point to the activation of apoptosis and ER stress. Data derived from the dynamic monitor showed that the abnormal regulation of PDI arise in20day after incubation (dpi), and reach the maximum at60dpi accompanying with apoptotic activation and endoplasmic reticulum.2. To investigate the possible co-localization between PDI protein and mutanted prion protein, HEK293cell co-transfected with mutanted PrP and human PDI plasm ids were subjected to immunoblot and immunofluorescence assay. The results showed that the wild-type PrP protein mainly distributing on cellular membrane and partially in the cytoplasm. Elsewise, the mutated prion protein experience different process of Traffic and metabolism. There are significant plaque-like accumulation in PrP-KDEL and PrP-PG15expressing HEK293cells. Cell viability and WB assay showed that mutanted prion protein can induce serious cytotoxicity, and trigger the activation of ER stress and apoptotic pathway. Additionally, there were specific co-localization between mutanted PrP proteins and PDI in endoplasmic reticulum.3. HEK293cells were transfected with expressing plasmids pcDNA3.1-PDI and/or pcDNA3.1-PrP-PG5, pcDNA3.1-PrP-KDEL, pcDNA3.1-PrP-PG15and were subjected to CCK-8and Western blot assay with the appropriate antibodies. The results showed that over-expression of PDI can relieve the cytotoxicity induced by mutanted prion protein, especially by PrP-KDEL, and inhibit the activation of Caspase-12. Meanwhile, using the silent oligos targeted PDI to knockdown the expression of endogenous PDI, results indicated that down-regulation of PDI can partially relieve the cytotoxicity induced by PrP-PG15, and inhibit the activation of Caspase-3and up-regulate the expression of Grp78protein.4. To assess the mechanism of expressing mutanted PrP proteins on cell viability and cytotoxicity, mitochondrial membrane potential analysis and outflux of Cyto.C from mitochondria were performed on various transfected HEK293cells. The results shown that mutanted PrP proteins can affect on the homeostasis of mitochondrial membrane potential and sequentially open the release of Cyto.C, and activate the pro-Caspase-3and apoptosis. In other side, mis-expression for PDI can partially block the mitochondrial dysfunction. Finally, we also detect the S-nitrosylated PDI proteins in infected or heredity toxic PrP proteins. The results indicated that misfolded prion proteins can induce the formation of SNO-PDI in infected brain tissues and transfected HEK293cells. Block of SNO-PDI formation can relieve the cytotoxicity induced by mutanted prion proteins.Taking together, in present study, we have revealed the relationships between mutanted prion proteins, PDI, ER stress and malfunction. With the263K infected brain tissues and transfected HEK293cell, we found the different role of PDI in cytotoxicity induced by different mutanted PrP plasmids. In the mechanism of PDI regulating cytotoxicity, we also proposed that mitochondrial dysfunction and S-nitrosylated modification of PDI mat feed the regulating feature in prion disease. |
PDF Full Text Request