ABSTRACT
Doppel (Dpl) is a homolog of normal cellular prion protein (PrPc) with unknown functions. Ectopic expression of Dpl in the central nervous system (CNS) causes neurotoxicity and this effect is rescued by the expression of PrPc. However, the molecular basis for the protective effect of PrPc remains unclear. Using a yeast two-hybrid system, we showed that Dpl binds the full-length 37-kDa laminin receptor precursor protein (LRP), one of the receptors of PrPc. The interaction was also validated by immunoprecipitation and immunoblotting using transfected cell lines and in vivo derived tissues. Further mapping experiments showed that although the middle fragment containing residues 100-220 of LRP was able to interact with Dpl, deletion of the N-terminal domain of the full-length LRP abolished its interaction with Dpl. These results suggest that while both PrPc and Dpl interact with LRP, the domains that are involved in the binding are not the same. Our results may have implications for the molecular mechanisms of Dpl-PrPc antagonism and physiological roles of Dpl.
Subject(s)
Prions/metabolism , Protein Precursors/metabolism , Receptors, Laminin/metabolism , Animals , Cell Line , Central Nervous System/metabolism , DNA, Complementary/metabolism , Female , GPI-Linked Proteins , Gene Deletion , Humans , Immunoblotting , Ligands , Male , Mice , Phenotype , Plasmids/metabolism , Precipitin Tests , Prions/chemistry , Protein Binding , Protein Precursors/chemistry , Protein Structure, Tertiary , Receptors, Laminin/chemistry , Recombinant Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Testis/metabolism , Testis/pathology , Transfection , Two-Hybrid System TechniquesABSTRACT
Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful for understanding the mysterious mechanism of conformational conversion process of prion diseases and proving the "protein-only" hypothesis. In this report, an engineered PrPsc-like conformation was produced from a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD). Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD, spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-like characteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increased fluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopy experiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high content beta-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit more soluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrPsc and will be helpful for further understanding the mechanism of conformational conversion process.
Subject(s)
PrPSc Proteins/chemistry , Prions/chemistry , Protein Engineering/methods , Amyloid/chemistry , Animals , Benzothiazoles , Cattle , Circular Dichroism , Coloring Agents/pharmacology , Congo Red/pharmacology , Electrophoresis, Polyacrylamide Gel , Endopeptidase K/chemistry , Microscopy , Microscopy, Electron , Plasmids/metabolism , PrPSc Proteins/metabolism , Protein Conformation , Protein Isoforms , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Spectrometry, Fluorescence , Thiazoles/chemistryABSTRACT
Prion protein has a key role in the occurrence of transmissible spongiform encephalopathy (TSE) and development of these diseases. Here, we provide a convenient procedure for on-column purification and refolding of the full-length mature bovine prion protein (bPrP) from Escherichia coli using immobilized metal (Ni) affinity chromatography, based on the metal-binding property of its unusual octarepeat sequences containing six tandem copies. Following extensive washing, the bPrP pellet was solubilized by guanidine hydrochloride and subjected to Ni-NTA agarose column. Purification and refolding were achieved by stepwise gradient washing with reduced guanidine hydrochloride concentrations. Triton X-100 and beta-mercaptoethanol were required in this rapid refolding process. The isolated prion protein was identified by monoclonal antibodies and its integrity was monitored by mass spectroscopy. Its correct folding was confirmed from circular dichroism (CD) experiments. Moreover, thioflavin T-binding assay showed that the recombinant bPrP could be transformed into amyloid fiber structures like that of the infectious prion isoform PrP(sc).
