Animals ; PrPSc Proteins ; metabolism ; pathogenicity ; Prion Diseases ; transmission ; Prions ; physiology

Doppel protein (abbreviation Dpl) is a newly recognized Glycosyl phosphatidyl inositol (GPI) anchored and highly glycosylated protein, which is similar to prion protein (PrP) in the chemical structure. The encoding gene of Dpl named PRND locates at the downstream of the prion protein gene (PRNP). These two proteins are different in physiological functions. The expression of Dpl focuses on testis tissue at the adult, and takes an important role in maintaining sperm integrality, normal fertility, and motion ability. We reviewed the biological characters, physiological functions of Dpl and its effects on male reproduction in order to provide theory guidance for the study on physiological function and male reproduction controlling.
Animals ; GPI-Linked Proteins ; Humans ; Male ; Prions ; metabolism ; physiology ; Reproduction ; physiology ; Testis ; growth & development ; metabolism

Animals ; China ; epidemiology ; Humans ; Prevalence ; Prion Diseases ; epidemiology ; metabolism ; prevention & control ; Prions ; metabolism

In order to explore the potential influences of the disulfide bridge on the physical and chemical properties of PrP protein, the expressed recombinant human wild-type PrP protein was purified for using in an established redox process for the reduction and oxidation of the ethanethiol group within PrP. Sedimentation tests illustrated that redox process remarkably promoted the aggregation of recombinant PrP. Thioflavin T binding assay revealed an enhanced fibrillization of the recombinant human PrP after redox process. Far-UV circular dichroism demonstrated that the PrP treated with redox process showed a significant p-sheet rich structure. Furthermore, PrP-specific Western blot identified that the recombinant PrP after redox possessed stronger proteinase K-resistance. Those data indicates that the formation of the disulfide bridge induces the alteration of the secondary structure and enhances the progresses of aggregation and fibrillization of PrP protein.
Amyloid ; chemistry ; Endopeptidase K ; metabolism ; Humans ; Oxidation-Reduction ; Prions ; chemistry ; metabolism ; Protein Multimerization ; Protein Structure, Secondary ; Proteolysis ; Sulfhydryl Compounds ; chemistry

Misfolded isoform of prion protein (PrP), termed scrapie PrP (PrP(Sc)), tends to aggregate into various fibril forms. Previously, we reported various conditions that affect aggregation of recombinant PrP into amyloids. Because amyloidogenesis of PrP is closely associated with transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, we investigated infectivity of recombinant PrP amyloids generated in vitro. Using cultured cell lines which overexpress cellular PrP of different species, we measured the level of de novo synthesized PrP(Sc) in cells inoculated with recombinant mouse PrP amyloids. While PrP-overexpressing cells were susceptible to mouse-adapted scrapie prions used as the positive control, demonstrating the species barrier effect, infection with amyloids made of truncated recombinant PrP (PrP[89-230]) failed to form and propagate PrP(Sc) even in the cells that express mouse cellular PrP. This suggests that infectivity of PrP amyloids generated in vitro is different from that of natural prions. Recombinant PrP (89-230) amyloids tested in the current study retain no or a minute level, if any, of prion infectivity.
Animals ; Cell Line ; Kidney/*metabolism/*pathology ; Mice ; PrPSc Proteins/*metabolism ; Prion Diseases/*metabolism/*pathology ; Prions/*metabolism ; Rabbits ; Recombinant Proteins/*metabolism ; Up-Regulation

To investigate changes of 14-3-3beta from apoptosis induced by PrP106-126 polypeptide, HeLa cell was incubated with PrP106-126 for 4h or 8h. Nucleus changes and the expression of PARP were detected differently by Hoechst staining and Western blotting. Expressing of protein and mRNA from 14-3-3beta was determined by Western blotting and Real-time PCR. The results show that typical nucleus pyknosis and chip of apoptosis and degradation of PARP were induced by PrP106-126 peptide in HeLa cells. Degradation of 14-3-3beta appeared in apoptosis groups induced by PrP106-126 peptide. However, 14-3-3beta mRNA did not display any changes in apoptosis groups. This study indicated that degradation of antiapoptosis protein 143-3beta induced by PrP106-126 peptide may be one of pathogenesis mechanism of prion disease.
14-3-3 Proteins ; metabolism ; Apoptosis ; drug effects ; HeLa Cells ; Humans ; Peptide Fragments ; pharmacology ; Prions ; pharmacology ; Proteolysis ; drug effects

In order to quantify the curing effects of phenanthridine on yeast prion, we introduced semi-denaturing agarose gel electrophoresis and fluorescence recovery after photobleaching techniques to quantify the curing effects of phenanthridine on yeast prion at the protein and cellular levels with the [PSI+] yeast strain expressing GFP-Sup35p (NGMC). The results showed that these two approaches could precisely quantify the curing effects of phenanthridine on [PSI+] cells. After a treatment for 1 through 5 days with phenanthridine, the curing rates of [PSI+] cells were 0%, 0%, 51.7%, 87.5% and 94.4%, respectively. Meanwhile, we quantified the sizes of Sup35p polymers in phenanthridine induced pink phenotype cells. The aggregation status in 1-2 days phenanthridine treated cells were similar to those in [PSI+] cells, while the aggregation status in 3-5 days phenanthridine treated cells were similar to those in [psi(-)] cells.
Computer Simulation ; Models, Biological ; Peptide Termination Factors ; metabolism ; Phenanthridines ; pharmacology ; Prions ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae ; cytology ; drug effects ; metabolism ; Saccharomyces cerevisiae Proteins ; metabolism

OBJECTIVETo study the potential interaction between PrP protein and glial fibrillary acidic protein (GFAP) and identify the binding region within PrP with GFAP.

METHODSThe supernatant of healthy and scrapie-infected hamsters' brain homogenate was prepared, while various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing or in-vitro translation system. The possible molecular interaction between PrP proteins and GFAP was tested by Pull-down and immunoprecipitation assays.

RESULTSBoth native PrP(C) and its protease-resistant isoform (PrP(Sc)) formed complexes with the native GFAP. The full-length recombinant PrP proteins interacted with GFAP. The domain responsible for interacting GFAP was located at C-terminal of PrP (residues 91 to 231).

CONCLUSIONThe studies of the association of PrP with GFAP may further provide insight into a potential role of GFAP in the biological function of PrP and the pathogenesis of prion disease.

Animals ; Brain ; metabolism ; Cricetinae ; Gene Deletion ; Glial Fibrillary Acidic Protein ; genetics ; metabolism ; Immunoprecipitation ; Mice ; Prions ; genetics ; metabolism ; Protein Binding ; Recombinant Proteins ; metabolism

Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that beta-amyloid1-42 oligomer causes neurotoxicity associated with Alzheimer's disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer's disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in beta-amyloid. Residues 23-39 and 93-119 in the prion protein were involved in binding to beta-amyloid1-40 and 1-42, and monomers of this protein interacted with prion protein residues 93-113 and 123-166. Furthermore, beta-amyloid antibodies against the C-terminus detected bound beta-amyloid1-42 at residues 23-40, 104-122 and 159-175. beta-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to beta-amyloid1-40 and 1-42. The 3D structure appears to be necessary for beta-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer's disease.
Amyloid beta-Peptides/*metabolism ; Electrophoresis ; Enzyme-Linked Immunosorbent Assay ; *Epitope Mapping ; Epitopes/*metabolism ; Humans ; *Immunoassay ; Prions/*metabolism ; Protein Binding ; Recombinant Proteins/metabolism

Prion proteins (PrPs) are infectious pathogens that cause a group of invariably fatal, neurodegenerative diseases, including Creutzfeldt-Jakob disease, by means of an entirely novel mechanism. They are produced by various species, including reptile, rodent, ruminant and mammals, during normal metabolic processes, but they can be slowly changed into pathogenic isoforms upon contact with other infectious PrP isoforms. This transmission can occur across species barriers. In the present study, phylogram for each PrP sequence was generated by PAUP* 4.0 program using Neighbor-Joining method with 1,000 times bootstrapping process for the phylogenetic analysis. The molecular dynamics (MD) simulations were performed by the SANDER module in the AMBER 7 package using Amber 99 force field. All the simulation process was conducted in the IBM p690 Supercomputing System in Korea Institute of Science and Technology Information. To reduce the calculation time, we used the Generalized Born (GB) model. We compared the sequences and structural characteristics of normal and pathogenic (E200K) human PrPs with those of other reptile, rodent, ruminant and mammalian PrPs. Phylogenetic analysis revealed that, although the turtle PrP sequence is the most distinct of the PrPs analyzed, it nonetheless retains five conserved secondary structural elements that are similar to those found in the mammalian PrPs, suggesting that these elements have important functions in vivo. The RMS deviation between the normal and E200K human PrPs was larger than that between the normal human and bovine PrPs, and all of the beta-sheet structures in human E200K PrP were very stable during MD simulations.
Animals ; Cattle ; *Computational Biology ; Humans ; Phylogeny ; Prions/*chemistry/classification/*genetics/isolation & purification ; Reptiles/metabolism ; Rodentia/metabolism ; Ruminants/metabolism ; Sequence Analysis, Protein ; Species Specificity