The biochemical essence of prion replication is the molecular multiplication of the disease-associated misfolded isoform of prion protein (PrP), termed PrP
Sc
, in a nucleic acid-free manner. PrP
Sc
...is generated by the protein misfolding process facilitated by conformational conversion of the host-encoded cellular PrP to PrP
Sc
. Evidence suggests that an auxiliary factor may play a role in PrP
Sc
propagation. We and others previously discovered that plasminogen interacts with PrP, while its functional role for PrP
Sc
propagation remained undetermined. In our recent in vitro PrP conversion study, we showed that plasminogen substantially stimulates PrP
Sc
propagation in a concentration-dependent manner by accelerating the rate of PrP
Sc
generation while depletion of plasminogen, destabilization of its structure and interference with the PrP-plasminogen interaction hinder PrP
Sc
propagation. Further investigation in cell culture models confirmed an increase of PrP
Sc
formation by plasminogen. Although molecular basis of the observed activity for plasminogen remain to be addressed, our results demonstrate that plasminogen is the first cellular protein auxiliary factor proven to stimulate PrP
Sc
propagation.
Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion ...protein (PrP
C) is required for prion replication, the influence of PrP
C abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP
C was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP
C supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP
C-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP
C is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP
Sc amplification by using concentrated PrP
C source and expands the use of this methodology.
The conformational change of cellular prion protein (PrP
) to its misfolded counterpart, termed PrP
, is mediated by a hypothesized cellular cofactor. This cofactor is believed to interact directly ...with certain amino acid residues of PrP
. When these are mutated into cationic amino acid residues, PrP
formation and prion replication halt in a dominant negative (DN) manner, presumably due to strong binding of the cofactor to mutated PrP
, designated as DN PrP mutants. Previous studies demonstrated that plasminogen and its kringle domains bind to PrP and accelerate PrP
generation. In this study, in vitro binding analysis of kringle domains of plasminogen to Q167R DN mutant PrP (PrPQ167R) was performed in parallel with the wild type (WT) and Q218K DN mutant PrP (PrPQ218K). The binding affinity of PrPQ167R was higher than that of WT PrP, but lower than that of PrPQ218K. Scatchard analysis further indicated that, like PrPQ218K and WT PrP, PrPQ167R interaction with plasminogen occurred at multiple sites, suggesting cooperativity in this interaction. Competitive binding analysis using
-lysine or
-arginine confirmed the increase of the specificity and binding affinity of the interaction as PrP acquired DN mutations. Circular dichroism spectroscopy demonstrated that the recombinant PrPs used in this study retained the α-helix-rich structure. The α-helix unfolding study revealed similar conformational stability for WT and DN-mutated PrPs. This study provides an additional piece of biochemical evidence concerning the interaction of plasminogen with DN mutant PrPs.
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 (PrP89-230) 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.
Inflammation as a major defense mechanism against pathogens is modulated by diverse microbial products. A variety of plant and microbial products interacting with Toll-like receptors initiate a wide ...spectrum of responses from phagocytosis to cytokine production, which modulates inflammation. Jasmonates are fatty acid-derived cyclopentanones produced by plants and lower eukaryotes that play an important role in the defense against insects. In this study, we are set up to define the molecular targets of J2 action. While the lipopolysaccharide (LPS) stimulation of macrophage cell line RAW264.7 induced TNF-α, IL-6, iNOS, and COX-2 that were associated with an increase in miR-155 and miR-146a, the J2 suppressed the induction of these inflammatory cytokines and enzymes as well as miR-155 in a dose-dependent manner. To assess the associations of miR-155 with inflammatory markers, we overexpressed miR-155 and found attenuation of COX-2 suppression with J2 treatment. Furthermore, J2 inhibited NF-κB, p65, and IκB but had no or only minimal effects on the mitogen-activated protein kinase pathway. In conclusion, the present study demonstrates that J2 suppresses LPS stimulation of RAW264.7 cells by targeting NF-κB pathways.
Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity ...rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in
cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with ＞95% purity. The purified recPrPs predominantly adopted an α-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.
Prion diseases are incurable neurodegenerative disorders. Our previous study demonstrated that polylysine was effective in prolonging the incubation period in a rodent model and in alleviating the ...scrapie prion protein (PrP(Sc)) burden in the brain at the terminal stage of the disease. Here, we report that intraperitoneal administration of polylysine suppresses the accumulation of prions in the spleen during the early stages of the disease. This study supports the congruence of PrP(Sc) inhibition by polylysine in both the spleen and brain.
Through the development of organic synthetic skill, chemicals that mimic signaling mediators such as steroid hormones have been exposed to the environment. Recently, it has become apparent that this ...circumstance should be further studied in the field of physiology. Estrogenic action of chronic low-dose nonylphenol (NP) and di-(2-ethylhexyl) phthalate (DEHP) in mouse uterus was assessed in this study. Ten to twelve-week-old female mice (CD-1) were fed drinking water containing NP (50 or $500{\mu}g/L$) or DEHP (133 or $1,330{\mu}g/L$) for 10 weeks. Uterine diameter, the thickness of myometrium and endometrium, and the height of luminal epithelial cells were measured and the number of glands were counted. The expression levels of the known $17{\beta}$-estradiol ($E_2$)-regulated genes were evaluated with real-time RT-PCR methodology. The ration of uterine weight to body weight increased in $133{\mu}g/L$ DEHP. Endometrial and myometrial thickness increased in 133 and $1,330{\mu}g/L$ DEHP treated groups, and in 50, $500{\mu}g/L$ NP and $133{\mu}g/L$ DEHP, respectively. The height of luminal epithelial cell decreased in NP groups. The numbers of luminal epithelial gland were decreased in NP groups but increased in $50{\mu}g/L$ DEHP group. The histological characters of glands were not different between groups. The mRNA expression profiles of the known $17{\beta}$-estradiol ($E_2$) downstream genes, Esr1, Esr2, Pgr, Lox, and Muc1, were also different between NP and DEHP groups. The expression levels dramatically increased in some genes by the NP or DEHP. Based on these results, it is suggested that the chronic low-dose NP or DEHP works as estrogen-like messengers in uterus with their own specific gene expression-regulation patterns.
Through the development of organic synthetic skill, chemicals that mimic signaling mediators such as steroid hormones have been exposed to the environment. Recently, it has become apparent that this ...circumstance should be further studied in the field of physiology. Estrogenic action of chronic low-dose nonylphenol (NP) and di-(2-ethylhexyl) phthalate (DEHP) in mouse uterus was assessed in this study. Ten to twelve-week-old female mice (CD-1) were fed drinking water containing NP (50 or 500 μg/L) or DEHP (133 or 1,330 μg/L) for 10 weeks. Uterine diameter, the thickness of myometrium and endometrium, and the height of luminal epithelial cells were measured and the number of glands were counted. The expression levels of the known 17β-estradiol (E
)-regulated genes were evaluated with real-time RT-PCR methodology. The ration of uterine weight to body weight increased in 133 μg/L DEHP. Endometrial and myometrial thickness increased in 133 and 1,330 μg/L DEHP treated groups, and in 50, 500 μg/L NP and 133 μg/L DEHP, respectively. The height of luminal epithelial cell decreased in NP groups. The numbers of luminal epithelial gland were decreased in NP groups but increased in 50 μg/L DEHP group. The histological characters of glands were not different between groups. The mRNA expression profiles of the known 17β-estradiol (E
) downstream genes,
, were also different between NP and DEHP groups. The expression levels dramatically increased in some genes by the NP or DEHP. Based on these results, it is suggested that the chronic low-dose NP or DEHP works as estrogen-like messengers in uterus with their own specific gene expression-regulation patterns.
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