Ionic Strength and Transition Metals Control PrPSc Protease Resistance and Conversion-inducing Activity

Nishina, Koren; Jenks, Samantha; Supattapone, Surachai

doi:10.1074/jbc.m406548200

Cited by 40 publications

(36 citation statements)

References 77 publications

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“…In contrast, previous studies using cell-free conversion 6 and rPrP fibril formation [7][8][9] , respectively, in the presence of denaturant or at low pH have shown that the rate of PrP-res formation was directly proportional to the PrP-sen concentration, presumably owing to the homogenous denaturation status of on QUIC reactions showed that salt is needed for rPrP-res formation and the sensitivity of this method was maximal at 500 mM NaCl at pH7. The requirement for salt in rPrP-res formation is consistent with the fact that salt is required for cell-free conversion in the absence of GdnHCl 10 and the maintenance of a protease-resistant PrP Sc conformation 11 . In addition, salt has also been reported to reduce the thermodynamic stability of the rHuPrP-sen 12 .…”

Section: Acknowledgmentsmentioning

confidence: 58%

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Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

Atarashi

Satoh

Sano

et al. 2011

Nat Med

539

482

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The development of technologies for the in vitro amplification of the abnormal conformers of prion protein (PrP Sc ) has generated the potential for a novel diagnostic assay for prion disease. Previously, we developed a new PrP Sc amplification assay designated quaking-induced conversion (QUIC), which involves intermittent, automated shaking of the substrate, soluble recombinant PrP. We further improved the rapidity and practicality of this method by combining it with thioflavin T fluorescence to monitor the amyloid fibril formation. This assay, termed "real-time QUIC (RT-QUIC)", allows within 48 h, the detection of ≥1 fg of PrP Sc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects, and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the ante-mortem evaluation of suspected CJD. Definitive ante-mortem confirmation of CJD requires the detection of PrP Sc in patient biopsy specimens, the practice of which is discouraged because it is both invasive and poses risks to health care personnel. Recently, however, in vitro PrP Sc amplification techniques, including protein misfolding cyclic amplification (PMCA) 5-7 , the amyloid seeding assay 8 , as well as QUIC have been reported to enable the direct and highly sensitive detection of PrP Sc in various tissues, including cerebrospinal fluid (CSF). QUIC assays involve the use of soluble recombinant PrP (rPrP-sen) as a substrate, which is seeded with PrP Sc , and then subjected to intermittent automated shaking. This technique can be performed more easily than the PMCA, which requires repeated sonication. Previous studies have demonstrated that QUIC assays correctly discriminated between normal and scrapie-infected CSF samples in both hamster and sheep prion disease models 9,10 . However, ultrasensitive PrP Sc detection in CSF from CJD subjects has not yet been accomplished. Accordingly, we further refined the QUIC 5 assay to improve its sensitivity and practicability, and then applied the technique in a blind pilot study to detect PrP Sc in CJD-CSF specimens.Given that a correlation between protease-resistant rPrP aggregate (rPrP-res) levels and thioflavin T (ThT) fluorescence had been shown previously 7 , we sought to determine the relative kinetics of rPrP-res formation by monitoring levels of ThT fluorescence in the QUIC assay. This was intended to minimize the time needed to detect rPrP-res. We first tested whether PrP Sc -dependent rPrP-res (rPrP-res (Sc) ) formation could be induced using a microplate reader with intermittent shaking. Human rPrP-sen (rHuPrP-sen) and a 10 -7 dilution of CJD (molecular subtype MM1) brain homogenate (BH) were used as the substrate and "seed", respectively. We conducted QUIC reactions at various concentrations (0, 0.25, 0.5 and 1.0 M) of guanidine-HCl (GdnHCl), because it h...

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Section: Acknowledgmentsmentioning

confidence: 58%

“…Shaking is thought to cause partial unfolding of a portion of the rPrP-sen by increasing the air-water interface 11 . Moreover, shaking enhances the interaction between rPrP-sen and PrP Sc , and promote the fragmentation of rPrP-res polymers 12 .…”

mentioning

confidence: 99%

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

Atarashi

Satoh

Sano

et al. 2011

Nat Med

539

482

View full text Add to dashboard Cite

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“…An NMR structure of the octapeptide repeat from residues 61 to 84 bound to pentosan polysulfate was recently released showing a series of loosely defined loops with hydrophobic exposed tryptophans (Taubner et al, 2009). Interestingly, Cu may also influence the protease resistance of PrP Sc molecules (Nishina et al, 2004), and PrP Sc formed in a Cu-free environment was 20 times more susceptible to proteinase-K digestion; addition of Cu restored normal protease resistance.…”

Section: Metal Ionsmentioning

confidence: 99%

Toward a Mechanism of Prion Misfolding and Structural Models of PrP^Sc: Current Knowledge and Future Directions

Guest

Plotkin

Cashman

2011

Journal of Toxicology and Environmental Health, Part A

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Despite extensive investigation, many features of prion protein misfolding remain enigmatic. Physicochemical variables known to influence misfolding are reviewed to help elucidate the mechanism of prionogenesis and identify salient features of PrP Sc , the misfolded conformer of the prion protein.Prospective work on refinement of candidate PrP Sc models based on thermodynamic considerations will help to complete atomic-scale structural details missing from experimental studies and may explain the basis for the templating activity of PrP Sc in disease.

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“…The conversion process is enhanced by RNA molecules, redox-active metals like copper, certain denaturants, sulfated glycans, solvents, pH, temperature, reducing agents, inhibition of glycosylation, and a combination of buffer conditions and metal ions (156,197,246 (43,79,136). However, astrocyte-specific expression of PrP C is sufficient to support the replication and neurotoxicity of infectious PrP Sc , even in the absence of neuronal PrP C expression, indicating the involvement of other neurotoxic molecules or mechanisms in this process (98).…”

Section: Deposits Of Prpmentioning

confidence: 99%

“…The majority of these metals induce the aggregation of purified or recombinant PrP C to a form resembling PrP Sc in certain characteristics (156). How- …”

Section: B Prp and Other Metals In Particular Ironmentioning

confidence: 99%

Redox Control of Prion and Disease Pathogenesis

Singh

Das

et al. 2010

Antioxidants & Redox Signaling

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Imbalance of brain metal homeostasis and associated oxidative stress by redox-active metals like iron and copper is an important trigger of neurotoxicity in several neurodegenerative conditions, including prion disorders. Whereas some reports attribute this to end-stage disease, others provide evidence for specific mechanisms leading to brain metal dyshomeostasis during disease progression. In prion disorders, imbalance of brain-iron homeostasis is observed before end-stage disease and worsens with disease progression, implicating ironinduced oxidative stress in disease pathogenesis. This is an unexpected observation, because the underlying cause of brain pathology in all prion disorders is PrP-scrapie (PrP Sc ), a b-sheet-rich conformation of a normal glycoprotein, the prion protein (PrP C ). Whether brain-iron dyshomeostasis occurs because of gain of toxic function by PrP Sc or loss of normal function of PrP C remains unclear. In this review, we summarize available evidence suggesting the involvement of oxidative stress in prion-disease pathogenesis. Subsequently, we review the biology of PrP C to highlight its possible role in maintaining brain metal homeostasis during health and the contribution of PrP Sc in inducing brain metal imbalance with disease progression. Finally, we discuss possible therapeutic avenues directed at restoring brain metal homeostasis and alleviating metal-induced oxidative stress in prion disorders. Antioxid. Redox Signal. 12, 1271-1294.

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Ionic Strength and Transition Metals Control PrPSc Protease Resistance and Conversion-inducing Activity

Cited by 40 publications

References 77 publications

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

Toward a Mechanism of Prion Misfolding and Structural Models of PrP^Sc: Current Knowledge and Future Directions

Redox Control of Prion and Disease Pathogenesis

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Ionic Strength and Transition Metals Control PrPSc Protease Resistance and Conversion-inducing Activity

Cited by 40 publications

References 77 publications

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

Toward a Mechanism of Prion Misfolding and Structural Models of PrPSc: Current Knowledge and Future Directions

Redox Control of Prion and Disease Pathogenesis

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Product

Resources

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Toward a Mechanism of Prion Misfolding and Structural Models of PrP^Sc: Current Knowledge and Future Directions