Old News is New News! Say that 5 times fast!

[Kathy]

Probably the most revealing study done in prion research was published in 2000 by: David R. Brown, Farida Hafiz, Leslie L. Glasssmith, Boon-Seng Wong, Ian M. Jones, Christine Clive and Stephen J. Haswell


Consequences of manganese replacement of copper for prion protein function and proteinase resistance , EMBO Journal Vol. 19, pp. 1180-1186, 2000.

Link: http://embojournal.npgjournals.com/cgi/content/full/19/6/1180?ijkey=sBpvL9/Ho3r0A

Quotes from this study entitled:

Although it has been established that PrPc is a copper-binding protein it is unknown whether the protein can bind other divalent cations. In this report we found that recombinant PrPc will bind manganese and nickel. Of these, manganese appeared to alter PrPc to a proteinase-resistant form that forms fibrils. Furthermore, PrPc expression influences uptake of manganese into cells. Therefore, we speculate that incorporation of manganese into PrPc may be one way in which PrPSc can be formed in vivo.

Recombinant full-length and deleted PrP was refolded as described with other divalent cations including calcium, magnesium, manganese, nickel, iron and zinc at concentrations of 5 mM.......

In order to determine whether manganese and nickel could replace copper when in competition, full-length PrP was refolded with either nickel and copper or manganese and copper in equimolar concentrations (5 mM). In these cases (Table II) copper was preferentially bound in place of nickel but manganese was bound equivalently with copper (i.e. two atoms each per molecule). When the concentration of manganese was decreased during refolding (5 mM copper, 0.5 mM manganese) the amount of manganese binding was reduced to one-tenth that of copper. These results suggest that manganese can substitute equivalently for copper in the holo-form of PrP.

...

Copper- and manganese-loaded PrP that had been left to age for 2 weeks at 4°C were assayed again for superoxide dismutase activity using the same assay. In this case, copper-loaded PrP showed the same activity as fresh material. However, the manganese-loaded PrP showed almost no activity, suggesting that some change had occurred in the protein during aging.

However, when PrP that had been aged for 2 weeks was treated with PK, manganese-refolded PrP showed increased PK resistance up to 25 µg/ml (Figure 4). Copper-refolded PrP could be completely degraded with 2 µg/ml PK...

Also, the CD spectrum of copper-refolded PrP that was aged for 2 weeks was no different to that of freshly prepared PrP. However, when manganese-refolded PrP was aged for 2 weeks, the CD spectrum showed strong changes at ~210 nm, consistent with greatly increased beta-sheet content. This suggests that manganese-refolded PrP underwent a spontaneous change in secondary structure when manganese was bound.

This suggests that manganese can enter cells through competition with copper at more sites than copper can compete with manganese.

The bands seen after PK treatment were more reminiscent of the bands seen after PK treatment of PrPSc than of those for the recombinant protein seen in Figure 4. These results suggest that cells can incorporate manganese into PrP and that some of the PrP is PK resistant.

However, more significant for this study is the finding that replacement of copper with other cations can lead to changes in secondary structure. It appears that PrP is less stable on binding manganese and quickly converts to a misfolded form. 

The observation that on binding manganese PrPc becomes proteinase resistant is probably the most important finding in the present report.

The finding that incorporation of manganese into PrP makes it proteinase resistant and abolishes its function is a long way from explaining sporadic prion disease. However, a current favourite among hypotheses concerning conversion of PrPc to PrPSc requires the formation of `seeds' or `nuclei' of proteinase-resistant PrP (Cohen and Prusiner, 1998). The formation of sufficient proteinase-resistant PrP to create such a seed is the limiting step in this model of PrPSc formation. Incorporation of manganese in place of copper in the holo-form of PrPc may represent one possible way in which the substance of a `seed' could form. ...

Although PrP can bind the same number of manganese atoms as copper atoms the resulting protein is aberrant. These results show for the first time a mechanism by which proteinase-resistant native PrPc can be expressed by cells.

Read it, and pass it on to everone you know. The link is above. Thanks.

 

[TimH]

Thank you, Kathy, for posting that!

 

[Kathy]

Well every day brings new news. This one showed up, for me, after I posted the above message, otherwise, I would have included it!!


Biochem Biophys Methods. 2005 Jun 30;63(3):213-21.

Automated PrPres amplification using indirect sonication.

Sarafoff NI, Bieschke J, Giese A, Weber P, Bertsch U, Kretzschmar HA.

Centre for Neuropathology, Prion Research Ludwig-Maxmilians-University Feodor-Lynen-Str. 23, 81377 Munich, Germany. [email protected].

Prions, which mainly consist of the scrapie isoform of the prion protein (PrP(Sc)), induce the misfolding of the physiological prion protein (PrP(C)). The Protein Misfolding Cyclic Amplification (PMCA), a process consisting of sonication and incubation, is one of the few methods thought to model autocatalytic prion replication and generation of proteinase K (PK)-resistant PrP (PrPres) in vitro. Here we show for the first time that the amplification may be achieved through direct as well as indirect sonication (water bath sonication using sealed sample containers), allowing the PMCA method to be automated. The automated method may serve as a valuable tool in high throughput screening for the diagnosis or compound identification for treatment of prion disease. The in vitro amplification process is weakly facilitated by divalent cations such as Mn, Zn and Ni, but not Cu, however, the presence of metal ions decreases the stability of PrPres against proteinase K digestion.