Prion diseases have been widely studied, but despite many great leaps in our knowledge of prion and protein misfolding diseases in general, many gaps remain. One example is the structural triggers or provocators of prion conversion found within the prion protein (PrP) itself. While factors interacting on PrP can promote or dissuade conversion, it is also evident that inherent properties of differing PrP (presumably especially conformation) have an effect on the likelihood of prion conversion. Previous work from our lab found that the substitution of histidine 95 with a tyrosine increased prion conversion, while the same individual substitution at four other copper-binding histidines of PrP had no effect on prion conversion propensity. Therefore, we focused on the role of mutations at this amino acid of the mouse PrP (MoPrP), H95, to explore if this increase in prion conversion is unique to the H95Y mutation and better understand how mutations here affect conversion. In this original work, we perform an amino acid scan at H95, replacing the histidine with every other common amino acid and comparing the prion conversion propensity. The results are remarkable with the residues with hydrophobic side chains increasing prion conversion by about 150% and residues with electrically-charged side chains decreasing prion conversion by about 75%, both compared to WT PrP. We continued and provide the first robust data that mutations on the prion protein at residue 95 (specifically H95D, H95E, H95K and H95R) decrease the propensity for cellular PrP (PrPC) to misfold into scrapie PrP (PrPSc), with transient transfection of mutant proteins into ScN2a cells. We next biochemically characterize PrP H95E and PrP H95Y, the most promising mutant for reducing and increasing conversion, respectively, in N2aPrP−/− cells stably transfected with the mutant PrPs. We conclude that PrP H95E and PrP H95Y are biochemically similar to PrP WT. Also with these generated stable cell lines, we use immunofluorescence to study the localization and trafficking of the mutant PrPs relative to organelle markers. The only difference we observed was relative to the endosomal recycling compartments, with PrP H95E co-localizing less compared to PrP WT and PrP H95Y co-localizing more compared to PrP WT. This is in agreement with other work that links the endosomal recycling compartments with prion conversion. More work is required to understand what structural changes, if any, occur on the PrP H95E mutant and to understand if this plays a role in conversion, similar to the work carried out on the PrP H95Y mutant. At this point we conclude that the replacement of PrP H95 with electrically charged side chains decreases the prion conversion propensity, and after a detailed study of PrP H95E conclude that this is likely due to reduced co-localization with the endosomal recycling compartments, but we cannot rule out the influence of structural changes and would like to continue studies along this line.

Identifying molecular determinants of prion conversion / Pischke, Kate Elizabeth. - (2017 Oct 31).

Identifying molecular determinants of prion conversion

Pischke, Kate Elizabeth
2017

Abstract

Prion diseases have been widely studied, but despite many great leaps in our knowledge of prion and protein misfolding diseases in general, many gaps remain. One example is the structural triggers or provocators of prion conversion found within the prion protein (PrP) itself. While factors interacting on PrP can promote or dissuade conversion, it is also evident that inherent properties of differing PrP (presumably especially conformation) have an effect on the likelihood of prion conversion. Previous work from our lab found that the substitution of histidine 95 with a tyrosine increased prion conversion, while the same individual substitution at four other copper-binding histidines of PrP had no effect on prion conversion propensity. Therefore, we focused on the role of mutations at this amino acid of the mouse PrP (MoPrP), H95, to explore if this increase in prion conversion is unique to the H95Y mutation and better understand how mutations here affect conversion. In this original work, we perform an amino acid scan at H95, replacing the histidine with every other common amino acid and comparing the prion conversion propensity. The results are remarkable with the residues with hydrophobic side chains increasing prion conversion by about 150% and residues with electrically-charged side chains decreasing prion conversion by about 75%, both compared to WT PrP. We continued and provide the first robust data that mutations on the prion protein at residue 95 (specifically H95D, H95E, H95K and H95R) decrease the propensity for cellular PrP (PrPC) to misfold into scrapie PrP (PrPSc), with transient transfection of mutant proteins into ScN2a cells. We next biochemically characterize PrP H95E and PrP H95Y, the most promising mutant for reducing and increasing conversion, respectively, in N2aPrP−/− cells stably transfected with the mutant PrPs. We conclude that PrP H95E and PrP H95Y are biochemically similar to PrP WT. Also with these generated stable cell lines, we use immunofluorescence to study the localization and trafficking of the mutant PrPs relative to organelle markers. The only difference we observed was relative to the endosomal recycling compartments, with PrP H95E co-localizing less compared to PrP WT and PrP H95Y co-localizing more compared to PrP WT. This is in agreement with other work that links the endosomal recycling compartments with prion conversion. More work is required to understand what structural changes, if any, occur on the PrP H95E mutant and to understand if this plays a role in conversion, similar to the work carried out on the PrP H95Y mutant. At this point we conclude that the replacement of PrP H95 with electrically charged side chains decreases the prion conversion propensity, and after a detailed study of PrP H95E conclude that this is likely due to reduced co-localization with the endosomal recycling compartments, but we cannot rule out the influence of structural changes and would like to continue studies along this line.
Legname, Giuseppe
Pischke, Kate Elizabeth
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11767/60502
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