Rapid ex vivo reverse genetics identifies the essential determinants of prion protein toxicity.

The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208 -H140 hydrogen bond ("H-latch") between the α2-α3 and ß2-α2 loops of PrPC . Importantly, GDL that suppresses the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced 19 individual PrPC variants to PrPC -deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27 ) within PrPC . Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-type PrPC . K27 may represent an actionable target for compounds aimed at preventing prion-related neurodegeneration.

Relationships of ligand binding, redox properties, and protonation in Coprinus cinereus peroxidase.

The pH dependence of the redox potentials and kinetics for CO association and dissociation was determined between pH 3.0 and 13.0 at 25 degrees C for the wild-type Coprinus cinereus fungal peroxidase and for a site-directed mutant in which Asp245, which is H-bonded to N delta of the imidazole of the proximal His183, was substituted with Asn. The determination of these functional properties allowed this information to be merged in a self-consistent fashion and to formulate for the first time a complete scheme employing the minimum number of groups required to describe the whole proton-linked behavior of both redox and ligand binding properties. The overall pH dependence can be accounted for by four redox- and ligand-linked groups. The proximal H-bond, which is strictly conserved in all peroxidases, will still be present in the site-specific mutant, but will no longer have an ionic character, and this event will bring about an alteration of redox equilibria and CO binding kinetics, envisaging a relevant role played by this H-bond also in modulating redox properties and ligand binding equilibria.