”Approach to study the interactome of the C-terminal domain of PrPC”

Introduction: Prion protein (PrPC) is a highly conserved glycoprotein, which can convert into a pathological isoform (PrPSc) causing TSEs. Although the mechanism conversion is not yet fully understood, it is known that α2 and α3 helices are especially sensitive to mutations associated with genetic prion diseases, suggesting that they may facilitate pathogenic conversion. Since most to the studies has been focused on the structural conversion propensities of H2-H3 PrP mutants, here we investigate H2-H3 interactors to further understand their role on PrP conversion.

Objective: We propose to analyze the interactions between WT PrPC and PrPC with deletions in H2-H3 domain with proteins expressed in the SNC.

Methods: The following PrP mutants were obtained by site directed mutagenesis: ∆183-185, ∆189-192, ∆198-200 and ∆205-207. We performed purification of PrP and PrP harbouring H2-H3 deletions, analyzing proper folding by in silico studies and Circular Dichroism (CD). A comparative analysis of the molecular interactions of WT PrPC and PrP deletant mutants is being performed using coimmunoprecipitation and WB.

Results and Discussion: Structural integrity of PrP WT and PrP (todas) was assessed by in silico molecular dynamics. CD also showed no alteration in WT and PrP_∆189-192 deletant mutant. Preliminary data showed APP, Syntaxin and P2X2 co-immunoprecipitating with WT PrP. This work opens a promising avenue for exploring new functions of PrP and profiling its interactions in critical regions for its conversion, to further elucidate the structural conversion mechanism into the pathological form.