Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

Abstract

Background:In vivo folding could play an essential role in prion neurodegenerations. Results: Artificial mutants causing labile PrP folds when expressed in cells originate toxic CtmPrP featured by the absence of the intramolecular disulfide bond. Conclusion: Oxidative folding impairment facilitates the formation of the toxic PrP forms. Significance: Unveiling the mechanism facilitating the formation of toxic PrP forms is crucial for the understanding and prevention of prion disorders.The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP C ) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.Prion disorders are dominant gain-of-function neurodegenerations whose pathogenesis is linked to misfolded forms of the cellular prion protein (PrP C ), 3 including the prion PrP Sc and the neurotoxic CtmPrP (1-4). PrPSc is an aggregated and proteaseresistant ␤-sheet-enriched conformer of PrP C , which self-perpetuates by the templating the conversion of cell surface PrP C (1, 4). In contrast, CtmPrP is an intracellular transmembrane form generated at the ER with neurotoxic properties (1,5,6). Despite that CtmPrP formation was associated with features of the ER translocation process several pathogenic mutations in the C-terminal domain such as H187R and E200K enhance its levels, suggesting a yet unexplored in vivo interplay between folding and the accumulation and action of this neurotoxic form (6 -8).Since the enunciation of the prion hypothesis, research has focused on the mechanism by which a native PrP C structure reorganizes and acquires self-propagative features like those of PrP Sc (9 -11). The PrP C native state was assigned to the fold adopted by the chain lacking the signal sequences and containing the disulfide bond and used as reference for testing the effect of pathogenic mutations and its conversion into active prions (10,(12)(13)(14)(15)(16). However, the in vivo folding of proteins segregating into the secretory route such as PrP is a complex process participated by the ER folding machinery. This machinery coordinates processing (signal sequences removal, addition of covalent modifications, binding of cofactors, etc.), avoids undesired aggregations, and permits the acquisition of correct structure. This global process involves m...

An Involvement of Oxidative Stress in Endoplasmic Reticulum Stress and Its Associated Diseases

“…Alterations in the disulfide bond formation or mispairing of cysteine residues results in protein misfolding or inability of the protein to attain its proper configuration [16]. Several reports have described the role of GSH in preventing non-native disulfide bond formation or the generation of misfolded proteins.…”

PrP charge structure encodes interdomain interactions

“…Mutants were generated using pcDNA4.1-HaPrP(1–254) as a template ( Table 1S ) 80 . Transient transfections were performed using Chinese hamster ovary (CHO) cells and Fugene 6 as a transfection reagent (Roche), as described 80 .…”

“…Mutants were generated using pcDNA4.1-HaPrP(1–254) as a template ( Table 1S ) 80 . Transient transfections were performed using Chinese hamster ovary (CHO) cells and Fugene 6 as a transfection reagent (Roche), as described 80 . After 40 h, the cells were harvested via in situ lysis in a cold RIPA buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate).…”

“…Samples were then centrifuged at 10000 × g for 30 min, and the pellets were re-dissolved in TNE buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA). Enzymatic digestions with PNGaseF (New England Biolabs) were performed for 1 h at 37 °C 80 . After digestion, the reactions were stopped by the addition of Laemmli buffer and PrP analyzed via immunoblotting using the POM17 antibody.…”

Protective effects of Danshen injection against erectile dysfunction via suppression of endoplasmic reticulum stress activation in a streptozotocin-induced diabetic rat model

“…It is well known that redox homeostasis has a critical role in protein synthesis and folding. The oxidizing environment in the ER lumen facilitates the formation of non-native disulfide bonds, which can lead to protein misfolding or protein inactivation [11]. Therefore, hyperglycemia-induced oxidative stress or an oxidizing environment may trigger ER stress, which was demonstrated in our previous study [12].…”