<i>In Vitro</i> Amplification of Protease-Resistant Prion Protein Requires Free Sulfhydryl Groups

Lucassen, Ralf; Nishina, Koren; Supattapone, Surachai

doi:10.1021/bi027218d

Cited by 87 publications

(76 citation statements)

References 34 publications

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“…Whether other components participate in their assembly and/or stabilization and whether these factors display different affinities for the WT or mutant chains remains to be elucidated (6). Additionally, this unknown lipid-bound conformation of thiol-free PrP could indeed function as the seed for the conversion of PrP C into PrP Sc , even in traces amounts (70,71). This could provide a mechanistic explanation for the spontaneous generation of pathogenic forms of PrP in sporadic human diseases.…”

Section: Discussionmentioning

confidence: 99%

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

Lisa

Domingo

Martínez

et al. 2012

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

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

Lisa

Domingo

Martínez

et al. 2012

Journal of Biological Chemistry

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“…Sonication then provides additional seeds for further 59 aggregate growth by fragmentation of these aggregates. Amplification has also been observed 60 without sonication (Saborio et al, 2001;Lucassen et al, 2003). However, we were able to 61…”

Section: Introduction 37mentioning

confidence: 57%

Generation of genuine prion infectivity by serial PMCA

Weber

Giese

Piening

et al. 2007

Veterinary Microbiology

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15Prions are the causative infectious agents of transmissible spongiform 16 encephalopathies (TSEs). They are thought to arise from misfolding and aggregation of the 17 prion protein (PrP). In serial transmission protein misfolding cyclic amplification (sPMCA) 18 experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the 19 structural conversion of cellular prion protein (PrP C ) as efficiently as PrP Sc from the brain of 20 scrapie-infected (263K) hamsters confirming an autocatalytic misfolding cascade as 21 postulated by the prion hypothesis. However, the fact that PrPres generated in vitro was 22 associated with approximately ten times less infectivity than an equivalent quantity of brain-23 derived PrPSc casts doubt on the "protein-only" hypothesis of prion propagation and backs 24 theories that suggest there are additional molecular species of infectious PrP or other agent-25 associated factors. By combining sPMCA with prion delivery on suitable carrier particles we 26 were able to resolve the apparent discrepancy between the amount of PrPres and infectivity 27 which we were then able to relate to differences in the size distribution of PrP aggregates and 28 consecutive differences in regard to biological clearance. These findings demonstrate that we 29 have designed an experimental set-up yielding in vitro generated prions that are 30 indistinguishable from prions isolated from scrapie-infected hamster brain in terms of 31 proteinase K resistance, autocatalytic conversion activity, and -most notably -specific 32 biological infectivity. 33 34

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“…These data suggest that factors affecting the stability of the disulfide bridge could have an impact on prion replication. Additional support for this hypothesis comes from experiments showing that in vitro amplification of PrP SC is dependent on the presence of free sulfhydryl groups (Lucassen et al, 2003). Finally, reshuffling of cysteine bridges from intramolecular to intermolecular forms has been proposed to play a role on PrP conversion and on the stabilization of the misfolded protein aggregates (Tompa et al, 2002).…”

Section: Discussionmentioning

confidence: 98%

The Disulfide Isomerase Grp58 Is a Protective Factor against Prion Neurotoxicity

Hetz¹,

Russelakis-Carneiro²,

Wälchli³

et al. 2005

J. Neurosci.

189

176

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Prion diseases are transmissible neurodegenerative disorders characterized by extensive neuronal apoptosis and accumulation of misfolded prion protein (PrP SC). Recent reports indicate that PrP SC induces neuronal apoptosis via activation of the endoplasmic reticulum (ER) stress pathway and activation of the ER resident caspase-12. Here, we investigate the relationship between prion replication and induction of ER stress during different stages of the disease in a murine scrapie model. The first alteration observed consists of the upregulation of the ER chaperone of the glucose-regulated protein Grp58, which was detected during the presymptomatic phase and followed closely the formation of PrP SC . An increase in Grp58 expression correlated with PrP SC accumulation at all stages of the disease in different brain areas, suggesting that this chaperone may play an important role in the cellular response to prion infection. Indeed, in vitro studies using N2a neuroblastoma cells demonstrated that inhibition of Grp58 expression with small interfering RNA led to a significant enhancement of PrP SC toxicity. Conversely, overexpression of Grp58 protected cells against PrP SC toxicity and decreased the rate of caspase-12 activation. Grp58 and PrP were shown to interact by coimmunoprecipitation, observing a higher interaction in cells infected with scrapie prions. Our data indicate that expression of Grp58 is an early cellular response to prion replication, acting as a neuroprotective factor against prion neurotoxicity. Our findings suggest that targeting Grp58 interaction may have applications for developing novel strategies for treatment and early diagnosis of prion diseases.

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In Vitro Amplification of Protease-Resistant Prion Protein Requires Free Sulfhydryl Groups

Cited by 87 publications

References 34 publications

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

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

Generation of genuine prion infectivity by serial PMCA

The Disulfide Isomerase Grp58 Is a Protective Factor against Prion Neurotoxicity

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