The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrPC) 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.
Introduction
Prion disorders are dominant gain-of-function neurodegenerations whose pathogenesis is linked to misfolded forms of the cellular prion protein (PrPC),
3
including the prion PrPSc and the neurotoxic CtmPrP (The abbreviations used are: PrP
prion protein, CtmPrP, transmembrane PrPC
Endo Hendo-β-N-acetylglucosaminidase H
ERendoplasmic reticulum
GPIglycosylphosphatidylinositol
PIPLCphosphatidylinositol phospholipase C
PKproteinase K
PNGase Fpeptide:N-glycosidase F
PrPCcellular PrP
PrPScaggregated and protease-resistant β-sheet-enriched conformer of PrPC
PrPDSPrP chain containing the combined M206S and M213S (M205S and M212S in mouse) substitution
PDIprotein disulfide isomerase
βCOPβ-coatomer protein.
1
, 2
, 3
, 4
). PrPSc is an aggregated and protease-resistant β-sheet-enriched conformer of PrPC, which self-perpetuates by the templating the conversion of cell surface PrPC (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
, 7
, 8
).Since the enunciation of the prion hypothesis, research has focused on the mechanism by which a native PrPC structure reorganizes and acquires self-propagative features like those of PrPSc (
9
, 10
, 11
). The PrPC 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 multiple transient protein-protein interactions with the nascent chains that can sense alterations resulting from environmental changes to the presence of mutations (17
, 18
, 19
). Any variation in the sequence of events can impact the final product, as for the doses of secretory and transmembrane PrP forms, and its fate (6
, 7
, 20
, 21
, 22
, 23
, - Drisaldi B.
- Stewart R.S.
- Adles C.
- Stewart L.R.
- Quaglio E.
- Biasini E.
- Fioriti L.
- Chiesa R.
- Harris D.A.
Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation.
J. Biol. Chem. 2003; 278: 21732-21743
24
, 25
, 26
, 27
, 28
, 29
, 30
, 31
, 32
, 33
).Metabolic studies addressing the effect of pathogenic mutations in the C-terminal domain of PrP as disease predisposition factors have reported a wide range of alterations in processing, trafficking, aggregation, accumulation, and toxicity which varied among experimental setups, as the cell line used and the background expression of wild-type (WT) PrPC (
20
, 21
, 23
, - Drisaldi B.
- Stewart R.S.
- Adles C.
- Stewart L.R.
- Quaglio E.
- Biasini E.
- Fioriti L.
- Chiesa R.
- Harris D.A.
Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation.
J. Biol. Chem. 2003; 278: 21732-21743
24
, 25
, 26
, 28
, 29
, 31
, 33
). These aberrancies contrast with structural reports in which the same pathogenic mutations do not impede the correct in vitro folding, but variably modify the stability, dynamics, and surface reactivity of the native state (12
, 13
, 14
, 15
, 16
, 34
, 35
). Indeed, aging factors such as oxidative modifications and exhaustion of the ER folding machinery which are not considered in structural studies may play fundamental roles in the formation of pathogenic PrP.Of the different mutations in the globular domain experimentally tested, substitutions of conserved methionines in α-helix 3 (hitherto PrPα3M) provoked the largest α-fold destabilization (
36
). In particular, singly or combined M206S and M213S replacements in rHaPrP(23–231) yielded extremely labile folds with enhanced aggregation capacity (36
). These mutations also mimicked the flexibility distortions impinged by sulfoxidation of such methionines found in the PrP chains in the conversion pathway (12
, 13
, 14
, 36
, 37
, 38
, 39
). Despite these interesting results, the effect of these substitutions had not been addressed in living systems.Here, we have used various cultured cells expressing PrPα3M mutants to investigate and model the role of in vivo folding in the synthesis and accumulation of PrP forms. Unexpectedly, we found that the PrPα3M expression is highly toxic and that such toxicity relates to the exclusive formation of CtmPrP due to impeded disulfide bond formation.
DISCUSSION
The efficient production of secreted PrPC starts with the co-translational translocation to the ER followed by a series of concerted processing including the cleavage of the N-terminal signal sequence, the addition of glycan chains at two facultative sites, the formation of an intramolecular disulfide bond, and a transamidation at the C terminus to add the GPI moiety (
1
). Of these post-translational modifications, glycosylation and disulfide bond formation depend on the cellular redox state (22
, 27
). Impairing the ER oxidative environment or mutating the cysteines yields intracellular, diglycosylated PrP chains lacking the disulfide bond, which resembles the PrPα3M mutants described here (22
, 27
, 30
, 32
). In this work we demonstrated the interplay between oxidative folding and the formation of the toxic CtmPrP. Introduction of polar substitutions at the α3 methionines, singly or combined, precluded the formation of the intramolecular disulfide bond and dictated the stabilization of highly toxic CtmPrP topologies that killed cells. Importantly, the absence of the disulfide bond in CtmPrP and its escape from the quality control checkpoints support the impairment of the oxidative folding as key pathogenic event for the of toxic PrP forms during normal aging.CtmPrP has been traditionally viewed as a transmembrane form whose translocation is governed by the hydrophobic region, resulting in a cytosolic N-terminal domain containing the signal sequence (
2
, 3
, 49
). However, recent experiments in cell cultures showed that CtmPrP lacked the N-terminal signal sequence, supporting a model of transbilayer post-translocation slippage as observed with recombinant PrP lacking disulfide bond and lipid vesicles (6
, 53
, 54
). Conditions favoring the slippage by trapping the hydrophobic region at the translocon as in A117V may either kinetically delay or impose a distance constraint impeding the formation of the disulfide bond. Also, mutations that may hinder oxidoreductase recognition such as α3M and Y216A may favor the insertion of the N-terminal domain as shown with recombinant chains lacking a disulfide bond (30
, 53
, 54
).Our results also suggest that conditions preventing PrP oxidative folding may favor the formation of CtmPrP and, consequently, promote its deleterious effects. Indeed, both maintenance of the levels and activity of ER oxidoreductases such as Grp58 and PdIa protect against the toxicity of misfolded PrP forms (
55
, 56
). On aging, both levels and activity of ER chaperones cause a decline of oxidative folding (- Watts J.C.
- Huo H.
- Bai Y.
- Ehsani S.
- Jeon A.H.
- Won A.H.
- Shi T.
- Daude N.
- Lau A.
- Young R.
- Xu L.
- Carlson G.A.
- Williams D.
- Westaway D.
- Schmitt-Ulms G.
Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones.
PLoS Pathog. 2009; 5: e1000608
57
). Because PrP3αM mutants were tailored to mimic the effects of Met sulfoxidation, conditions favoring such modification on nascent chains would also facilitate the formation of CtmPrP. In this line, ER stress exhaustion concurs with an overproduction of reactive oxygen species which are the major oxidants of Met residues, as those exposed in nascent unfolded chains (58
). Taken together, both decreased efficiency of the oxidative folding and increased probability of sulfoxidation of Met may then explain the aging-associated accumulation of CtmPrP forms in sporadic and inherited diseases.The role of disulfide bonds in PrP biology has mainly focused on their contribution to the generation of prions. The analysis of highly pure preparations of PrP27–30, the protease-resistant core of PrPSc, indicated that all Cys were forming disulfide bonds (
2
, 59
). But those samples lack CtmPrP forms due to the proteinase K digestion step used in the purification. The first evidence indirectly suggesting a pathogenic role for free thiols was provided by deletion mutants (60
). Mice expressing PrPΔ177-200 and PrPΔ201–217 that are unable to forms intramolecular disulfide bonds developed signs and lesions characteristic of neuronal storage disorders. In these mice, truncated PrP was detergent-insoluble in detergent, PK-sensitive, and with migration properties resembling those of PrPα3M mutants. Also, Cys mutants used in cellular studies resulted in PrP forms sharing key properties with PrPα3M mutants, but their topology and toxicity were not addressed (22
, 27
, 30
, 32
).The finding that free thiols lead to the formation of CtmPrP has several implications. From the structural point of view, the C-terminal domain has to expand its known conformational repertoire to accommodate the absence of the α2–α3 constraint and a double tether to the membrane (
51
, 52
, 61
). Also, the fibrillation of CtmPrP may be impeded by the diglycosylation (52
, 62
). But the detergent insolubility of CtmPrP suggests that it may populate distinct aggregate states, adding more structural complexity. Functionally, the aggregation of PrPα3M mutants suggests that CtmPrP may exert its toxic function through an oligomeric thiol trap. Such traps have been described in the regulation of IgM and adiponectin secretions (63
, 64
, 65
, 66
, 67
, 68
, 69
). Importantly, given the validation of the PrP chains by the ER quality control systems the assembly of such oligomeric traps must take place upon delivery to a different environment (20
). 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 PrPC into PrPSc, 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.Acknowledgments
We thank Rosa Sánchez for technical assistance and Silvia Zorrilla for advice.
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Article Info
Publication History
Published online: October 26, 2012
Received in revised form:
September 6,
2012
Received:
July 6,
2012
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© 2012 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
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- Withdrawal: Failure of prion protein oxidative folding guides the formation of toxic transmembrane formsJournal of Biological ChemistryVol. 292Issue 49Open Access
