Probing amyloid fibril secondary structures by infrared nanospectroscopy: experimental and theoretical considerations.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillary structure with a higher ß-sheet content than their native structure. Attenuated total reflection Fourier transform infrared spectroscopy only provides bulk analysis of a sample therefore it is impossible to discriminate between different aggregated structures. To overcome this limitation, near-field techniques like AFM-IR have emerged in the last twenty years to allow infrared nanospectroscopy. This technique obtains IR spectra with a spatial resolution of ten nanometres, the size of isolated fibrils. Here, we present essential practical considerations to avoid misinterpretations and artefacts during these analyses. Effects of polarization of the incident IR laser, illumination configuration and coating of the AFM probes are discussed, including the advantages and drawbacks of their use. This approach will improve interpretation of AFM-IR spectra especially for the determination of secondary structures of species not accessible using classical ATR-FTIR.

A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP.

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of ß-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

Symmetry-breaking transitions in the early steps of protein self-assembly.

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly ß-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aß(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

Prion strain-dependent tropism is maintained between spleen and granuloma and relies on lymphofollicular structures.

In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.

Membrane interaction of off-pathway prion oligomers and lipid-induced on-pathway intermediates during prion conversion: A clue for neurotoxicity.

Soluble oligomers of prion proteins (PrP), produced during amyloid aggregation, have emerged as the primary neurotoxic species, instead of the fibrillar end-products, in transmissible spongiform encephalopathies. However, whether the membrane is among their direct targets, that mediate the downstream adverse effects, remains a question of debate. Recently, questions arise from the formation of membrane-active oligomeric species generated during the ß-aggregation pathway, either in solution, or in lipid environment. In the present study, we characterized membrane interaction of off-pathway oligomers from recombinant prion protein generated along the amyloid aggregation and compared to lipid-induced intermediates produced during lipid-accelerated fibrillation. Using calcein-leakage assay, we show that the soluble prion oligomers are the most potent in producing leakage with negatively charged vesicles. Binding affinities, conformational states, mode of action of the different PrP assemblies were determined by thioflavin T binding-static light scattering experiments on DOPC/DOPS vesicles, as well as by FTIR-ATR spectroscopy and specular neutron reflectivity onto the corresponding supported lipid bilayers. Our results indicate that the off-pathway PrP oligomers interact with lipid membrane via a distinct mechanism, compared to the inserted lipid-induced intermediates. Thus, separate neurotoxic mechanisms could exist following the puzzling intermediates generated in the different cell compartments. These results not only reveal an important regulation of lipid membrane on PrP behavior but may also provide clues for designing stage-specific and prion-targeted therapy.

Generating Bona Fide Mammalian Prions with Internal Deletions.

UNLABELLED: Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. IMPORTANCE: Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

Information content in data sets for a nucleated-polymerization model.

We illustrate the use of statistical tools (asymptotic theories of standard error quantification using appropriate statistical models, bootstrapping, and model comparison techniques) in addition to sensitivity analysis that may be employed to determine the information content in data sets. We do this in the context of recent models [S. Prigent, A. Ballesta, F. Charles, N. Lenuzza, P. Gabriel, L.M. Tine, H. Rezaei, and M. Doumic, An efficient kinetic model for assemblies of amyloid fibrils and its application to polyglutamine aggregation, PLoS ONE 7 (2012), e43273. doi:10.1371/journal.pone.0043273.] for nucleated polymerization in proteins, about which very little is known regarding the underlying mechanisms; thus, the methodology we develop here may be of great help to experimentalists. We conclude that the investigated data sets will support with reasonable levels of uncertainty only the estimation of the parameters related to the early steps of the aggregation process.

The role of interruptions in polyQ in the pathology of SCA1.

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.

An efficient kinetic model for assemblies of amyloid fibrils and its application to polyglutamine aggregation.

Protein polymerization consists in the aggregation of single monomers into polymers that may fragment. Fibrils assembly is a key process in amyloid diseases. Up to now, protein aggregation was commonly mathematically simulated by a polymer size-structured ordinary differential equations (ODE) system, which is infinite by definition and therefore leads to high computational costs. Moreover, this Ordinary Differential Equation-based modeling approach implies biological assumptions that may be difficult to justify in the general case. For example, whereas several ordinary differential equation models use the assumption that polymerization would occur at a constant rate independently of polymer size, it cannot be applied to certain protein aggregation mechanisms. Here, we propose a novel and efficient analytical method, capable of modelling and simulating amyloid aggregation processes. This alternative approach consists of an integro-Partial Differential Equation (PDE) model of coalescence-fragmentation type that was mathematically derived from the infinite differential system by asymptotic analysis. To illustrate the efficiency of our approach, we applied it to aggregation experiments on polyglutamine polymers that are involved in Huntington's disease. Our model demonstrates the existence of a monomeric structural intermediate [Formula: see text] acting as a nucleus and deriving from a non polymerizing monomer ([Formula: see text]). Furthermore, we compared our model to previously published works carried out in different contexts and proved its accuracy to describe other amyloid aggregation processes.

Mechanistic insights into cellular alteration of prion by poly-D-lysine: the role of H2H3 domain.

Misfolding of the prion protein (PrP) is the central feature of prion diseases. The conversion of the normal α-helical PrP(C) into a pathological ß-enriched PrP(Sc) constitutes an early event in the infectious process. Several hypotheses, involving different regions of the protein, endeavor to delineate the structural mechanism underlying this change of conformation. All current working hypotheses, however, are based on biophysical and modeling studies, the biological relevance of which still needs to be assessed. We have studied the effect of positively charged polymers on the conversion, using polylysine as a model system, and have investigated a possible mechanism of structural stabilization. We have shown that poly-D-lysine removes proteinase K-resistant PrP from prion-infected SN56 neuroblastoma cells without affecting PrP(C). The effect is enantiospecific since the levorotary isomer, poly-L-lysine, has a markedly weaker effect, likely because of its higher susceptibility to degradation. In vitro cross-linking and NMR studies confirm a direct interaction between polylysine and PrP, which mainly maps to the PrP region containing helices 2 and 3 (H2H3). Interaction prevents conformational conversion and protein aggregation. Our results establish a central role of H2H3 in PrP(Sc) amyloidogenesis and replication and provide biological relevance for the pathological misfolding of this domain.

NMR structure of a viral peptide inserted in artificial membranes: a view on the early steps of the birnavirus entry process.

Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5-8 and 2-4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by (1)H NMR studies provide structural insights of the pep46 domain interaction. In CDCl(3)/CD(3)OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.

In vitro and in vivo neurotoxicity of prion protein oligomers.

The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.

Sequential generation of two structurally distinct ovine prion protein soluble oligomers displaying different biochemical reactivities.

In pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. In prion diseases the lethal evolution is associated with formation of PrP(Sc), a misfolded and amyloid form of the normal cellular prion protein PrP. Although several molecular mechanisms were proposed to account for the propagation of the infectious agent, the events responsible for cell death are still unclear. The correlation between PrP(C) expression level and the rate of disease evolution on one side, and the fact that PrP(Sc) deposition in brain did not strictly correlate with the apparition of clinical symptoms on the other side, suggested a potential role for diffusible oligomers in neuronal death. To get better insight into the molecular mechanisms of PrP(C) oligomerization, we studied the heat-induced oligomerization pathway of the full-length recombinant ovine PrP at acidic pH. This led to the irreversible formation of two well-identified soluble oligomers that could be recovered by size-exclusion chromatography. Both oligomers displayed higher beta-sheet content when compared to the monomer. A sequential two-step multimolecular process accounted for the rate of their formation and their ratio partition, both depending on the initial protein concentration. Small-angle X-ray scattering allowed the determination of the molecular masses for each oligomer, 12mer and 36mer, as well as their distinct oblate shapes. The two species differed in accessibility of polypeptide chain epitopes and of pepsin-sensitive bonds, in a way suggesting distinct conformations for their monomeric unit. The conversion pathway leading to these novel oligomers, displaying contrasted biochemical reactivities, might be a clue to unravel their biological roles.

Heme as an optical probe of a conformational transition of ovine recPrP.

The prion protein occurs as a globular domain and a leading fragment whose structure is not well-defined. For the ovine species, all of the tryptophan residues are in the initial fragment, while the globular domain is rich in tyrosine residues. Using heme as a spectroscopic probe, we have studied the recombinant prion protein before and after a temperature-induced conformational change. As for most heme proteins, the absorption spectrum of heme-CO displays a red shift upon binding to the protein, and both the Y and W fluorescence are highly quenched. Flash photolysis kinetics of the PrP-heme-CO complex shows a low yield for the bimolecular phase, indicating a pocket around the hemes. By comparing the holoprotein and the truncated sequence corresponding to the globular domain, the stoichiometry was determined to be five hemes for the globular domain and two hemes for the leading fragment. At high temperature, the hemes are released; upon cooling, only two hemes bind, and only the tryptophan fluorescence is quenched; this would indicate that the globular domain has formed a more compact structure, which is inert with respect to the hydrophobic probe. The final state of polymerization is perturbed if the synthetic peptide "N3" (PrP residues 142-166, which include the first helix) is added to the prion protein solution; the temperature cycle no longer reduces the number of heme binding sites. This would indicate that the peptide may alter or inhibit the polymer formation.