Assembly of natural and recombinant prion protein into fibrils

2005 ◽  
Vol 386 (6) ◽  
pp. 569-580 ◽  
Author(s):  
Karl-Werner Leffers ◽  
Holger Wille ◽  
Jan Stöhr ◽  
Erika Junger ◽  
Stanley B. Prusiner ◽  
...  
Keyword(s):  
Prion Protein ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Fibril Formation ◽  
Full Length ◽  
Low Concentrations ◽  
Recombinant Prion Protein ◽  
Β Sheet

AbstractThe conversion of the α-helical, cellular isoform of the prion protein (PrPC) to the insoluble, β-sheet-rich, infectious, disease-causing isoform (PrPSc) is the fundamental event in the prion diseases. The C-terminal fragment of PrPSc(PrP 27–30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrPSc, PrP 27–30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrPScfrom PrPCand the assembly of rods from PrP 27–30, we solubilized Syrian hamster (sol SHa) PrP 27–30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27–30 adopted a β-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27–30 in a soluble, β-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27–30, but also with native SHaPrPC. Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90–231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27–30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27–30 or PrPCwere bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89–230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.

scholarly journals In vitro conversion and seeded fibrillization of posttranslationally modified prion protein

2011 ◽  
Vol 392 (5) ◽  
Author(s):  
Jan Stöhr ◽  
Kerstin Elfrink ◽  
Nicole Weinmann ◽  
Holger Wille ◽  
Dieter Willbold ◽  
...  
Keyword(s):  
Prion Protein ◽  
Posttranslational Modifications ◽  
Prion Diseases ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Full Length ◽  
Proteinase K

AbstractThe conversion of the cellular isoform of the prion protein (PrPC) into the pathologic isoform (PrPSc) is the key event in prion diseases. To study the conversion process, anin vitrosystem based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrPCisolated from Chinese hamster ovary cells (CHO-PrPC) was examined. CHO-PrPCharbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrPCwere compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrPCinto an aggregated, β-sheet-rich PrPSc-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrPSc. Compared to recPrP (90-231), fibril formation with CHO-PrPCrequires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrPScpurified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. Thein vivosituation can be simulated closer with CHO-PrPCbecause the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.

scholarly journals Amyloid fibrils from the N-terminal prion protein fragment are infectious

2016 ◽  
Vol 113 (48) ◽  
pp. 13851-13856 ◽  
Author(s):  
Jin-Kyu Choi ◽  
Ignazio Cali ◽  
Krystyna Surewicz ◽  
Qingzhong Kong ◽  
Pierluigi Gambetti ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Disease ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Strong Support ◽  
Proteinase K ◽  
Molecular Architecture ◽  
Core Mapping ◽  
Scrapie Strains ◽  
Β Sheet

Recombinant C-terminally truncated prion protein PrP23-144 (which corresponds to the Y145Stop PrP variant associated with a Gerstmann–Sträussler–Scheinker-like prion disease) spontaneously forms amyloid fibrils with a parallel in-register β-sheet architecture and β-sheet core mapping to residues ∼112–139. Here we report that mice (bothtga20and wild type) inoculated with a murine (moPrP23-144) version of these fibrils develop clinical prion disease with a 100% attack rate. Remarkably, even though fibrils in the inoculum lack the entire C-terminal domain of PrP, brains of clinically sick mice accumulate longer proteinase K-resistant (PrPres) fragments of ∼17–32 kDa, similar to those observed in classical scrapie strains. Shorter, Gerstmann–Sträussler–Scheinker-like PrPresfragments are also present. The evidence that moPrP23-144 amyloid fibrils generated in the absence of any cofactors are bona fide prions provides a strong support for the protein-only hypothesis of prion diseases in its pure form, arguing against the notion that nonproteinaceous cofactors are obligatory structural components of all infectious prions. Furthermore, our finding that a relatively short β-sheet core of PrP23-144 fibrils (residues ∼112–139) with a parallel in-register organization of β-strands is capable of seeding the conversion of full-length prion protein to the infectious form has important implications for the ongoing debate regarding structural aspects of prion protein conversion and molecular architecture of mammalian prions.

An Engineered PrPsc-like Molecule from the Chimera of Mammalian Prion Protein and Yeast Ure2p Prion-inducing Domain

2004 ◽  
Vol 36 (2) ◽  
pp. 128-132
Author(s):  
Shao-Man Yin ◽  
Man-Sun Sy ◽  
Po Tien
Keyword(s):  
Prion Protein ◽  
Amyloid Fibrils ◽  
Physiological Condition ◽  
Prion Diseases ◽  
Cd Spectroscopy ◽  
Conversion Process ◽  
Fibrillar Morphology ◽  
Thioflavine T ◽  
Conformational Conversion ◽  
Β Sheet

Abstract Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful for understanding the mysterious mechanism of conformational conversion process of prion diseases and proving the “protein-only” hypothesis. In this report, an engineered PrPsc-like conformation was produced from a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD). Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD, spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-like characteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increased fluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopy experiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high content β-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit more soluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrPsc and will be helpful for further understanding the mechanism of conformational conversion process.

scholarly journals High-Pressure Response of Amyloid Folds

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 202 ◽  
Author(s):  
Joan Torrent ◽  
Davy Martin ◽  
Angélique Igel-Egalon ◽  
Vincent Béringue ◽  
Human Rezaei
Keyword(s):  
High Pressure ◽  
Prion Protein ◽  
Amyloid Fibrils ◽  
Quaternary Structure ◽  
Prion Diseases ◽  
Structural Diversity ◽  
Structural Form ◽  
Volumetric Features ◽  
Protein Fibril ◽  
Recombinant Prion Protein

The abnormal protein aggregates in progressive neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and prion diseases, adopt a generic structural form called amyloid fibrils. The precise amyloid fold can differ between patients and these differences are related to distinct neuropathological phenotypes of the diseases. A key focus in current research is the molecular mechanism governing such structural diversity, known as amyloid polymorphism. In this review, we focus on our recent work on recombinant prion protein (recPrP) and the use of pressure as a variable for perturbing protein structure. We suggest that the amyloid polymorphism is based on volumetric features. Accordingly, pressure is the thermodynamic parameter that fits best to exploit volume differences within the states of a chemical reaction, since it shifts the equilibrium constant to the state that has the smaller volume. In this context, there are analogies with the process of correct protein folding, the high pressure-induced effects of which have been studied for more than a century and which provides a valuable source of inspiration. We present a short overview of this background and review our recent results regarding the folding, misfolding, and aggregation-disaggregation of recPrP under pressure. We present preliminary experiments aimed at identifying how prion protein fibril diversity is related to the quaternary structure by using pressure and varying protein sequences. Finally, we consider outstanding questions and testable mechanistic hypotheses regarding the multiplicity of states in the amyloid fold.

scholarly journals Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

2021 ◽  
Vol 22 (10) ◽  
pp. 5075
Author(s):  
Mantas Ziaunys ◽  
Andrius Sakalauskas ◽  
Kamile Mikalauskaite ◽  
Ruta Snieckute ◽  
Vytautas Smirnovas
Keyword(s):  
Protein Aggregation ◽  
Prion Protein ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Morphological Properties ◽  
Large Sample Size ◽  
Structural Variations ◽  
Temperature Dependent ◽  
Protein Fibrils ◽  
Propagation Properties

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.

scholarly journals Altered toxicity of the prion protein peptide PrP106–126 carrying the Ala117→Val mutation

2000 ◽  
Vol 346 (3) ◽  
pp. 785-791 ◽  
Author(s):  
David R. BROWN
Keyword(s):  
Point Mutation ◽  
Prion Protein ◽  
Prion Diseases ◽  
Point Mutations ◽  
Human Sequence ◽  
Gene Coding ◽  
Normal Human ◽  
Microtubule Formation ◽  
Β Sheet ◽  
Prion Protein Peptide

The inherited prion diseases such as Gerstmann-Sträussler-Scheinker syndrome (GSS) are linked to point mutations in the gene coding for the cellular isoform of the prion protein (PrPC). One particular point mutation A117V (Ala117 → Val) is linked to a variable pathology that usually includes deposition of neurofibrillary tangles. A prion protein peptide carrying this point mutation [PrP106-126(117V)] was generated and compared with a peptide based on the normal human sequence [PrP106-126(117A)]. The inclusion of this point mutation increased the toxicity of PrP106-126 which could be linked to an increased β-sheet content. An assay of microtubule formation in the presence of tau indicated that PrP106-126 decreased the rate of microtubule formation that could be related to the displacement of tau. PrP106-126 carrying the 117 mutation was more efficient at inhibiting microtubule formation. These results suggest a possible mechanism of toxicity for protein carrying this mutation via destabilization of the cytoskeleton and deposition of tau in filaments, as observed in GSS.

scholarly journals Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation

2004 ◽  
Vol 377 (3) ◽  
pp. 709-716 ◽  
Author(s):  
Emma T. A. S. JAIKARAN ◽  
Melanie R. NILSSON ◽  
Anne CLARK
Keyword(s):  
Type 2 Diabetes ◽  
Amyloid Fibrils ◽  
Secretory Granules ◽  
Pancreatic Beta Cell ◽  
Islet Amyloid Polypeptide ◽  
Fibril Formation ◽  
Islet Amyloid ◽  
Β Sheet

Islet amyloid polypeptide (IAPP), or ‘amylin’, is co-stored with insulin in secretory granules of pancreatic islet β-cells. In Type 2 diabetes, IAPP converts into a β-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop β-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.

scholarly journals Assessment of the PrP c Amino-Terminal Domain in Prion Species Barriers

2016 ◽  
Vol 90 (23) ◽  
pp. 10752-10761 ◽  
Author(s):  
Kristen A. Davenport ◽  
Davin M. Henderson ◽  
Candace K. Mathiason ◽  
Edward A. Hoover
Keyword(s):  
Prion Protein ◽  
Bank Vole ◽  
Protein Misfolding ◽  
Prion Diseases ◽  
Full Length ◽  
Spongiform Encephalopathy ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Human Sequence ◽  
Amino Terminal Domain

ABSTRACT Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but they appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrP c , to a pathogenic, amyloid isoform, scrapie prion protein, PrP Sc . We examined the role of the PrP c amino-terminal domain (N-terminal domain [NTD], amino acids [aa] 23 to 90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa 23 to 231) or truncated (aa 90 to 231) PrP c . We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrP c , but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrP c s were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrP c . The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrP c to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrP c molecule. IMPORTANCE We demonstrate that the amino-terminal domain of the normal prion protein, PrP c , hinders seeded conversion of bovine and white-tailed deer PrP c s to the prion forms, but it facilitates conversion of the human and bank vole PrP c s to the prion forms. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrP c s requires interaction with the rest of the molecule to facilitate conversion by CWD prions. These data suggest that interactions of the amino-terminal domain with the rest of the PrP c molecule play an important role in the susceptibility of humans to CWD prions.

scholarly journals Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

2020 ◽  
Vol 295 (21) ◽  
pp. 7544-7553 ◽  
Author(s):  
Dexter N. Dean ◽  
Jennifer C. Lee
Keyword(s):  
Alternative Splicing ◽  
Amyloid Fibrils ◽  
Limited Proteolysis ◽  
Amyloid Formation ◽  
Functional Amyloid ◽  
Melanin Biosynthesis ◽  
Functional Amyloids ◽  
Fibril Morphology ◽  
Amyloid Assembly ◽  
Β Sheet

The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar β-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a “nucleator” of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

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