Cell-free formation of protease-resistant prion protein

Kocisko, David A.; Come, Jon H.; Priola, Suzette A.; Chesebro, Bruce; Raymond, Gregory J.; Lansbury, Peter T.; Caughey, Byron

doi:10.1038/370471a0

Cited by 814 publications

(593 citation statements)

References 26 publications

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“…Conversion efficacy is monitored by apparition of labeled, protease-resistant PrP Sc after 1-3 days incubation at 37˚C [50,111]. In a recent work, such a converting activity was reported to be dependent upon the size of PrP Sc aggregates used as seeds [182].…”

Section: Cell-free Systemsmentioning

confidence: 99%

Prion agent diversity and species barrier

2008

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-Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP Sc , a misfolded, aggregated form of the host-encoded cellular prion protein (PrP C ) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP Sc and neuropathology. Prion strains are thought to be enciphered within distinct PrP Sc conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.prion / strain / misfolding / species barrier / PrP Table of Mammalian prion diseases or transmissible spongiform encephalopathies (TSE) form a group of related, invariably fatal neurodegenerative disorders of both animals and humans. The brain pathology consists of spongiosis, astrocytosis, neuronal loss and neural tissue from affected individuals contains an infectious agent, the prion, setting these diseases apart from other neurodegenerative diseases. Prion replication is thought to involve in essence the self-perpetuating conversion of the host-encoded cellular prion protein (PrP C ) into a misfolded form (PrP Sc ) that tends to aggregate and may be neurotoxic (for reviews [1,157]). Prion replication may also occur in lymphoid tissues but is there poorly if not pathogenic (for review [126], [133]). PrP C is a protein with two variably occupied glycosylation sites. It is attached at the outer of the plasma membrane by a glycosylphosphatidylinositol anchor. Its secondary structure is rich in alpha-helix and the protein is likely to be in a monomeric state in mild detergents. While its precise physiological function has not been assigned, PrP C is essential for prion replication and neurotoxicity to occur [57,129]. Upon infection, PrP C is refolded -without apparent post-translational modification -into beta-sheet -rich PrP Sc , initially in the presence of exogenous PrP Sc and then by an autocatalytic process. It leads to the formation of aggregates, sometimes of amyloid type, that can be differentiated from PrP C because of their partial resistance to protease digestion and of their insolubility into non-denaturing detergents [153]. Proteinase K, the most commonly used protease, di...

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Section: Cell-free Systemsmentioning

confidence: 99%

Prion agent diversity and species barrier

2008

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“…Whatever the nature of the conversion process, an important experimental advance [62] has been the demonstration that incubation of 35S-labeled biosynthetic hamster PrP c with about 50 fold excess of PrP sc from scrapie-infected hamster brain resulted in the conversion of some labeled protein into the partly proteinase K-resistant state characteristic for hamster PrP so. To achieve this conversion, 35S-labeled PrP c and PrP sc were partly denatured with 3M guanidinium chloride prior to incubation in a lower, critical concentration of the denaturant.…”

Section: Conversion Of Prp C To Prp Scmentioning

confidence: 99%

Molecular biology of transmissible spongiform encephalopathies

1996

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The prion, the transmissible agent that causes spongiform encephalopathies such as serapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and identical with PrP so, a modified form of the normal host protein PrP ¢ which is encoded by the single cop~v gene Prnp. The 'protein only" hypothesis proposes that PrP ~c, when introduced into a normal host, causes the conversion of PrP c into prpS~; it therefore predicts that an animal devoid of PrP c should be resistant to prion diseases. We generated homozygous Prnp °1° ('PrP knockout') mice and showed that, after inoculation with prions, they remained free of scrapie for at least 2 years while wild-type controls all died within 6 months. There was no propagation of prions in the Prnp °t° animals. Surprisingly, heterozygous Prnp °t+ mice, which express PrP c at about half the normal level, also showed enhanced resistance to scrapie disease despite high levels of infectious agent and PrP ~c in the brain early on. After introduction of murine PrP transgenes Prnp °t° mice became highly susceptible to mouse but not to hamster prions, while the insertion of Syrian hamster PrP transgenes rendered them susceptible to hamster but to a much lesser extent to mouse prions. These complementation experiments paved the way to the application of reverse genetics. We have prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and have found that PrP lacking 48 amino proximal amino acids, which comprise four of the five octa repeats of PrP, is still biologically active.

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“…In addition to their different stabilities to proteolytic degradation, the secondary, tertiary, and quaternary structures of PrP C and PrP Sc also differ (3)(4)(5)(6)(7). PrP C is monomeric and highly ␣-helical (5), whereas PrP Sc adopts a multimeric arrangement and contains a large amount of ␤-structure and less helical structure.…”

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confidence: 99%

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

Martins

Frosoni

Martínez

et al. 2006

Journal of Biological Chemistry

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Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP C , into a misfolded, protease-resistant form, PrP Sc . Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106 -120 from the so-called amyloidogenic core of PrP (residues 106 -126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65°C. Under partially denaturing conditions (65°C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65°C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65°C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65°C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP Sc can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106 -120 of PrP and does not require other cellular factors or a PrP Sc template.The conformational conversion of the normal cellular isoform of the prion protein, PrP C , 4 into an abnormal pathological isoform, PrP Sc , underlies a group of fatal neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases, which includes Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle (1). Neuropathologically, prion diseases are characterized by neuronal loss and astrogliosis and often by spongiform degeneration of the brain and deposition of amyloid plaques (1). The unique feature of these diseases is that, in addition to sporadic and inherited forms, they may be acquired by transmission of an infectious agent. The "proteinonly" hypothesis of prion propagation postulates that the abnormal isoform, PrP Sc , acts as a transmissible agent of the disease and self-propagates its pathological conformation using PrP C as a substrate (1).PrP Sc is defined as an aggregated form of PrP that is largely resistant to proteinase K (PK) digestion under conditions in which PrP C and most other proteins are readily degraded (2). In addition to their different stabilities to proteolytic degradation, the secondary, tertiary, and quaternary structures of PrP C and PrP Sc also differ (3-7...

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Cell-free formation of protease-resistant prion protein

Cited by 814 publications

References 26 publications

Prion agent diversity and species barrier

Prion agent diversity and species barrier

Molecular biology of transmissible spongiform encephalopathies

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

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