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Baskakov IV. Role of sialylation in prion disease pathogenesis and prion structure. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2020; 175:31-52. [PMID: 32958238 DOI: 10.1016/bs.pmbts.2020.07.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of a sialoglycoprotein called the prion protein or PrPC. Sialylation of the prion protein, a terminal modification of N-linked glycans, was discovered more than 30 years ago, yet the role of sialylation in prion pathogenesis is not well understood. This chapter summarizes current knowledge on the role of sialylation of the prion protein in prion diseases. First, we discuss recent data suggesting that sialylation of PrPSc N-linked glycans determines the fate of prion infection in an organism and control prion lymphotropism. Second, emerging evidence pointing out at the role N-glycans in neuroinflammation are discussed. Thirds, this chapter reviews a mechanism postulating that sialylated N-linked glycans are important players in defining strain-specific structures. A new hypothesis according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms is discussed. Finally, this chapter explain how N-glycan sialylation control the prion replication and strain interference. In summary, comprehensive review of our knowledge on N-linked glycans and their sialylation provided in this chapter helps to answer important questions of prion biology that have been puzzling for years.
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Affiliation(s)
- Ilia V Baskakov
- Department of Anatomy and Neurobiology, and Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD, United States.
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Makarava N, Chang JCY, Molesworth K, Baskakov IV. Posttranslational modifications define course of prion strain adaptation and disease phenotype. J Clin Invest 2020; 130:4382-4395. [PMID: 32484800 PMCID: PMC7410085 DOI: 10.1172/jci138677] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Accepted: 05/13/2020] [Indexed: 12/13/2022] Open
Abstract
Posttranslational modifications are a common feature of proteins associated with neurodegenerative diseases including prion protein (PrPC), tau, and α-synuclein. Alternative self-propagating protein states or strains give rise to different disease phenotypes and display strain-specific subsets of posttranslational modifications. The relationships between strain-specific structure, posttranslational modifications, and disease phenotype are poorly understood. We previously reported that among hundreds of PrPC sialoglycoforms expressed by a cell, individual prion strains recruited PrPC molecules selectively, according to the sialylation status of their N-linked glycans. Here we report that transmission of a prion strain to a new host is accompanied by a dramatic shift in the selectivity of recruitment of PrPC sialoglycoforms, giving rise to a self-propagating scrapie isoform (PrPSc) with a unique sialoglycoform signature and disease phenotype. The newly emerged strain has the shortest incubation time to disease and is characterized by colocalization of PrPSc with microglia and a very profound proinflammatory response, features that are linked to a unique sialoglycoform composition of PrPSc. The current work provides experimental support for the hypothesis that strain-specific patterns of PrPSc sialoglycoforms formed as a result of selective recruitment dictate strain-specific disease phenotypes. This work suggests a causative relationship between a strain-specific structure, posttranslational modifications, and disease phenotype.
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Affiliation(s)
- Natallia Makarava
- Center for Biomedical Engineering and Technology and
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Jennifer Chen-Yu Chang
- Center for Biomedical Engineering and Technology and
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Kara Molesworth
- Center for Biomedical Engineering and Technology and
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Ilia V. Baskakov
- Center for Biomedical Engineering and Technology and
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, USA
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Makarava N, Chang JCY, Baskakov IV. Region-Specific Sialylation Pattern of Prion Strains Provides Novel Insight into Prion Neurotropism. Int J Mol Sci 2020; 21:ijms21030828. [PMID: 32012886 PMCID: PMC7037812 DOI: 10.3390/ijms21030828] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 01/10/2020] [Accepted: 01/23/2020] [Indexed: 12/14/2022] Open
Abstract
Mammalian prions are unconventional infectious agents that invade and replicate in an organism by recruiting a normal form of a prion protein (PrPC) and converting it into misfolded, disease-associated state referred to as PrPSc. PrPC is posttranslationally modified with two N-linked glycans. Prion strains replicate by selecting substrates from a large pool of PrPC sialoglycoforms expressed by a host. Brain regions have different vulnerability to prion infection, however, molecular mechanisms underlying selective vulnerability is not well understood. Toward addressing this question, the current study looked into a possibility that sialylation of PrPSc might be involved in defining selective vulnerability of brain regions. The current work found that in 22L -infected animals, PrPSc is indeed sialylated in a region dependent manner. PrPSc in hippocampus and cortex was more sialylated than PrPSc from thalamus and stem. Similar trends were also observed in brain materials from RML- and ME7-infected animals. The current study established that PrPSc sialylation status is indeed region-specific. Together with previous studies demonstrating that low sialylation status accelerates prion replication, this work suggests that high vulnerability of certain brain region to prion infection could be attributed to their low sialylation status.
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Affiliation(s)
- Natallia Makarava
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD 21201, USA; (N.M.); (J.C.-Y.C.)
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Jennifer Chen-Yu Chang
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD 21201, USA; (N.M.); (J.C.-Y.C.)
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Ilia V. Baskakov
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD 21201, USA; (N.M.); (J.C.-Y.C.)
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
- Correspondence:
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Region-specific glial homeostatic signature in prion diseases is replaced by a uniform neuroinflammation signature, common for brain regions and prion strains with different cell tropism. Neurobiol Dis 2020; 137:104783. [PMID: 32001329 DOI: 10.1016/j.nbd.2020.104783] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Revised: 01/21/2020] [Accepted: 01/25/2020] [Indexed: 02/08/2023] Open
Abstract
Chronic neuroinflammation is recognized as a major neuropathological hallmark in a broad spectrum of neurodegenerative diseases including Alzheimer's, Parkinson's, Frontal Temporal Dementia, Amyotrophic Lateral Sclerosis, and prion diseases. Both microglia and astrocytes exhibit region-specific homeostatic transcriptional identities, which under chronic neurodegeneration, transform into reactive phenotypes in a region- and disease-specific manner. Little is known about region-specific identity of glia in prion diseases. The current study was designed to determine whether the region-specific homeostatic signature of glia changes with the progression of prion diseases, and whether these changes occur in a region-dependent or universal manner. Also of interest was whether different prion strains give rise to different reactive phenotypes. To answer these questions, we analyzed gene expression in the thalamus, cortex, hypothalamus and hippocampus of mice infected with 22L and ME7 prion strains using a Nanostring Neuroinflammation panel at the subclinical, early clinical and advanced stages of the disease. We found that at the preclinical stage of the disease, the region-specific homeostatic identities were preserved. However, with the appearance of clinical signs, the region-specific signatures were partially lost and replaced with a neuroinflammation signature. While the same sets of genes were activated by both prion strains, the timing of neuroinflammation and the degree of activation in different brain regions was strain-specific. Changes in astrocyte function scored at the top of the activated pathways. Moreover, clustering analysis suggested that the astrocyte function pathway responded to prion infection prior to the Activated Microglia or Neuron and Neurotransmission pathways. The current work established neuroinflammation gene expression signature associated with prion diseases. Our results illustrate that with the disease progression, the region-specific homeostatic transcriptome signatures are replaced by the region-independent neuroinflammation signature, which is common for prion strains with different cell tropism. The prion-associated neuroinflammation signature identified in the current study overlapped only partially with the microglia degenerative phenotype and the disease-associated microglia phenotype reported for animal models of other neurodegenerative diseases.
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Baskakov IV, Katorcha E, Makarava N. Prion Strain-Specific Structure and Pathology: A View from the Perspective of Glycobiology. Viruses 2018; 10:v10120723. [PMID: 30567302 PMCID: PMC6315442 DOI: 10.3390/v10120723] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2018] [Revised: 12/13/2018] [Accepted: 12/15/2018] [Indexed: 01/15/2023] Open
Abstract
Prion diseases display multiple disease phenotypes characterized by diverse clinical symptoms, different brain regions affected by the disease, distinct cell tropism and diverse PrPSc deposition patterns. The diversity of disease phenotypes within the same host is attributed to the ability of PrPC to acquire multiple, alternative, conformationally distinct, self-replicating PrPSc states referred to as prion strains or subtypes. Structural diversity of PrPSc strains has been well documented, yet the question of how different PrPSc structures elicit multiple disease phenotypes remains poorly understood. The current article reviews emerging evidence suggesting that carbohydrates in the form of sialylated N-linked glycans, which are a constitutive part of PrPSc, are important players in defining strain-specific structures and disease phenotypes. This article introduces a new hypothesis, according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms that form strain-specific patterns of carbohydrate epitopes on PrPSc surface and contribute to defining the disease phenotype and outcomes.
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Affiliation(s)
- Ilia V Baskakov
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MA 21201, USA.
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MA 21201, USA.
| | - Elizaveta Katorcha
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MA 21201, USA.
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MA 21201, USA.
| | - Natallia Makarava
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MA 21201, USA.
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MA 21201, USA.
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Makarava N, Savtchenko R, Lasch P, Beekes M, Baskakov IV. Preserving prion strain identity upon replication of prions in vitro using recombinant prion protein. Acta Neuropathol Commun 2018; 6:92. [PMID: 30208966 PMCID: PMC6134792 DOI: 10.1186/s40478-018-0597-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2018] [Accepted: 09/06/2018] [Indexed: 11/24/2022] Open
Abstract
Last decade witnessed an enormous progress in generating authentic infectious prions or PrPSc in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrPSc with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrPSc gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate. Up to now, it remains unclear whether prion strain identity can be preserved upon replication in rPrP. The current study reports that faithful replication of hamster strain SSLOW could be achieved in vitro using rPrP as a substrate. We found that a mixture of phosphatidylethanolamine (PE) and synthetic nucleic acid polyA was sufficient for stable replication of hamster brain-derived SSLOW PrPSc in serial Protein Misfolding Cyclic Amplification (sPMCA) that uses hamster rPrP as a substrate. The disease phenotype generated in hamsters upon transmission of recombinant PrPSc produced in vitro was strikingly similar to the original SSLOW diseases phenotype with respect to the incubation time to disease, as well as clinical, neuropathological and biochemical features. Infrared microspectroscopy (IR-MSP) indicated that PrPSc produced in animals upon transmission of recombinant PrPSc is structurally similar if not identical to the original SSLOW PrPSc. The current study is the first to demonstrate that rPrP can support replication of brain-derived PrPSc while preserving its strain identity. In addition, the current work is the first to document that successful propagation of a hamster strain could be achieved in vitro using hamster rPrP.
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Baskakov IV, Katorcha E. Multifaceted Role of Sialylation in Prion Diseases. Front Neurosci 2016; 10:358. [PMID: 27551257 PMCID: PMC4976111 DOI: 10.3389/fnins.2016.00358] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 07/18/2016] [Indexed: 11/13/2022] Open
Abstract
Mammalian prion or PrP(Sc) is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of a sialoglycoprotein called the prion protein, or PrP(C). Sialylation of the prion protein N-linked glycans was discovered more than 30 years ago, yet the role of sialylation in prion pathogenesis remains poorly understood. Recent years have witnessed extraordinary growth in interest in sialylation and established a critical role for sialic acids in host invasion and host-pathogen interactions. This review article summarizes current knowledge on the role of sialylation of the prion protein in prion diseases. First, we discuss the correlation between sialylation of PrP(Sc) glycans and prion infectivity and describe the factors that control sialylation of PrP(Sc). Second, we explain how glycan sialylation contributes to the prion replication barrier, defines strain-specific glycoform ratios, and imposes constraints for PrP(Sc) structure. Third, several topics, including a possible role for sialylation in animal-to-human prion transmission, prion lymphotropism, toxicity, strain interference, and normal function of PrP(C), are critically reviewed. Finally, a metabolic hypothesis on the role of sialylation in the etiology of sporadic prion diseases is proposed.
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Affiliation(s)
- Ilia V. Baskakov
- Department of Anatomy and Neurobiology, Center for Biomedical Engineering and Technology, University of Maryland School of MedicineBaltimore, MD, USA
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Sano K, Atarashi R, Nishida N. Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion. Prion 2016; 9:237-43. [PMID: 26284507 PMCID: PMC4601500 DOI: 10.1080/19336896.2015.1062201] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrPSc). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrPSc results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrPSc. However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrPSc can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.
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Affiliation(s)
- Kazunori Sano
- a Department of Physiology and Pharmacology; Faculty of Pharmaceutical Sciences; Fukuoka University ; Fukuoka, Japan
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Saijo E, Hughson AG, Raymond GJ, Suzuki A, Horiuchi M, Caughey B. PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains. J Virol 2016; 90:4905-4913. [PMID: 26937029 PMCID: PMC4859706 DOI: 10.1128/jvi.00088-16] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2016] [Accepted: 02/19/2016] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED Understanding the structure of PrP(Sc) and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrP(Sc), we purified proteinase-resistant PrP(Sc) (PrP(RES)) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrP(Sc) specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrP(RES), even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrP(Sc)-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrP(Sc) multimers. IMPORTANCE It has long been apparent that prion strains can have different conformations near the N terminus of the PrP(Sc) protease-resistant core. Here, we show that a C-terminal conformational PrP(Sc)-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrP(Sc) also contribute to the phenotypic distinction between prion strains.
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Affiliation(s)
- Eri Saijo
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA
| | - Andrew G Hughson
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA
| | - Gregory J Raymond
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA
| | - Akio Suzuki
- Laboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
| | - Motohiro Horiuchi
- Laboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
| | - Byron Caughey
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA
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Abstract
Prion diseases are a heterogeneous class of fatal neurodegenerative disorders associated with misfolding of host cellular prion protein (PrP(C)) into a pathological isoform, termed PrP(Sc). Prion diseases affect various mammals, including humans, and effective treatments are not available. Prion diseases are distinguished from other protein misfolding disorders - such as Alzheimer's or Parkinson's disease - in that they are infectious. Prion diseases occur sporadically without any known exposure to infected material, and hereditary cases resulting from rare mutations in the prion protein have also been documented. The mechanistic underpinnings of prion and other neurodegenerative disorders remain poorly understood. Various proteomics techniques have been instrumental in early PrP(Sc) detection, biomarker discovery, elucidation of PrP(Sc) structure and mapping of biochemical pathways affected by pathogenesis. Moving forward, proteomics approaches will likely become more integrated into the clinical and research settings for the rapid diagnosis and characterization of prion pathogenesis.
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Affiliation(s)
- Roger A Moore
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIH,NIAID, Hamilton, MT 59840, USA
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Katorcha E, Makarava N, Savtchenko R, Baskakov IV. Sialylation of the prion protein glycans controls prion replication rate and glycoform ratio. Sci Rep 2015; 5:16912. [PMID: 26576925 PMCID: PMC4649626 DOI: 10.1038/srep16912] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2015] [Accepted: 10/21/2015] [Indexed: 11/09/2022] Open
Abstract
Prion or PrP(Sc) is a proteinaceous infectious agent that consists of a misfolded and aggregated form of a sialoglycoprotein called prion protein or PrP(C). PrP(C) has two sialylated N-linked carbohydrates. In PrP(Sc), the glycans are directed outward, with the terminal sialic acid residues creating a negative charge on the surface of prion particles. The current study proposes a new hypothesis that electrostatic repulsion between sialic residues creates structural constraints that control prion replication and PrP(Sc) glycoform ratio. In support of this hypothesis, here we show that diglycosylated PrP(C) molecules that have more sialic groups per molecule than monoglycosylated PrP(C) were preferentially excluded from conversion. However, when partially desialylated PrP(C) was used as a substrate, recruitment of three glycoforms into PrP(Sc) was found to be proportional to their respective populations in the substrate. In addition, hypersialylated molecules were also excluded from conversion in the strains with the strongest structural constraints, a strategy that helped reduce electrostatic repulsion. Moreover, as predicted by the hypothesis, partial desialylation of PrP(C) significantly increased the replication rate. This study illustrates that sialylation of N-linked glycans creates a prion replication barrier that controls replication rate and glycoform ratios and has broad implications.
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Affiliation(s)
- Elizaveta Katorcha
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland, 21201 United States of America.,Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America
| | - Natallia Makarava
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland, 21201 United States of America.,Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America
| | - Regina Savtchenko
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland, 21201 United States of America.,Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America
| | - Ilia V Baskakov
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland, 21201 United States of America.,Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America
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Okada H, Masujin K, Miyazawa K, Yokoyama T. Transmissibility of H-Type Bovine Spongiform Encephalopathy to Hamster PrP Transgenic Mice. PLoS One 2015; 10:e0138977. [PMID: 26466381 PMCID: PMC4605493 DOI: 10.1371/journal.pone.0138977] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2015] [Accepted: 09/07/2015] [Indexed: 11/18/2022] Open
Abstract
Two distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE) can be distinguished from classical (C-) BSE found in cattle based on biochemical signatures of disease-associated prion protein (PrPSc). H-BSE is transmissible to wild-type mice—with infected mice showing a long survival period that is close to their normal lifespan—but not to hamsters. Therefore, rodent-adapted H-BSE with a short survival period would be useful for analyzing H-BSE characteristics. In this study, we investigated the transmissibility of H-BSE to hamster prion protein transgenic (TgHaNSE) mice with long survival periods. Although none of the TgHaNSE mice manifested the disease during their lifespan, PrPSc accumulation was observed in some areas of the brain after the first passage. With subsequent passages, TgHaNSE mice developed the disease with a mean survival period of 220 days. The molecular characteristics of proteinase K-resistant PrPSc (PrPres) in the brain were identical to those observed in first-passage mice. The distribution of immunolabeled PrPSc in the brains of TgHaNSE mice differed between those infected with H-BSE as compared to C-BSE or L-BSE, and the molecular properties of PrPres in TgHaNSE mice infected with H-BSE differed from those of the original isolate. The strain-specific electromobility, glycoform profiles, and proteolytic cleavage sites of H-BSE in TgHaNSE mice were indistinguishable from those of C-BSE, in which the diglycosylated form was predominant. These findings indicate that strain-specific pathogenic characteristics and molecular features of PrPres in the brain are altered during cross-species transmission. Typical H-BSE features were restored after back passage from TgHaNSE to bovinized transgenic mice, indicating that the H-BSE strain was propagated in TgHaNSE mice. This could result from the overexpression of the hamster prion protein.
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Affiliation(s)
- Hiroyuki Okada
- National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan
- * E-mail: (HO); (KM)
| | - Kentaro Masujin
- National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan
- * E-mail: (HO); (KM)
| | - Kohtaro Miyazawa
- National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan
| | - Takashi Yokoyama
- National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan
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Daus ML. Techniques to elucidate the conformation of prions. World J Biol Chem 2015; 6:218-222. [PMID: 26322176 PMCID: PMC4549762 DOI: 10.4331/wjbc.v6.i3.218] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Revised: 05/04/2015] [Accepted: 06/16/2015] [Indexed: 02/05/2023] Open
Abstract
Proteinaceous infectious particles (prions) are unique pathogens as they are devoid of any coding nucleic acid. Whilst it is assumed that prion disease is transmitted by a misfolded isoform of the cellular prion protein, the structural insight of prions is still vague and research for high resolution structural information of prions is still ongoing. In this review, techniques that may contribute to the clarification of the conformation of prions are presented and discussed.
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Simoneau S, Thomzig A, Ruchoux MM, Vignier N, Daus ML, Poleggi A, Lebon P, Freire S, Durand V, Graziano S, Galeno R, Cardone F, Comoy E, Pocchiari M, Beekes M, Deslys JP, Fournier JG. Synthetic scrapie infectivity: interaction between recombinant PrP and scrapie brain-derived RNA. Virulence 2015; 6:132-44. [PMID: 25585171 DOI: 10.4161/21505594.2014.989795] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
The key molecular event in human cerebral proteinopathies, which include Alzheimer's, Parkinson's and Huntington's diseases, is the structural conversion of a specific host protein into a β-sheet-rich conformer. With regards to this common mechanism, it appears difficult to explain the outstanding infectious properties attributed to PrP(Sc), the hallmark of another intriguing family of cerebral proteinopathies known as transmissible spongiform encephalopathies (TSE) or prion diseases. The infectious PrP(Sc) or "prion" is thought to be composed solely of a misfolded form of the otherwise harmless cellular prion protein (PrP(c)). To gain insight into this unique situation, we used the 263K scrapie hamster model to search for a putative PrP(Sc)-associated factor that contributes to the infectivity of PrP(Sc) amyloid. In a rigorously controlled set of experiments that included several bioassays, we showed that originally innocuous recombinant prion protein (recPrP) equivalent to PrP(c) is capable of initiating prion disease in hamsters when it is converted to a prion-like conformation (β-sheet-rich) in the presence of RNA purified from scrapie-associated fibril (SAF) preparations. Analysis of the recPrP-RNA infectious mixture reveals the presence of 2 populations of small RNAs of approximately 27 and 55 nucleotides. These unprecedented findings are discussed in light of the distinct relationship that may exist between this RNA material and the 2 biological properties, infectivity and strain features, attributed to prion amyloid.
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Affiliation(s)
- Steve Simoneau
- a Division of Prions and Related Diseases (SEPIA); Institute of Emerging Diseases and Innovative Therapies (iMETI); CEA ; Fontenay-aux-Roses, France
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15
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Klimova N, Makarava N, Baskakov IV. The diversity and relationship of prion protein self-replicating states. Virus Res 2014; 207:113-9. [PMID: 25312451 DOI: 10.1016/j.virusres.2014.10.002] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2014] [Revised: 08/15/2014] [Accepted: 10/01/2014] [Indexed: 10/24/2022]
Abstract
It has become evident that the prion protein (PrP) can form a diverse range of self-replicating structures in addition to bona fide PrP(Sc) or strain-specific PrP(Sc) variants. Some self-replicating states can be only produced in vitro, whereas others can be formed in vivo and in vitro. While transmissible, not all states that replicate in vivo are truly pathogenic. Some of them can replicate silently without causing symptoms or clinical diseases. In the current article we discuss the data on PK-digestion patterns of different self-replicating PrP states in connection with other structural data available to date and assess possible relationships between different self-replicating states. Even though different self-replicating PrP states appear to have significantly different global folding patterns, it seems that the C-terminal region exhibits a cross-β-sheet structure in all self-replicating states, as this region acquires the proteolytically most stable conformation. We also discuss the possibility of the transformation of self-replicating states and triggering of PrP(Sc) formation within the frame of the deformed templating model. The spread of silent self-replicating states is of a particular concern because they can lead to transmissible prion disease. Moreover, examples on how different replication requirements favor different states are discussed. This knowledge can help in designing conditions for selective amplification of a particular PrP state in vitro.
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Affiliation(s)
- Nina Klimova
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201, USA; Department of Anatomy and Neurobiology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201, USA
| | - Natallia Makarava
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201, USA; Department of Anatomy and Neurobiology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201, USA
| | - Ilia V Baskakov
- Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201, USA; Department of Anatomy and Neurobiology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201, USA.
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16
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Conformational properties of prion strains can be transmitted to recombinant prion protein fibrils in real-time quaking-induced conversion. J Virol 2014; 88:11791-801. [PMID: 25078700 DOI: 10.1128/jvi.00585-14] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrP(Sc)). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses Escherichia coli-derived recombinant PrP (rPrP) for the sensitive detection of PrP(Sc). To investigate whether the properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP fibrils) using RT-QUIC, we examined the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L strain. In the first round of the reaction, there were differences in the secondary structures, especially in bands attributed to β-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fib(Ch)) and 22L-seeded (1st-rPrP-fib(22L)) rPrP fibrils. Of note, specific identifying characteristics of the two rPrP fibril types seen in the β-sheets resembled those of the original PrP(Sc). Furthermore, the conformational stability of 1st-rPrP-fib(Ch) was significantly higher than that of 1st-rPrP-fib(22L), as with Chandler and 22L PrP(Sc). The survival periods of mice inoculated with 1st-rPrP-fib(Ch) or 1st-rPrP-fib(22L) were significantly shorter than those of mice inoculated with mixtures from the mock 1st-round RT-QUIC procedure. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP fibrils became predominant probably because of their high growth rate. Together, these findings show that at least some strain-specific conformational properties can be transmitted to rPrP fibrils and unknown cofactors or environmental conditions may be required for further conservation. Importance: The phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrP(Sc)). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how strain-specific conformations are transmitted. In this study, we investigated whether the properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated by real-time quaking-induced conversion (RT-QUIC), a recently developed in vitro PrP(Sc) formation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.
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Haïk S, Brandel JP. Infectious prion diseases in humans: cannibalism, iatrogenicity and zoonoses. INFECTION GENETICS AND EVOLUTION 2014; 26:303-12. [PMID: 24956437 DOI: 10.1016/j.meegid.2014.06.010] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/17/2014] [Revised: 06/10/2014] [Accepted: 06/13/2014] [Indexed: 12/24/2022]
Abstract
In contrast with other neurodegenerative disorders associated to protein misfolding, human prion diseases include infectious forms (also called transmitted forms) such as kuru, iatrogenic Creutzfeldt-Jakob disease and variant Creutzfeldt-Jakob disease. The transmissible agent is thought to be solely composed of the abnormal isoform (PrP(Sc)) of the host-encoded prion protein that accumulated in the central nervous system of affected individuals. Compared to its normal counterpart, PrP(Sc) is β-sheet enriched and aggregated and its propagation is based on an autocatalytic conversion process. Increasing evidence supports the view that conformational variations of PrP(Sc) encoded the biological properties of the various prion strains that have been isolated by transmission studies in experimental models. Infectious forms of human prion diseases played a pivotal role in the emergence of the prion concept and in the characterization of the very unconventional properties of prions. They provide a unique model to understand how prion strains are selected and propagate in humans. Here, we review and discuss how genetic factors interplay with strain properties and route of transmission to influence disease susceptibility, incubation period and phenotypic expression in the light of the kuru epidemics due to ritual endocannibalism, the various series iatrogenic diseases secondary to extractive growth hormone treatment or dura mater graft and the epidemics of variant Creutzfeldt-Jakob disease linked to dietary exposure to the agent of bovine spongiform encephalopathy.
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Affiliation(s)
- Stéphane Haïk
- Sorbonne Universités, UPMC Univ Paris 06 UMR S 1127, Inserm, U 1127, CNRS UMR 7225, ICM, F-75013 Paris, France; AP-HP, Groupe hospitalier Pitié-Salpêtrière, Cellule Nationale de Référence des Maladies de Creutzfeldt-Jakob, F-75013 Paris, France; Centre National de Référence des Agents Transmissibles Non Conventionnels, F-75013 Paris, France.
| | - Jean-Philippe Brandel
- Sorbonne Universités, UPMC Univ Paris 06 UMR S 1127, Inserm, U 1127, CNRS UMR 7225, ICM, F-75013 Paris, France; AP-HP, Groupe hospitalier Pitié-Salpêtrière, Cellule Nationale de Référence des Maladies de Creutzfeldt-Jakob, F-75013 Paris, France; Centre National de Référence des Agents Transmissibles Non Conventionnels, F-75013 Paris, France
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18
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Prion protein misfolding, strains, and neurotoxicity: an update from studies on Mammalian prions. Int J Cell Biol 2013; 2013:910314. [PMID: 24454379 PMCID: PMC3884631 DOI: 10.1155/2013/910314] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2013] [Revised: 11/10/2013] [Accepted: 11/11/2013] [Indexed: 11/17/2022] Open
Abstract
Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders affecting humans and other mammalian species. The central event in TSE pathogenesis is the conformational conversion of the cellular prion protein, PrPC, into the aggregate, β-sheet rich, amyloidogenic form, PrPSc. Increasing evidence indicates that distinct PrPSc conformers, forming distinct ordered aggregates, can encipher the phenotypic TSE variants related to prion strains. Prion strains are TSE isolates that, after inoculation into syngenic hosts, cause disease with distinct characteristics, such as incubation period, pattern of PrPSc distribution, and regional severity of histopathological changes in the brain. In analogy with other amyloid forming proteins, PrPSc toxicity is thought to derive from the existence of various intermediate structures prior to the amyloid fiber formation and/or their specific interaction with membranes. The latter appears particularly relevant for the pathogenesis of TSEs associated with GPI-anchored PrPSc, which involves major cellular membrane distortions in neurons. In this review, we update the current knowledge on the molecular mechanisms underlying three fundamental aspects of the basic biology of prions such as the putative mechanism of prion protein conversion to the pathogenic form PrPSc and its propagation, the molecular basis of prion strains, and the mechanism of induced neurotoxicity by PrPSc aggregates.
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Daus ML, Wagenführ K, Thomzig A, Boerner S, Hermann P, Hermelink A, Beekes M, Lasch P. Infrared microspectroscopy detects protein misfolding cyclic amplification (PMCA)-induced conformational alterations in hamster scrapie progeny seeds. J Biol Chem 2013; 288:35068-80. [PMID: 24163371 DOI: 10.1074/jbc.m113.497131] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP(C)) into Proteinase K-resistant, infectious PrP particles (PrP(TSE)), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP(C) substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP(C) substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.
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Affiliation(s)
- Martin L Daus
- From FG 14-AG 5: Unconventional Pathogens and Their Inactivation, Applied Infection Control and Hospital Hygiene, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany and
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Laferrière F, Tixador P, Moudjou M, Chapuis J, Sibille P, Herzog L, Reine F, Jaumain E, Laude H, Rezaei H, Béringue V. Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics. PLoS Pathog 2013; 9:e1003702. [PMID: 24130496 PMCID: PMC3795044 DOI: 10.1371/journal.ppat.1003702] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2013] [Accepted: 08/27/2013] [Indexed: 11/18/2022] Open
Abstract
Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP(Sc), an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP(C)). Stable variations in PrP(Sc) conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP(Sc) quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP(Sc) quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP(Sc). To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP(Sc) tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP(Sc) aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP(Sc) quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.
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Affiliation(s)
- Florent Laferrière
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Philippe Tixador
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Mohammed Moudjou
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Jérôme Chapuis
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Pierre Sibille
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Laetitia Herzog
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Fabienne Reine
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Emilie Jaumain
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Hubert Laude
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Human Rezaei
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Vincent Béringue
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
- * E-mail:
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21
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Campisi E, Cardone F, Graziano S, Galeno R, Pocchiari M. Role of proteomics in understanding prion infection. Expert Rev Proteomics 2013; 9:649-66. [PMID: 23256675 DOI: 10.1586/epr.12.58] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Transmissible spongiform encephalopathies or prion diseases are fatal neurodegenerative pathologies characterized by the autocatalytic misfolding and polymerization of a cellular glycoprotein (cellular prion protein [PrP(C)]) that accumulates in the CNS and leads to neurodegeneration. The detailed mechanics of PrP(C) conversion to its pathological isoform (PrP(TSE)) are unclear but one or more exogenous factors are likely involved in the process of PrP misfolding. In the last 20 years, proteomic investigations have identified several endogenous proteins that interact with PrP(C), PrP(TSE) or both, which are possibly involved in the prion pathogenetic process. However, current approaches have not yet produced convincing conclusions on the biological value of such PrP interactors. Future advancements in the comprehension of the molecular pathogenesis of prion diseases, in experimental techniques and in data analysis procedures, together with a boost in more productive international collaborations, are therefore needed to improve the understanding on the role of PrP interactors. Finally, the advancement of 'omics' techniques in prion diseases will contribute to the development of novel diagnostic tests and effective drugs.
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Affiliation(s)
- Edmondo Campisi
- Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy
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22
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Boerner S, Wagenführ K, Daus ML, Thomzig A, Beekes M. Towards further reduction and replacement of animal bioassays in prion research by cell and protein misfolding cyclic amplification assays. Lab Anim 2013; 47:106-15. [PMID: 23479773 DOI: 10.1177/0023677213476856] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Laboratory animals have long since been used extensively in bioassays for prions in order to quantify, usually in terms of median infective doses [ID50], how infectious these pathogens are in vivo. The identification of aberrant prion protein as the main component and self-replicating principle of prions has given rise to alternative approaches for prion titration. Such approaches often use protein misfolding cyclic amplification (PMCA) for the cell-free biochemical measurement of prion-associated seeding activity, or cell assays for the titration of in vitro infectivity. However, median seeding and cell culture infective doses (SD50 and CCID50, respectively) of prions are neither formally congruent nor definitely representative for ID50 titres in animals and can be therefore only tentatively translated into the latter. This may potentially impede the acceptance and use of alternative methods to animal bioassays in prion research. Thus, we suggest performing PMCA and cell assays jointly, and to check whether these profoundly different test principles deliver consistent results in order to strengthen the reliability and credibility of prion ID50 assessments by in vitro methods. With regard to this rationale, we describe three pairs of PMCA and glial cell assays for different hamster-adapted prion agents (the frequently used 263K scrapie strain, and 22A-H scrapie and BSE-H). In addition, we report on the adaptation of quantitative PMCA to human variant Creutzfeldt-Jakob disease (vCJD) prions on steel wires for prion disinfection studies. Our rationale and methodology can be systematically extended to other types of prions and used to further reduce or replace prion bioassays in rodents.
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Affiliation(s)
- Susann Boerner
- Work Group Unconventional Pathogens and Their Inactivation, Division of Applied Infection Control and Hospital Hygiene, Department of Infectious Diseases, Robert Koch-Institut, 13353 Berlin, Germany
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23
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Miller LM, Bourassa MW, Smith RJ. FTIR spectroscopic imaging of protein aggregation in living cells. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2013; 1828:2339-46. [PMID: 23357359 DOI: 10.1016/j.bbamem.2013.01.014] [Citation(s) in RCA: 206] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Accepted: 01/16/2013] [Indexed: 01/22/2023]
Abstract
Protein misfolding and aggregation are the hallmark of a number of diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and the prion diseases. In all cases, a naturally-occurring protein misfolds and forms aggregates that are thought to disrupt cell function through a wide range of mechanisms that are yet to be fully unraveled. Fourier transform infrared (FTIR) spectroscopy is a technique that is sensitive to the secondary structure of proteins and has been widely used to investigate the process of misfolding and aggregate formation. This review focuses on how FTIR spectroscopy and spectroscopic microscopy are being used to evaluate the structural changes in disease-related proteins both in vitro and directly within cells and tissues. Finally, ongoing technological advances will be presented that are enabling time-resolved FTIR imaging of protein aggregation directly within living cells, which can provide insight into the structural intermediates, time scale, and mechanisms of cell toxicity associated with aggregate formation. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.
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Affiliation(s)
- Lisa M Miller
- Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973, USA.
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24
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Singh J, Sabareesan A, Mathew M, Udgaonkar JB. Development of the Structural Core and of Conformational Heterogeneity during the Conversion of Oligomers of the Mouse Prion Protein to Worm-like Amyloid Fibrils. J Mol Biol 2012; 423:217-31. [DOI: 10.1016/j.jmb.2012.06.040] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2012] [Revised: 06/15/2012] [Accepted: 06/28/2012] [Indexed: 10/28/2022]
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25
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Daus ML, Beekes M. Chronic wasting disease: fingerprinting the culprit in risk assessments. Prion 2012; 6:17-22. [PMID: 22453172 DOI: 10.4161/pri.6.1.17776] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Transmissible spongiform encephalopathies (prion diseases) in animals may be associated with a zoonotic risk potential for humans as shown by the occurrence of variant Creutzfeldt-Jakob disease in the wake of the bovine spongiform encephalopathy epidemic. Thus, the increasing exposure of humans in North America to cervid prions of chronic wasting disease (CWD) in elk and deer has prompted comprehensive risk assessments. The susceptibility of humans to CWD infections is currently under investigation in different studies using macaques as primate models. The necessity for such studies was recently reinforced when disease-associated prion protein and its seeding activity were detected in muscles of clinically inconspicuous CWD-infected white-tailed deer. Increasing evidence points to the existence of different CWD strains, and CWD prions may also change or newly emerge over time. Therefore, CWD isolates examined in macaques should be characterized as precisely as possible for their molecular identity. On this basis other CWD field samples collected in the past, present or future could be systematically compared with macaque-tested inocula in order to assess whether they are covered by the ongoing risk assessments in primates. CWD typing by Fourier transform-infrared spectroscopy of pathological prion protein may provide a method of choice for this purpose.
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Affiliation(s)
- Martin L Daus
- P24-Transmissible Spongiform Encephalopathies, Robert Koch-Institut, Berlin, Germany
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26
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Bessen RA, Robinson CJ, Seelig DM, Watschke CP, Lowe D, Shearin H, Martinka S, Babcock AM. Transmission of chronic wasting disease identifies a prion strain causing cachexia and heart infection in hamsters. PLoS One 2011; 6:e28026. [PMID: 22174765 PMCID: PMC3236201 DOI: 10.1371/journal.pone.0028026] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2011] [Accepted: 10/30/2011] [Indexed: 01/20/2023] Open
Abstract
Chronic wasting disease (CWD) is an emerging prion disease of free-ranging and captive cervids in North America. In this study we established a rodent model for CWD in Syrian golden hamsters that resemble key features of the disease in cervids including cachexia and infection of cardiac muscle. Following one to three serial passages of CWD from white-tailed deer into transgenic mice expressing the hamster prion protein gene, CWD was subsequently passaged into Syrian golden hamsters. In one passage line there were preclinical changes in locomotor activity and a loss of body mass prior to onset of subtle neurological symptoms around 340 days. The clinical symptoms included a prominent wasting disease, similar to cachexia, with a prolonged duration. Other features of CWD in hamsters that were similar to cervid CWD included the brain distribution of the disease-specific isoform of the prion protein, PrPSc, prion infection of the central and peripheral neuroendocrine system, and PrPSc deposition in cardiac muscle. There was also prominent PrPSc deposition in the nasal mucosa on the edge of the olfactory sensory epithelium with the lumen of the nasal airway that could have implications for CWD shedding into nasal secretions and disease transmission. Since the mechanism of wasting disease in prion diseases is unknown this hamster CWD model could provide a means to investigate the physiological basis of cachexia, which we propose is due to a prion-induced endocrinopathy. This prion disease phenotype has not been described in hamsters and we designate it as the ‘wasting’ or WST strain of hamster CWD.
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Affiliation(s)
- Richard A Bessen
- Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America.
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Haïk S, Brandel JP. Biochemical and strain properties of CJD prions: complexity versus simplicity. J Neurochem 2011; 119:251-61. [PMID: 21790605 DOI: 10.1111/j.1471-4159.2011.07399.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrP(Sc)), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrP(C)). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrP(Sc). In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.
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Affiliation(s)
- Stéphane Haïk
- Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière (CRICM), UMRS 975, Equipe "Alzheimer's and Prion Diseases", Paris, France.
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Moore RA, Timmes AG, Wilmarth PA, Safronetz D, Priola SA. Identification and removal of proteins that co-purify with infectious prion protein improves the analysis of its secondary structure. Proteomics 2011; 11:3853-65. [PMID: 21805638 DOI: 10.1002/pmic.201100253] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2011] [Revised: 05/30/2011] [Accepted: 06/27/2011] [Indexed: 12/13/2022]
Abstract
Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP(Sc) ) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, β-sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP(Sc) secondary structure. We have used FTIR and LC-MS/MS to analyze enriched PrP(Sc) from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrP(Sc) . Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn and β-sheet structures attributed to PrP(Sc) . The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652 cm(-1) in the FTIR spectrum associated with PrP(Sc) . However, even the removal of more than 99% of the ferritin from PrP(Sc) did not completely abolish absorbance at 1652 cm(-1) . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP(Sc) and suggest that the α-helical, loop/turn and β-sheet secondary structure that remains following their removal are derived from PrP(Sc) itself.
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Affiliation(s)
- Roger A Moore
- Rocky Mountain Laboratories/Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th St., Hamilton, MT 59840, USA.
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Baron GS, Hughson AG, Raymond GJ, Offerdahl DK, Barton KA, Raymond LD, Dorward DW, Caughey B. Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra. Biochemistry 2011; 50:4479-90. [PMID: 21539311 PMCID: PMC3101284 DOI: 10.1021/bi2003907] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent β-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP(res) with GPI- and glycan-deficient PrP(res) produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP(res) revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to β-sheets. These PrP(res) spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP(res) were nearly indistinguishable. With ME7 PrP(res), modest differences between the wild-type and anchorless spectra were detected, notably an ∼2 cm(-1) shift in an apparent β-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP(res), at least relative to the differences observed between strains, but can subtly affect turns and certain β-sheet components. Recently reported H-D exchange analyses of anchorless PrP(res) preparations strongly suggested the presence of strain-dependent, solvent-inaccessible β-core structures throughout most of the C-terminal half of PrP(res) molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP(res).
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Affiliation(s)
- Gerald S. Baron
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Andrew G. Hughson
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Gregory J. Raymond
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Danielle K. Offerdahl
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Kelly A. Barton
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Lynne D. Raymond
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - David W. Dorward
- Microscopy Unit, Research Technology Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Byron Caughey
- Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
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Somerville RA, Gentles N. Characterization of the effect of heat on agent strains of the transmissible spongiform encephalopathies. J Gen Virol 2011; 92:1738-1748. [PMID: 21471321 DOI: 10.1099/vir.0.030452-0] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
The causal agents of the transmissible spongiform encephalopathy (TSE) diseases, sometimes called prion diseases, are characterized by high resistance to inactivation with heat. Results from thermal inactivation experiments on nine TSE strains, seven passaged in two PrP genotypes, showed differences in sensitivity to heat inactivation ranging over 17 °C. In addition, the rate of inactivation with increasing temperature varied between TSE models. In some cases passage in an alternative PrP genotype had little effect on the resulting inactivation properties, but for others the infectious agent was inactivated at lower temperatures. No strain with higher thermostability properties was selected. The effect of mixing two TSE strains, to see whether their properties were affected through interaction with each other, was also examined. The results showed that both strains behaved as expected from the behaviour of the unmixed controls, and that the strain responsible for inducing TSE disease could be identified. There was no evidence of a direct effect on intrinsic strain properties. Overall, the results illustrate the diversity in properties of TSE strains. They require intrinsic molecular properties of TSE agents to accommodate high resistance to inactivation and a mechanism, independent of the host, to directly encode these differences. These findings are more readily reconciled with models of TSE agents with two separate components, one of which is independent of the host and comprises a TSE-specific nucleic acid, than with models based solely on conformational changes to a host protein.
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Affiliation(s)
- Robert A Somerville
- Neuropathogenesis Division, The Roslin Institute and Royal (Dick) Veterinary School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, EH25 9PS, Scotland, UK
| | - Nicola Gentles
- Neuropathogenesis Division, The Roslin Institute and Royal (Dick) Veterinary School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, EH25 9PS, Scotland, UK
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Shu Y, Masujin K, Okada H, Iwamaru Y, Imamura M, Matsuura Y, Mohri S, Yokoyama T. Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies. Prion 2011; 5:103-8. [PMID: 21597334 DOI: 10.4161/pri.5.2.15847] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Atypical forms of bovine spongiform encephalopathy (BSE) may be caused by different prions from classical BSE (C-BSE). In this study, we examined the susceptibility of mice overexpressing mouse and hamster chimeric prion protein (PrP) to L-type atypical BSE (L-BSE). None of the transgenic mice showed susceptibility to L-BSE, except mice overexpressing hamster PrP. We also examined the transmission properties of L-BSE in hamsters. The incubation period of hamsters intracerebrally inoculated with L-BSE was 576.8 days, and that of the subsequent passage was decreased to 208 days. Although the lesion and glycoform profiles and relative proteinase K resistant core fragment of the abnormal isoform of PrP (PrPcore) of L-BSE were similar to that of C-BSE, the deposition of the abnormal isoform of PrP (PrPSc) and the molecular weight of PrPcore of L-BSE was different from than that of C-BSE. In hamster models, some prion strain characteristics of L-BSE were indistinguishable from those of C-BSE.
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Affiliation(s)
- Yujing Shu
- Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan
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Didonna A, Vaccari L, Bek A, Legname G. Infrared microspectroscopy: a multiple-screening platform for investigating single-cell biochemical perturbations upon prion infection. ACS Chem Neurosci 2011; 2:160-74. [PMID: 22778865 DOI: 10.1021/cn1000952] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2010] [Accepted: 12/08/2010] [Indexed: 12/15/2022] Open
Abstract
Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP(Sc)) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrP(C), into nascent PrP(Sc). The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level.
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Affiliation(s)
- Alessandro Didonna
- Laboratory of Prion Biology, Neurobiology Sector, Scuola Internazionale Superiore di Studi Avanzati (SISSA), via Bonomea 265, I-34136 Trieste, Italy
| | - Lisa Vaccari
- ELETTRA Synchrotron Light Laboratory, S.S. 14 Km. 163.5, 34149 Basovizza, Trieste, Italy
| | - Alpan Bek
- CBM S.c.r.l., Consorzio per il Centro di Biomedicina Molecolare—Center for Molecular Biomedicine, Area Science Park—Basovizza SS 14, Km 163.5, I-34149 Trieste (TS), Italy
| | - Giuseppe Legname
- Laboratory of Prion Biology, Neurobiology Sector, Scuola Internazionale Superiore di Studi Avanzati (SISSA), via Bonomea 265, I-34136 Trieste, Italy
- ELETTRA Synchrotron Light Laboratory, S.S. 14 Km. 163.5, 34149 Basovizza, Trieste, Italy
- CBM S.c.r.l., Consorzio per il Centro di Biomedicina Molecolare—Center for Molecular Biomedicine, Area Science Park—Basovizza SS 14, Km 163.5, I-34149 Trieste (TS), Italy
- Italian Institute of Technology, SISSA Unit, Via Bonomea 265, 34136 Trieste, Italy
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Seuberlich T, Heim D, Zurbriggen A. Atypical transmissible spongiform encephalopathies in ruminants: a challenge for disease surveillance and control. J Vet Diagn Invest 2011; 22:823-42. [PMID: 21088166 DOI: 10.1177/104063871002200601] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Since 1987, when bovine spongiform encephalopathy (BSE) emerged as a novel disease in cattle, enormous efforts were undertaken to monitor and control the disease in ruminants worldwide. The driving force was its high economic impact, which resulted from trade restrictions and the loss of consumer confidence in beef products, the latter because BSE turned out to be a fatal zoonosis, causing variant Creutzfeldt-Jakob disease in human beings. The ban on meat and bone meal in livestock feed and the removal of specified risk materials from the food chain were the main measures to successfully prevent infection in cattle and to protect human beings from BSE exposure. However, although BSE is now under control, previously unknown, so-called atypical transmissible spongiform encephalopathies (TSEs) in cattle and small ruminants have been identified by enhanced disease surveillance. This report briefly reviews and summarizes the current level of knowledge on the spectrum of TSEs in cattle and small ruminants and addresses the question of the extent to which such atypical TSEs have an effect on disease surveillance and control strategies.
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Affiliation(s)
- Torsten Seuberlich
- NeuroCentre, National and OIE Reference Laboratories for BSE and Scrapie, DCR-VPH, Bremgartenstrasse 109a, CH-3001 Berne, Switzerland.
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Silva JL, Vieira TCRG, Gomes MPB, Rangel LP, Scapin SMN, Cordeiro Y. Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion. Methods 2010; 53:306-17. [PMID: 21145399 DOI: 10.1016/j.ymeth.2010.12.002] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2010] [Accepted: 12/02/2010] [Indexed: 11/17/2022] Open
Abstract
The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies.
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Affiliation(s)
- Jerson L Silva
- Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Brazil.
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Abstract
We investigated the susceptibilities of Syrian golden hamsters to transmissible spongiform encephalopathy agents from cattle. We report efficient transmission of the L-type atypical bovine spongiform encephalopathy (BSE) agent into hamsters. Importantly, hamsters were also susceptible to the transmissible mink encephalopathy agent from cattle, which has molecular features similar to those of the L-type BSE agent, as also shown in bovinized transgenic mice. In sharp contrast, hamsters could not be infected with classical or H-type BSE agents from cattle. However, previous adaptation of the classical BSE agent in wild-type mice led to efficient transmission. Thus, this study demonstrates the existence of distinct "strain barriers" upon the transmission of bovine prions in hamsters.
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Abstract
While prions share the ability to propagate strain information with nucleic acid-based pathogens, it is unclear how they mutate and acquire fitness in the absence of this informational component. Because prion diseases occur as epidemics, understanding this mechanism is of paramount importance for implementing control strategies to limit their spread and for evaluating their zoonotic potential. Here we review emerging evidence indicating how prion protein primary structures, in concert with PrP(Sc) conformational compatibility, determine prion strain mutation.
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Affiliation(s)
- Glenn C Telling
- Department of Microbiology, Immunology and Molecular Genetics, Sanders Brown Center on Aging, Department of Neurology, University of Kentucky Medical Center, Lexington, KY, USA.
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Bosch A, Prieto C, Serra DO, Martina P, Stämmbler M, Naumann D, Schmitt J, Yantorno O. Type-IV pili spectroscopic markers: applications in the quantification of piliation levels in Moraxella bovis cells by a FT-IR ANN-based model. JOURNAL OF BIOPHOTONICS 2010; 3:522-533. [PMID: 20422659 DOI: 10.1002/jbio.201000027] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2023]
Abstract
Type-IV pili are cell surface organelles found in a wide variety of Gram-negative bacteria. They have traditionally been detected by electron microscopy and ELISA techniques. However, these methodologies are not appropriate for the rapid discrimination and quantification of piliated and nonpiliated cells in industrial or field conditions. Here, the analysis of FT-IR spectra of piliated, nonpiliated and sheared Moraxella bovis cells, together with purified pili suspensions spectra, allowed the identification of 3 IR regions associated to spectroscopic markers of Type-IV pili: 1750-1600, 1450-1350 and 1280-950 cm(-1). Such IR-specific markers were found for piliated cells grown in different culture systems (liquid or solid media), independently of the strain or pili serotype. They were also sensitive to pili expression levels. Therefore, on the bases of these specific spectral features, an FT-IR ANN-based model was developed to classify piliation levels in 5 distinct groups. An overall classification rate of almost 90% demonstrates the strong potential of the ANN system developed to monitor M. bovis cultures in vaccine production.
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Affiliation(s)
- Alejandra Bosch
- Centro de Investigación y Desarrollo en Fermentaciones Industriales CINDEFI, UNLP, CONICET La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
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Schulz-Schaeffer WJ. The synaptic pathology of alpha-synuclein aggregation in dementia with Lewy bodies, Parkinson's disease and Parkinson's disease dementia. Acta Neuropathol 2010; 120:131-43. [PMID: 20563819 PMCID: PMC2892607 DOI: 10.1007/s00401-010-0711-0] [Citation(s) in RCA: 437] [Impact Index Per Article: 29.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2010] [Revised: 05/31/2010] [Accepted: 06/11/2010] [Indexed: 12/16/2022]
Abstract
Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are usually associated with loss of dopaminergic neurons. Loss of substantia nigra neurons and presence of Lewy body inclusions in some of the remaining neurons are the hallmark pathology seen in the final stages of the disease. Attempts to correlate Lewy body pathology to either cell death or severity of clinical symptoms, however, have not been successful. While the pathophysiology of the neurodegenerative process can hardly be explained by Lewy bodies, the clinical symptoms do indicate a degenerative process located at the presynapse resulting in a neurotransmitter deficiency. Recently it was shown that 90% or even more of alpha-synuclein aggregates in DLB cases were located at the presynapses in the form of very small deposits. In parallel, dendritic spines are retracted, whereas the presynapses are relatively preserved, suggesting a neurotransmitter deprivation. The same alpha-synuclein pathology can be demonstrated for PD. These findings give rise to the notion that not cell death but rather alpha-synuclein aggregate-related synaptic dysfunction causes the neurodegeneration. This opens new perspectives for understanding PD and DLB. If presynaptic alpha-synuclein aggregation, not neuronal loss, is the key issue of the neurodegenerative process, then PD and DLB may eventually be treatable in the future. The disease may progress via trans-synaptical spread, suggesting that stem cell transplants are of limited use. Future therapies may focus on the regeneration of synapses.
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Affiliation(s)
- Walter J Schulz-Schaeffer
- Department of Neuropathology, University Medical Center Göttingen, Robert-Koch-Str. 40, Göttingen, Germany.
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Ostapchenko VG, Sawaya MR, Makarava N, Savtchenko R, Nilsson KPR, Eisenberg D, Baskakov IV. Two amyloid States of the prion protein display significantly different folding patterns. J Mol Biol 2010; 400:908-21. [PMID: 20553730 DOI: 10.1016/j.jmb.2010.05.051] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2010] [Revised: 04/27/2010] [Accepted: 05/21/2010] [Indexed: 12/16/2022]
Abstract
It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question, we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein. Several biophysical techniques including X-ray diffraction, CD, Fourier transform infrared spectroscopy (FTIR), hydrogen-deuterium exchange, proteinase K digestion, and binding of a conformation-sensitive fluorescence dye revealed that R- and S-fibrils have substantially different secondary, tertiary, and quaternary structures. While both states displayed a 4. 8-A meridional X-ray diffraction typical for amyloid cross-beta-spines, they showed markedly different equatorial profiles, suggesting different folding pattern of beta-strands. The experiments on hydrogen-deuterium exchange monitored by FTIR revealed that only small fractions of amide protons were protected in R- or S-fibrils, an argument for the dynamic nature of their cross-beta-structure. Despite this fact, both amyloid states were found to be very stable conformationally as judged from temperature-induced denaturation monitored by FTIR and the conformation-sensitive dye. Upon heating to 80 degrees C, only local unfolding was revealed, while individual state-specific cross-beta features were preserved. The current studies demonstrated that the two amyloid states formed by the same amino acid sequence exhibited significantly different folding patterns that presumably reflect two different architectures of cross-beta-structure. Both S- and R-fibrils, however, shared high conformational stability, arguing that the energy landscape for protein folding and aggregation can contain several deep free-energy minima.
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Affiliation(s)
- Valeriy G Ostapchenko
- Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, MD 21201, USA
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Tixador P, Herzog L, Reine F, Jaumain E, Chapuis J, Le Dur A, Laude H, Béringue V. The physical relationship between infectivity and prion protein aggregates is strain-dependent. PLoS Pathog 2010; 6:e1000859. [PMID: 20419156 PMCID: PMC2855332 DOI: 10.1371/journal.ppat.1000859] [Citation(s) in RCA: 133] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2009] [Accepted: 03/16/2010] [Indexed: 11/18/2022] Open
Abstract
Prions are unconventional infectious agents thought to be primarily composed of PrP(Sc), a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP(C)). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP(Sc) conformation could encode this 'strain' diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP(Sc) aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP(Sc) aggregates from PrP(C). The distribution of PrP(Sc) and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP(Sc) peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12-30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP(Sc) aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.
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Affiliation(s)
- Philippe Tixador
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Laëtitia Herzog
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Fabienne Reine
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Emilie Jaumain
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Jérôme Chapuis
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Annick Le Dur
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
| | - Hubert Laude
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
- * E-mail: (HL); (VB)
| | - Vincent Béringue
- INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
- * E-mail: (HL); (VB)
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Pigmented creatine deposits in Amyotrophic Lateral Sclerosis central nervous system tissues identified by synchrotron Fourier Transform Infrared microspectroscopy and X-ray fluorescence spectromicroscopy. Neuroscience 2010; 166:1119-28. [DOI: 10.1016/j.neuroscience.2010.01.017] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2009] [Revised: 01/06/2010] [Accepted: 01/08/2008] [Indexed: 11/18/2022]
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Ritchie DL, Boyle A, McConnell I, Head MW, Ironside JW, Bruce ME. Transmissions of variant Creutzfeldt-Jakob disease from brain and lymphoreticular tissue show uniform and conserved bovine spongiform encephalopathy-related phenotypic properties on primary and secondary passage in wild-type mice. J Gen Virol 2009; 90:3075-3082. [PMID: 19656962 DOI: 10.1099/vir.0.013227-0] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Prion strains are defined by their biological properties after transmission to wild-type mice, specifically by their incubation periods and patterns of vacuolar pathology ('lesion profiles'). Preliminary results from transmissions of variant Creutzfeldt-Jakob disease (vCJD) to wild-type mice provided the first compelling evidence for the close similarity of the vCJD agent to the agent causing bovine spongiform encephalopathy (BSE). Complete results from this investigation, including the transmission characteristics of vCJD from brain and peripheral tissues of 10 cases (after primary transmission and subsequent mouse-to-mouse passage), have now been analysed. All 10 vCJD sources resulted in consistent incubation periods and lesion profiles, suggesting that all 10 patients were infected with the same strain of agent. Incubation periods suggested that infectious titres may be subject to regional variation within the brain. Comparison of incubation periods and lesion profiles from transmission of brain and peripheral tissues showed no evidence of tissue-specific modification in the biological properties of the agent. Analysis of the protease-resistant prion protein (PrP(res)) by Western blotting from primary and subsequent passages in mice showed a glycosylation pattern closely resembling that of vCJD in humans, the so-called BSE 'glycoform signature'. Minor variations in PrP(res) fragment size were evident between mouse strains carrying different alleles of the gene encoding PrP both in primary transmissions and on further passages of vCJD brain. Overall, the results closely resembled those of previously reported transmissions of BSE in the same mouse strains, consistent with BSE being the origin of all of these vCJD cases.
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Affiliation(s)
- Diane L Ritchie
- National CJD Surveillance Unit, School of Molecular and Clinical Medicine (Pathology), University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
| | - Aileen Boyle
- Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, The Roslin Institute, Roslin Biocentre, Roslin EH25 9PS, Midlothian, UK
| | - Irene McConnell
- Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, The Roslin Institute, Roslin Biocentre, Roslin EH25 9PS, Midlothian, UK
| | - Mark W Head
- National CJD Surveillance Unit, School of Molecular and Clinical Medicine (Pathology), University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
| | - James W Ironside
- National CJD Surveillance Unit, School of Molecular and Clinical Medicine (Pathology), University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
| | - Moira E Bruce
- Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, The Roslin Institute, Roslin Biocentre, Roslin EH25 9PS, Midlothian, UK
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Baskakov IV. Switching in amyloid structure within individual fibrils: implication for strain adaptation, species barrier and strain classification. FEBS Lett 2009; 583:2618-22. [PMID: 19482025 DOI: 10.1016/j.febslet.2009.05.044] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2009] [Revised: 05/19/2009] [Accepted: 05/25/2009] [Indexed: 01/09/2023]
Abstract
Amyloid fibrils are highly ordered crystal-like structures. It is generally assumed that individual amyloid fibrils consist of conformationally uniform cross-beta-sheet structures that enable the amyloids to replicate their individual conformations via a template-dependent mechanism. Recent studies revealed that amyloids are capable of accommodating a global conformational switch from one amyloid strain to another within individual fibrils. The current review highlights the high adaptation potential of amyloid structures and discusses the implication of these findings for several emerging issues including prion strain adaptation (i.e. gradual change in strain structure). It also proposes that the catalytic activity of an amyloid structure should be separated from its templating effect, and raises the question of strain classification according to their promiscuous or species-specific nature.
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Affiliation(s)
- Ilia V Baskakov
- Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.
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Moore RA, Taubner LM, Priola SA. Prion protein misfolding and disease. Curr Opin Struct Biol 2009; 19:14-22. [PMID: 19157856 DOI: 10.1016/j.sbi.2008.12.007] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2008] [Revised: 12/02/2008] [Accepted: 12/04/2008] [Indexed: 12/21/2022]
Abstract
Transmissible spongiform encephalopathies (TSEs or prion diseases) are a rare group of invariably fatal neurodegenerative disorders that affect humans and other mammals. TSEs are protein misfolding diseases that involve the accumulation of an abnormally aggregated form of the normal host prion protein (PrP). They are unique among protein misfolding disorders in that they are transmissible and have different strains of infectious agents that are associated with unique phenotypes in vivo. A wealth of biological and biophysical evidence now suggests that the molecular basis for prion diseases may be encoded by protein conformation. The purpose of this review is to provide an overview of the existing structural information for PrP within the context of what is known about the biology of prion disease.
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Affiliation(s)
- Roger A Moore
- Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, NIAID, NIH, 903 S. 4th Street, Hamilton, MT 59840, United States.
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Green KM, Castilla J, Seward TS, Napier DL, Jewell JE, Soto C, Telling GC. Accelerated high fidelity prion amplification within and across prion species barriers. PLoS Pathog 2008; 4:e1000139. [PMID: 18769716 PMCID: PMC2516356 DOI: 10.1371/journal.ppat.1000139] [Citation(s) in RCA: 101] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2008] [Accepted: 08/01/2008] [Indexed: 11/28/2022] Open
Abstract
Experimental obstacles have impeded our ability to study prion transmission within and, more particularly, between species. Here, we used cervid prion protein expressed in brain extracts of transgenic mice, referred to as Tg(CerPrP), as a substrate for in vitro generation of chronic wasting disease (CWD) prions by protein misfolding cyclic amplification (PMCA). Characterization of this infectivity in Tg(CerPrP) mice demonstrated that serial PMCA resulted in the high fidelity amplification of CWD prions with apparently unaltered properties. Using similar methods to amplify mouse RML prions and characterize the resulting novel cervid prions, we show that serial PMCA abrogated a transmission barrier that required several hundred days of adaptation and subsequent stabilization in Tg(CerPrP) mice. While both approaches produced cervid prions with characteristics distinct from CWD, the subtly different properties of the resulting individual prion isolates indicated that adaptation of mouse RML prions generated multiple strains following inter-species transmission. Our studies demonstrate that combined transgenic mouse and PMCA approaches not only expedite intra- and inter-species prion transmission, but also provide a facile means of generating and characterizing novel prion strains.
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Affiliation(s)
- Kristi M. Green
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
| | - Joaquín Castilla
- Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America
| | - Tanya S. Seward
- Sanders Brown Center on Aging, University of Kentucky, Lexington, Kentucky, United States of America
| | - Dana L. Napier
- Sanders Brown Center on Aging, University of Kentucky, Lexington, Kentucky, United States of America
| | - Jean E. Jewell
- Department of Veterinary Sciences, University of Wyoming, Laramie, Wyoming, United States of America
| | - Claudio Soto
- Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America
| | - Glenn C. Telling
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
- Sanders Brown Center on Aging, University of Kentucky, Lexington, Kentucky, United States of America
- Department of Neurology, University of Kentucky, Lexington, Kentucky, United States of America
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Scrapie prion protein structural constraints obtained by limited proteolysis and mass spectrometry. J Mol Biol 2008; 382:88-98. [PMID: 18621059 DOI: 10.1016/j.jmb.2008.06.070] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2008] [Revised: 05/16/2008] [Accepted: 06/24/2008] [Indexed: 01/30/2023]
Abstract
Elucidation of the structure of scrapie prion protein (PrP(Sc)), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP(Sc). Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in beta-strands. We treated PrP(Sc) isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP(Sc) was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrP(Sc) confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP(Sc) are susceptible to PK. Our results indicate that besides the "classic" amino-terminal PK cleavage points, PrP(Sc) contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short beta-sheet stretches connected by loops and turns.
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Makarava N, Baskakov IV. The same primary structure of the prion protein yields two distinct self-propagating states. J Biol Chem 2008; 283:15988-96. [PMID: 18400757 DOI: 10.1074/jbc.m800562200] [Citation(s) in RCA: 94] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The question of whether distinct self-propagating structures could be formed within the same amino acid sequence in the absence of external cofactors or templates has important implications for a number of issues, including the origin of prion strains and the engineering of smart, self-assembling peptide-based biomaterials. In the current study, we showed that chemically identical prion protein can give rise to conformationally distinct, self-propagating amyloid structures in the absence of cellular cofactors, post-translational modification, or PrP(Sc)-specified templates. Even more surprising, two self-replicating states were produced under identical solvent conditions, but under different shaking modes. Individual prion conformations were inherited by daughter fibrils in seeding experiments conducted under alternative shaking modes, illustrating the high fidelity of fibrillation reactions. Our study showed that the ability to acquire conformationally different self-propagating structures is an intrinsic ability of protein fibrillation and strongly supports the hypothesis that conformational variation in self-propagating protein states underlies prion strain diversity.
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Affiliation(s)
- Natallia Makarava
- Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA
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Baron T, Bencsik A, Biacabe AG, Morignat E, Bessen RA. Phenotypic similarity of transmissible mink encephalopathy in cattle and L-type bovine spongiform encephalopathy in a mouse model. Emerg Infect Dis 2008. [PMID: 18258040 PMCID: PMC2876762 DOI: 10.3201/eid13112.070635] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
L-type BSE is a more likely candidate for the origin of TME than typical BSE. Transmissible mink encepholapathy (TME) is a foodborne transmissible spongiform encephalopathy (TSE) of ranch-raised mink; infection with a ruminant TSE has been proposed as the cause, but the precise origin of TME is unknown. To compare the phenotypes of each TSE, bovine-passaged TME isolate and 3 distinct natural bovine spongiform encephalopathy (BSE) agents (typical BSE, H-type BSE, and L-type BSE) were inoculated into an ovine transgenic mouse line (TgOvPrP4). Transgenic mice were susceptible to infection with bovine-passaged TME, typical BSE, and L-type BSE but not to H-type BSE. Based on survival periods, brain lesions profiles, disease-associated prion protein brain distribution, and biochemical properties of protease-resistant prion protein, typical BSE had a distint phenotype in ovine transgenic mice compared to L-type BSE and bovine TME. The similar phenotypic properties of L-type BSE and bovine TME in TgOvPrP4 mice suggest that L-type BSE is a much more likely candidate for the origin of TME than is typical BSE.
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Affiliation(s)
- Thierry Baron
- Agence Française de Sécurité Sanitaire des Aliments-Lyon, Lyon, France.
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Lemmer K, Mielke M, Kratzel C, Joncic M, Oezel M, Pauli G, Beekes M. Decontamination of surgical instruments from prions. II. In vivo findings with a model system for testing the removal of scrapie infectivity from steel surfaces. J Gen Virol 2008; 89:348-358. [PMID: 18089760 DOI: 10.1099/vir.0.83396-0] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The unusual resistance of agents causing transmissible spongiform encephalopathies (TSEs) to chemical or thermal inactivation requires special decontamination procedures in order to prevent accidental transmission of these pathogens by surgical instruments. In the search for effective, instrument-compatible and routinely applicable decontamination procedures, a previous study [Lemmer, K., Mielke, M., Pauli, G. & Beekes, M. (2004). J Gen Virol 85, 3805-3816] identified promising reagents in an in vitro carrier assay using steel wires contaminated with the disease-associated prion protein, PrP(Sc). In the follow-up study presented here, these reagents were validated for their decontamination potential in vivo. Steel wires initially loaded with >or=3 x 10(5) LD(50) of 263K scrapie infectivity were implanted into the brains of hamsters after treatment for decontamination and subsequently monitored for their potential to trigger clinical disease or subclinical cerebral PrP(Sc) deposition within an observation period of 500 days. It was found that routinely usable reagents such as a commercially available alkaline cleaner (pH 12.2) applied for 1 h at 23 degrees C or for 10 min at 55 degrees C and a mixture of 0.2 % SDS and 0.3 % NaOH (pH 12.8) applied for 5 or 10 min at 23 degrees C achieved removal of 263K scrapie infectivity below the threshold of detection (titre reduction of >or=5.5 log(10) units). The increasing use during the past few years of similar model systems by different research groups will facilitate comparison and integration of findings on the decontamination of steel surfaces from prions. Methods identified as highly effective in the 263K steel wire model need to be validated for human TSE agents on different types of instrument surfaces.
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Affiliation(s)
- Karin Lemmer
- P24, Transmissible Spongiform Encephalopathies, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
| | - Martin Mielke
- FG 14, Applied Infection Control and Hospital Hygiene, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
| | - Christine Kratzel
- P24, Transmissible Spongiform Encephalopathies, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
| | - Marion Joncic
- P24, Transmissible Spongiform Encephalopathies, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
| | - Muhsin Oezel
- ZBS4, Centre for Biological Safety - Imaging Techniques for Rapid Morphology-Based Diagnostics of Infectious Organisms, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
| | - Georg Pauli
- ZBS1, Centre for Biological Safety - Highly Pathogenic Viruses, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
| | - Michael Beekes
- P24, Transmissible Spongiform Encephalopathies, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
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50
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Grassi J, Maillet S, Simon S, Morel N. Progress and limits of TSE diagnostic tools. Vet Res 2008; 39:33. [PMID: 18284910 DOI: 10.1051/vetres:2008009] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2007] [Accepted: 02/05/2008] [Indexed: 11/14/2022] Open
Abstract
Following the two "mad cow" crises of 1996 and 2000, there was an urgent need for rapid and sensitive diagnostic methods to identify animals infected with the bovine spongiform encephalopathy (BSE) agent. This stimulated research in the field of prion diagnosis and led to the establishment of numerous so-called "rapid tests" which have been in use in Europe since 2001 for monitoring at-risk populations (rendering plants) and animals slaughtered for human consumption (slaughterhouse). These rapid tests have played a critical role in the management of the mad cow crisis by allowing the removal of prion infected carcasses from the human food chain, and by allowing a precise epidemiological monitoring of the BSE epizootic. They are all based on the detection of the abnormal form of the prion protein (PrP(Sc) or PrP(res)) in brain tissues and consequently are only suitable for post-mortem diagnosis. Since it is now very clear that variant Creutzfeldt-Jakob disease (vCJD) can be transmitted by blood transfusion, the development of a blood test for the diagnosis of vCJD is a top priority. Although significant progress has been made in this direction, including the development of the protein misfolding cyclic amplification (PMCA) technology, at the time this paper was written, this objective had not yet been achieved. This is the most important challenge for the years to come in this field of prion research.
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Affiliation(s)
- Jacques Grassi
- Service de Pharmacologie et d'Immunoanalyse, 91191 Gif-sur-Yvette, France.
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