Theodor ("Ted") Otto Diener (* 28 February 1921 in Zürich, Switzerland; † 28 March 2023 in Beltsville, MD, USA) pioneered research on viroids while working at the Plant Virology Laboratory, ...Agricultural Research Service, USDA, in Beltsville. He coined the name viroid and defined viroids' important features like the infectivity of naked single-stranded RNA without protein-coding capacity. During scientific meetings in the 1970s and 1980s, viroids were often discussed at conferences together with other "subviral pathogens". This term includes what are now called satellite RNAs and prions. Satellite RNAs depend on a helper virus and have linear or, in the case of virusoids, circular RNA genomes. Prions, proteinaceous infectious particles, are the agents of scrapie, kuru and some other diseases. Many satellite RNAs, like viroids, are non-coding and exert their function by thermodynamically or kinetically controlled folding, while prions are solely host-encoded proteins that cause disease by misfolding, aggregation and transmission of their conformations into infectious prion isoforms. In this memorial, we will recall the work of Ted Diener on subviral pathogens.
Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 ...DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.
Synthetic Mammalian Prions Legname, Giuseppe; Baskakov, Ilia V.; Nguyen, Hoang-Oanh B. ...
Science (American Association for the Advancement of Science),
07/2004, Letnik:
305, Številka:
5684
Journal Article
Recenzirano
Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of β sheet-rich structures. Fibrils consisting of recMoPrP(89-230) ...were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.
Prion detection by an amyloid seeding assay Colby, David W; Zhang, Qiang; Wang, Shuyi ...
Proceedings of the National Academy of Sciences - PNAS,
12/2007, Letnik:
104, Številka:
52
Journal Article
Recenzirano
Odprti dostop
Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially ...purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrPSc, the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt-Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was almost equal to1 fg. Some prion strains consist largely of protease-sensitive PrPSc (sPrPSc), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrPSc and protease-resistant PrPSc that does not rely on protease digestion or immunodetection.
In a brief historical description, it is shown that the prion model was developed from the biochemical and biophysical properties of the scrapie infectious agent. The biochemical properties of the ...prion protein which is the major, if not only, component of the prion are outlined in detail. PrP is a host-encoded protein which exists as PrPC (cellular) in the non-infected host, and as PrPSc (scrapie) as the major component of the scrapie infectious agent. An overview of the purification techniques is given. Although chemically identical, the biophysical features of PrPSc are drastically different in respect to solubility, structure, and stability; furthermore, specific lipids and a polyglucose scaffold were found in prions, whereas for nucleic acids their absence could be proven. The structure of recombinant PrP in solution is known from spectroscopic studies and with high resolution from NMR analysis. Structural models of PrPSc were derived recently from electron microscopic analysis of two-dimensional crystals. Conformational transitions of PrP in vitro were studied with different techniques in order to mimic the natural PrPC to PrPSc conversion. Spontaneous transitions can be induced by solvent changes, but at present infectivity cannot be induced in vitro.
To defend against invading pathogens, plants possess RNA silencing mechanisms involving small RNAs (miRNAs, siRNAs). Also viroids – plant infectious, non-coding, unencapsidated RNA – cause the ...production of viroid-specific small RNAs (vsRNA), but viroids do escape the cytoplasmic silencing mechanism. Viroids with minor sequence variations can produce different symptoms in infected plants, suggesting an involvement of vsRNAs in symptom production. We analyzed by deep sequencing the spectrum of vsRNAs induced by the PSTVd strain AS1, which causes strong symptoms such as dwarfing and necrosis upon infection of tomato plants cv Rutgers. Indeed, vsRNAs found with highest frequency mapped to the pathogenicity-modulating domain of PSTVd, supporting an involvement of vsRNAs in symptom production. Furthermore, in PSTVd AS1-infected plants the accumulation of some endogenous miRNAs, which are involved in leaf development via regulation of transcription factors, is suppressed. The latter finding supports the hypothesis that a miRNA-dependent (mis)regula-tion of transcription factors causes the viroid symptoms.
Abstract
Viroids were described 47 years ago as the smallest RNA molecules capable of infecting plants and autonomously self-replicating without an encoded protein. Work on viroids initiated the ...development of a number of innovative methods. Novel chromatographic and gelelectrophoretic methods were developed for the purification and characterization of viroids; these methods were later used in molecular biology, gene technology and in prion research. Theoretical and experimental studies of RNA folding demonstrated the general biological importance of metastable structures, and nuclear magnetic resonance spectroscopy of viroid RNA showed the partially covalent nature of hydrogen bonds in biological macromolecules. RNA biochemistry and molecular biology profited from viroid research, such as in the detection of RNA as template of DNA-dependent polymerases and in mechanisms of gene silencing. Viroids, the first circular RNA detected in nature, are important for studies on the much wider spectrum of circular RNAs and other non-coding RNAs.
Mechanisms of prion protein assembly into amyloid Stöhr, Jan; Weinmann, Nicole; Wille, Holger ...
Proceedings of the National Academy of Sciences - PNAS,
02/2008, Letnik:
105, Številka:
7
Journal Article
Recenzirano
Odprti dostop
The conversion of the α-helical, cellular isoform of the prion protein (PrPC) to the insoluble, β-sheet-rich, infectious, disease-causing isoform (PrPSc) is the key event in prion diseases. In an ...earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, α-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.
Structural changes of membrane-anchored native PrPC Elfrink, Kerstin; Ollesch, Julian; Stöhr, Jan ...
Proceedings of the National Academy of Sciences - PNAS,
08/2008, Letnik:
105, Številka:
31
Journal Article
Recenzirano
Odprti dostop
Misfolding and subsequent aggregation of endogeneous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the ...posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular β-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity, is thought to be a crucial event in the development of prion diseases.
We investigated the therapeutic effects of two different versions of Aβ₁₋₁₅ ₍₁₆₎ liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetra-palmitoylated ...amyloid 1-15 peptide (palmAβ₁₋₁₅), or with amyloid 1-16 peptide (PEG-Aβ₁₋₁₆) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmAβ₁₋₁₅ liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-Aβ₁₋₁₆ had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmAβ₁₋₁₅, whereas those elicited by PEG-Aβ₁₋₁₆ were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly β-sheet conformation of palmAβ₁₋₁₅ and random coil of PEG-Aβ₁₋₁₆. We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a "conformational" disease, implying that antibodies against amyloid sequences in the β-sheet conformation are preferred as potential therapeutic agents.
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