Several sporadic and genetic diseases are caused by protein misfolding. These include cystic fibrosis and other devastating diseases of childhood as well as Alzheimer's, Parkinson's and other ...debilitating maladies of the elderly. A unified view of the molecular and cellular pathogenesis of these conditions has led to the search for chemical chaperones that can slow, arrest or revert disease progression. Molecules are now emerging that link our biophysical insights with our therapeutic aspirations.
Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt ...a cross-β structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform ( PrPSc) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right- or left-handed β-helical models. From a study of 119 all-β folds observed in globular proteins, we have now determined that, if PrPScfollows a known protein fold, it adopts either a β-sandwich or parallel β-helical architecture. With increasing evidence arguing for parallel β-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed β-helical fold. Left-handed β-helices readily form trimers, providing a natural template for a trimeric model of PrPSc. This trimeric model accommodates the PrP sequence from residues 89-175 in a β-helical conformation with the C terminus (residues 176-227), retaining the disulfide-linked α-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed β-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed β-helical model for PrPScmay provide important clues to the structure of filaments found in some other neurodegenerative diseases.
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.
A conformational isoform of the mammalian prion protein $(\Pr P^{Sc} )$ is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction ...patterns from infectious prions that show cross-ß diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of β strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four β-strands. We found that recombinant (ree) PrP amyloid differs substantially from highly infectious brainderived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked β-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.
Protease-sensitive synthetic prions Colby, David W; Wain, Rachel; Baskakov, Ilia V ...
PLOS pathogens,
01/2010, Letnik:
6, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Prions arise when the cellular prion protein (PrP(C)) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc). Frequently, PrP(Sc) is ...protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc) and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc). These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc).
Prion diseases in humans and animals are invariably fatal. Prions are composed of a disease-causing isoform (PrPSc) of the normal host prion protein (PrPC) and replicate by stimulating the conversion ...of PrPCinto nascent PrPSc. We report here that tricyclic derivatives of acridine and phenothiazine exhibit half-maximal inhibition of PrPScformation at effective concentrations (EC50) between 0.3 µM and 3 µM in cultured cells chronically infected with prions. The EC50for chlorpromazine was 3 µM, whereas quinacrine was 10 times more potent. A variety of 9-substituted, acridine-based analogues of quinacrine were synthesized, which demonstrated variable antiprion potencies similar to those of chlorpromazine and emphasized the importance of the side chain in mediating the inhibition of PrPScformation. Thus, our studies show that tricyclic compounds with an aliphatic side chain at the middle ring moiety constitute a new class of antiprion reagents. Because quinacrine and chlorpromazine have been used in humans for many years as antimalarial and antipsychotic drugs, respectively, and are known to pass the blood-brain barrier, we suggest that they are immediate candidates for the treatment of Creutzfeldt-Jakob disease and other prion diseases.
Pathway Complexity of Prion Protein Assembly into Amyloid Baskakov, Ilia V; Legname, Giuseppe; Baldwin, Michael A ...
Journal of biological chemistry/The Journal of biological chemistry,
06/2002, Letnik:
277, Številka:
24
Journal Article
Recenzirano
Odprti dostop
In vivo under pathological conditions, the normal cellular form of the prion protein, PrP C (residues 23â231), misfolds to the pathogenic isoform PrP Sc , a β-rich aggregated pathogenic multimer. ...Proteinase K digestion of PrP Sc leads to a proteolytically resistant core, PrP 27â30 (residues 90â231), that can form amyloid fibrils. To study the kinetic
pathways of amyloid formation in vitro , we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP Sc and found that it can adopt two non-native abnormal isoforms, a β-oligomer and an amyloid fibril. Several lines of kinetic
data suggest that the β-oligomer is not on the pathway to amyloid formation. The preferences for forming either a β-oligomer
or amyloid can be dictated by experimental conditions, with acidic pH similar to that seen in endocytic vesicles favoring
the β-oligomer and neutral pH favoring amyloid. Although both abnormal isoforms have high β-sheet content and bind 1-anilinonaphthalene-8-sulfonate,
they are dissimilar structurally. Multiple pathways of misfolding and the formation of distinct β-sheet-rich abnormal isoforms
may explain the difficulties in refolding PrP Sc in vitro , the need for a PrP Sc template, and the significant variation in disease presentation and neuropathology.
On passaging synthetic prions, two isolates emerged with incubation times differing by nearly 100 days. Using conformational-stability assays, we determined the guanidine hydrochloride (Gdn-HCI) ...concentration required to denature 50% of disease-causing prion protein ($PrP^{Sc}$) molecules, denoted as the $Gdn\cdotHCl_{1/2}$ value. For the two prion isolates enciphering shorter and longer incubation times, $Gdn\cdotHCl_{1/2}$ values of 2.9 and 3.7 M, respectively, were found. Intrigued by this result, we measured the conformational stabilities of 30 prion isolates from synthetic and naturally occurring sources that had been passaged in mice. When the incubation times were plotted as a function of the $Gdn\cdotHCl_{1/2}$ values, a linear relationship was found with a correlation coefficient of 0.93. These findings demonstrate that (i) less stable prions replicate more rapidly than do stable prions, and (ii) a continuum of $PrP^{Sc}$ structural states enciphers a multitude of incubation-time phenotypes. Our data argue that cellular machinery must exist for propagating a large number of different $PrP^{Sc}$ conformers, each of which enciphers a distinct biological phenotype as reflected by a specific incubation time. The biophysical explanation for the unprecedented plasticity of $PrP^{Sc}$ remains to be determined.
Infantile hemangiomas (IHs) are the most common tumors of childhood. Unlike other tumors, they have the unique ability to involute after proliferation, often leading primary care providers to assume ...they will resolve without intervention or consequence. Unfortunately, a subset of IHs rapidly develop complications, resulting in pain, functional impairment, or permanent disfigurement. As a result, the primary clinician has the task of determining which lesions require early consultation with a specialist. Although several recent reviews have been published, this clinical report is the first based on input from individuals representing the many specialties involved in the treatment of IH. Its purpose is to update the pediatric community regarding recent discoveries in IH pathogenesis, treatment, and clinical associations and to provide a basis for clinical decision-making in the management of IH.
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