The vibrational Raman optical activity (ROA) spectrum of a polypeptide in a model beta-sheet conformation, that of poly(l-lysine), was measured for the first time, and the alpha-helix --> beta-sheet transition monitored as a function of temperature in H(2)O and D(2)O. Although no significant population of a disordered backbone state was detected at intermediate temperatures, some side chain bands not present in either the alpha-helix or beta-sheet state were observed. The observation of ROA bands in the extended amide III region assigned to beta-turns suggests that, under our experimental conditions, beta-sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-lysine) are different from those observed in typical beta-sheet proteins and may be characteristic of an extended flat multistranded beta-sheet, which is unlike the more irregular and twisted beta-sheet found in most proteins. However, a reduced isoform of the truncated ovine prion protein PrP(94-233) that is rich in beta-sheet shows amide I and amide II ROA bands similar to those of beta-sheet poly(L-lysine), which suggests that the C-terminal domain of the prion protein is able to support unusually flat beta-sheets. A principal component analysis (PCA) that identifies protein structural types from ROA band patterns provides a useful representation of the structural relationships among the polypeptide and protein states considered in the study.
The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.
Avian infectious bronchitis virus (IBV) is a member of theCoronaviridae (order Nidovirales) (9), members of which are enveloped viruses with single-stranded, positive-sense RNA genomes that are 5Ј capped and 3Ј polyadenylated (30, 63). The 5Ј two-thirds of the coronavirus genome encodes the replicase gene producing two polyproteins, Rep1a and Rep1ab, the latter resulting from a Ϫ1 frameshift (7). The remaining proteins, which include the nucleoprotein (N), are expressed from a nested set of subgenomic mRNAs (sgRNAs) that are produced via a discontinuous transcription mechanism (6, 30). Each of these sgRNAs has a short nontranslated leader sequence (64 nucleotides for IBV) derived from the 5Ј end of the genome. Present within the leader sequence is a consensus sequence, which we have termed the transcription-associated sequence (TAS) (24). The TAS contains a conserved core motif, which in the case of IBV is CUUAACAA, which is also located in the genome, proximal to the start site for each sgRNA. For different coronaviruses, the core sequence varies and can be present more than once per TAS.N protein, the virus RNA binding protein, is one of the most abundant viral proteins in an infected cell (31). Several functions have been postulated for the coronavirus N protein throughout the virus life cycle (31); primarily, it complexes with the genomic RNA to form a ribonucleocapsid structure (17) and associates with the M protein (19, 39) to form the viral core (48). While N protein is required in trans to rescue the full-length clone of IBV (8) and a porcine coronavirus transmissible gastroenteritis virus clone (82), it is not required for others (1,71,72). Certainly, in the case of the rescue of the full-length clone of severe acute respiratory syndrome coronavirus, the presence of N protein increases viral titers compared to rescue performed in the absence of N transcript (83), suggesting that N protein may be involved in the efficiency of replication but that it is not essential.Based on amino acid sequence comparisons, three domains have been identified in the murine coronavirus, mouse hepatitis virus (MHV) N protein (46), of which the central domain (domain II) was identified as a potential RNA binding site (35, 40) capable of binding both coronavirus-and non-coronavirusderived RNA sequences (35,68). However, whether this binding occurs with equal or different affinity is uncertain (14,35,49). N protein has been shown to associate with several motifs on viral RNA, including the leader RNA sequence, with particular affinity for the core sequence of the TAS (2, 41), sequences at the 3Ј end of the genomic RNA (84), and the packaging signal (37). How these sequences promote N binding is unknown.Several coronavirus N proteins have been shown to be phosphorylated, including IBV, MHV, and transmissible gastroenteritis virus N proteins, although the precise sites were not identified (31). The role of phosphorylation in the virus life cycle is unknown, although the phosphorylation state of N protein has been predicted to pl...
We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2 -fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrP Sc than for recombinant PrP C and inhibited the accumulation of PrP res in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from nonspecific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, ␣-helix-rich and ␤-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the ␤-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the ␣-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the ␣-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is ␤-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.
The prion protein, PrPC, is a small, cell-surface glycoprotein notable primarily for its critical role in pathogenesis of the neurodegenerative disorders known as prion diseases. A hallmark of prion diseases is the conversion of PrPC into an abnormally folded isoform, which provides a template for further pathogenic conversion of PrPC, allowing disease to spread from cell to cell and, in some circumstances, to transfer to a new host. In addition to the putative neurotoxicity caused by the misfolded form(s), loss of normal PrPC function could be an integral part of the neurodegenerative processes and, consequently, significant research efforts have been directed toward determining the physiological functions of PrPC. In this review, we first summarise important aspects of the biochemistry of PrPC before moving on to address the current understanding of the various proposed functions of the protein, including details of the underlying molecular mechanisms potentially involved in these functions. Over years of study, PrPC has been associated with a wide array of different cellular processes and many interacting partners have been suggested. However, recent studies have cast doubt on the previously well-established links between PrPC and processes such as stress-protection, copper homeostasis and neuronal excitability. Instead, the functions best-supported by the current literature include regulation of myelin maintenance and of processes linked to cellular differentiation, including proliferation, adhesion, and control of cell morphology. Intriguing connections have also been made between PrPC and the modulation of circadian rhythm, glucose homeostasis, immune function and cellular iron uptake, all of which warrant further investigation.
In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP Sc produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP Sc recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a ␤-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP Sc , and the other to classical PrP Sc . Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP Sc with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.Keywords: prion protein; amyloid fibrils; conformational transition; proteinase K; Creutzfeldt-Jakob disease Several severe neurodegenerative diseases including Creutzfeldt-Jakob disease (CJD), Gerstmann-StrausslerSheinker disease, and fatal familial insomnia are associated with misfolding and aggregation of the prion protein (Prusiner 2001). Prion maladies manifest themselves in infectious, familial, and sporadic forms. To explain the infectious form of the prion diseases, the "protein-only hypothesis" postulates that an abnormal isoform of the prion protein, PrP Sc , acts as an infectious agent and propagates its pathological conformation in an autocatalytic manner using the normal isoform of the same protein, PrP C , as a substrate (Prusiner 1982). PrP C and PrP Sc differ substantially in their conformations. Unlike PrP C , PrP Sc is a multimeric assembly characterized by enhanced resistance to proteinase K (PK) digestion and by an increase in the amount of ␤-sheet structure Reprint requests to: Ilia V. Baskakov, 725 W. Lombard Street, Baltimore, MD 21201, USA; e-mail: Baskakov@umbi.umd.edu; fax: (410) 706-8184.Abbreviations: CJD, Creutzfeldt-Jakob disease; spCJD, sporadic CJD; PrP, prion protein; PrP C , the normal, cellular isoform of PrP; PrP Sc , the abnormal, infections isoform of PrP; rPrP, recombinant PrP; rPrP 89-230, recombinant PrP encompassing residues 89-230; rPrP 23-230, full-length recombinant PrP; ␣-rPrP 89-230, ␣-helical isoform of rPrP 89-231; ␣-rPrP 23-230, ␣-helical isoform of rPrP 23-231; PK, proteinase K; PrP 27-30, PK-resistant core of classical PrP ...
Tau is a microtubule-associated protein responsible mainly for stabilizing the neuronal microtubule network in the brain. Under normal conditions, tau is highly soluble and adopts an “unfolded” conformation. However, it undergoes conformational changes resulting in a less soluble form with weakened microtubule stabilizing properties. Altered tau forms characteristic pathogenic inclusions in Alzheimer's disease and related tauopathies. Although, tau hyperphosphorylation is widely considered to be the major trigger of tau malfunction, tau undergoes several post-translational modifications at lysine residues including acetylation, methylation, ubiquitylation, SUMOylation, and glycation. We are only beginning to define the site-specific impact of each type of lysine modification on tau biology as well as the possible interplay between them, but, like phosphorylation, these modifications are likely to play critical roles in tau's normal and pathobiology. This review summarizes the latest findings focusing on lysine post-translational modifications that occur at both endogenous tau protein and pathological tau forms in AD and other tauopathies. In addition, it highlights the significance of a site-dependent approach of studying tau post-translational modifications under normal and pathological conditions.
The human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies, but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the misfolding pathway that leads to the formation of ␤-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP to that of the valine 129 variant. We provide evidence for differences in the folding behavior between the two variants at the early stages of oligomer formation. We show that Met 129 has a higher propensity to form ␤-sheet-rich oligomers, whereas Val 129 has a higher tendency to fold into ␣-helical-rich monomers. An equimolar mixture of both variants displayed an intermidate folding behavior. We show that the oligomers of both variants are initially a mixture of ␣-and ␤-rich conformers that evolve with time to an increasingly homogeneous ␤-rich form. This maturation process, which involves no further change in proteinase K resistance, occurs more rapidly in the Met 129 form than the Val 129 form. Although the involvement of such ␤-rich oligomers in prion pathogenesis is speculative, the misfolding behavior could, in part, explain the higher susceptibility of individuals that are methionine homozygote to both sporadic and variant CreutzfeldtJakob disease.
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