Chaperone-supervised conversion of prion protein to its protease-resistant form

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.     

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0.1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.     

Site-specific insertion of a SINE-like element, Cp1, into centromeric tandem repeats from Chironomus pallidivittatus

A SINE-like dispersed element, Cp1, from the dipteran Chironomus pallidivittatus was found to show site-specific insertion into two different centromeric tandem repeats. The insertions result in identical target site duplications of nine base-pairs. In contrast, extracentromeric Cp1 elements, which are polymorphic and degenerate, are previously known to be surrounded by different target site duplications. The intracentromeric Cp1 is uniform in structure and contains a single pol III unit, upstream of which 87 bp arms of a palindrome surround a 103 bp unique sequence. The numbers of Cp1 elements per centromere were determined in microdissected material and were found to be in the range of five to ten units per centromere. The well-defined insertion properties, correlated to chromosomal localization, suggest that Cp1 is likely to be a component of importance for the centromere. Similarities of Cp1 and its parts to functionally identified centromeres in Saccharomyces cerevisiae and Schizosaccharomyces pombe are discussed.     

Molecular characterization of a Plasmodium chabaudi erythrocyte membrane-associated protein with glutamate-rich tandem repeats

BACKGROUND: The fourth American College of Chest Physicians Consensus Conference on Antithrombotic Therapy recently published guidelines that included recommendations regarding the management of excessive anticoagulation. Limited data are available to support these recommendations. OBJECTIVES: To assess management and outcomes of excessive anticoagulation in a group model health maintenance organization, compare management with the published guidelines, and analyze the cost of treatment strategies. METHODS: A search of computerized laboratory information identified patients with an international normalized ratio (INR) of greater than 6.0 during the 9-month study. Pertinent data were collected through a retrospective medical record review. Information was concurrently collected for cost analyses. RESULTS: The analysis included 301 episodes of excessive anticoagulation among 248 patients. Most (83%) episodes of elevated INRs were managed conservatively by a temporary discontinuation of warfarin sodium therapy until the INR was in a therapeutic range. Conservative management resulted in no sequelae in 212 (85.1%) of 249 episodes. Two episodes (0.8%) of major bleeding evolved in patients managed conservatively. No sequelae were documented in 23 (44%) of 52 episodes of phytonadione (vitamin K1) administration. Sixteen (31%) episodes of major bleeding were documented, but bleeding occurred before phytonadione administration in all cases. Administering phytonadione resulted in hospital admission for 3 patients--2 (3.8%) because of thromboembolism and 1 (1.9%) for the administration of heparin sodium. Cost-effectiveness analysis determined that treatment with phytonadione is 7 times more costly than conservative management when INRs are between 6.0 and 10.0. CONCLUSIONS: Most episodes of excessive anticoagulation were not managed per consensus guidelines. The higher the INR, the more likely were interventions to adhere to the guidelines. Administering phytonadione to patients with a moderate elevation of INRs (6.0-10.0) may be unnecessary. Based on this study, conservative management is a viable option.     

A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion

The yeast [PSI+] factor propagates by a prion-like mechanism involving self-replicating Sup35p amyloids. We identified multiple Sup35p mutants that either are poorly recruited into, or cause curing of, wildtype amyloids in vivo. In vitro, these mutants showed markedly decreased rates of amyloid formation, strongly supporting the protein-only prion hypothesis. Kinetic analysis suggests that the prion state replicates by accelerating slow conformational changes rather than by providing stable nuclei. Strikingly, our mutations map exclusively within a short glutamine/asparagine-rich region of Sup35p, and all but one occur at polar residues. Even after replacement of this region with polyglutamine, Sup35p retains its ability to form amyloids. These and other considerations suggest similarities between the prion-like propagation of [PSI+] and polyglutamine-mediated pathogenesis of several neurodegenerative diseases.     

Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites

Treatment of rabbit sarcoplasmic reticulum Ca2+-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp351 and ending in the Lys605-Arg615 region, well before the conserved sequences generally described as constituting the "hinge" region of this P-type ATPase (residues 670-760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca2+-ATPase as follows: high sensitivity of peptidic bond Arg505-Ala506 to trypsin cleavage, and high reactivity of lysine residue Lys515 toward the fluorescent label fluorescein 5'-isothiocyanate. Regarding functional properties, these fragments retain the ability to bind nucleotides, although with reduced affinity compared with intact Ca2+-ATPase. The fragments also bind Nd3+ ions, leaving open the possibility that these fragments could contain the metal-binding site(s) responsible for the inhibitory effect of lanthanide ions on ATPase activity. The p29/30 soluble domain, like similar proteolytic fragments that can be obtained from other P-type ATPases, may be useful for obtaining three-dimensional structural information on the cytosolic portion of these ATPases, with or without bound nucleotides. From our findings we infer that a real hinge region with conformational flexibility is located at the C-terminal boundary of p29/30 (rather than in the conserved region of residues 670-760); we also propose that the ATP-binding cleft is mainly located within the p29/30 domain, with the phosphorylation site strategically located at the N-terminal border of this domain.     

Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells

The central causative event in infectious, familial, and sporadic forms of prion disease is thought to be a conformational change that converts the cellular isoform of the prion protein (PrPC) into the scrapie isoform (PrPSc) that is the primary constituent of infectious prion particles. To provide a model system for analyzing the mechanistic details of this critical transformation, we have previously prepared cultured Chinese hamster ovary cells that stably express mouse PrP molecules carrying mutations homologous to those seen in familial prion diseases of humans. In the present work, we have analyzed the kinetics with which a PrP molecule containing an insertional mutation associated with Creutzfeldt-Jakob disease acquires several biochemical properties characteristic of PrPSc. Within 10 min of pulse labeling, the mutant protein undergoes a molecular alteration that is detectable by a change in Triton X-114 phase partitioning and phenyl-Sepharose binding. After 30 min of labeling, a detergent-insoluble and protease-sensitive form of the protein appears. After a chase period of several hours, the protein becomes protease-resistant. Incubation of cells at 18 degrees C or treatment with brefeldin A inhibits acquisition of detergent insolubility and protease resistance but does not affect Triton X-114 partitioning and phenyl-Sepharose binding. Our results support a model in which conversion of mutant PrPs to a PrPSc-like state proceeds in a stepwise fashion via a series of identifiable biochemical intermediates, with the earliest step occurring during or very soon after synthesis of the polypeptide in the endoplasmic reticulum.     

The integrin alpha IIb-beta 3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit

Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469). Sf9 insect cells were infected with the corresponding baculovirus, and the produced soluble recombinant proteins were purified using an RGD-like affinity column. The bound receptor fragments were specifically eluted with RGDS and existed as a heterodimeric complex (rec alpha IIbH-t beta 3) with an apparent M(r) of 160,000. In an immunocapture assay, the monoclonal antibody pl-55, which only recognizes the functionally active form of alpha IIb-beta 3, bound to the purified complex. Rec alpha IIbH-t beta 3 specifically bound 125I-fibrinogen with an affinity comparable with that of purified platelet alpha IIb-beta 3. Electron micrographs of rotary-shadowed rec alpha IIbH-t beta 3 showed that the complex had the characteristic globular head, but the two rodlike tails were 4-6 nm shorter than those found in intact alpha IIb-beta 3. Thus, the cysteine-rich repeats of beta 3 are not required for assembly, stability, and functional activity of this integrin.     

Attention to orientation, size, luminance, and color: Attentional failure within the form domain.

Whether Ss can attend to physical dimensions of objects without access to semantic information about them was examined. Ss decided which of 2 laterally presented pictures, a target and a distractor, had the same orientation (Exp 1), size (Exp 2), luminance (Exp 3), or color (Exp 4) as a reference picture. In each experiment, the matching stimuli were either physically identical, semantically related, or semantically unrelated. The reference stimulus and the distractor were either semantically related or unrelated. When matching was based on orientation or on size, performance was facilitated when the matching stimuli were semantically related, and it was disrupted when the distractor was semantically related to the reference stimulus. Semantic effects were eliminated when matching was based on luminance or color. The results are discussed in terms of physiological data, form and surface information, and global and local processing. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The N-terminal half of initiation factor IF3 is folded as a stable independent domain

Initiation factor IF3 plays an essential role in the initiation of protein translation by binding to the 30S ribosomal subunit and selecting a proper tRNA(fMet)/initiation codon complex. The domain structure of IF3 from Escherichia coli has been investigated by limited proteolysis followed by mass spectrometry and protein sequencing of the resulting peptides. This analysis revealed a highly segmented structure with two independent domains connected by a charged linker peptide, highly susceptible to proteolytic cleavage. The N-terminal domain is very stable and comparison of its 2-D NMR spectrum with that of intact IF3 revealed that it retains its three-dimensional fold.     

Rapid trisomy diagnosis (21, 18, and 13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis

PURPOSE AND METHODS: Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. This requires lengthy laboratory procedures and high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid and inexpensive and may potentially be suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction (F-PCR) using polymorphic small tandem repeats (STRs). RESULTS: In this paper we present data demonstrating that fluorescent PCR amplification of STRs can be used for rapid diagnosis of trisomy 21, trisomy 18, and trisomy 13 and can be successfully applied to both prenatal diagnosis and diagnosis of single cells. This study also reports significant numbers of prenatal diagnoses using quantitative fluorescent PCR. CONCLUSIONS: We believe that further studies of greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for trisomy diagnosis from single cells using multiple STR markers for either preimplantation genetic diagnosis or, potentially, diagnosis from fetal cells isolated from maternal blood.     

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), a neural member of the EGF family of differentiation factors, is implicated in neurite formation

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline-rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.     

Unexpected consequences of deletion of the first two repeats of the ligand-binding domain from the low density lipoprotein receptor. Evidence from a human mutation

Heterozygosity for a 5-kilobase (kb) deletion of the first two ligand-binding repeats (exons 2 and 3) of the low density lipoprotein (LDL) receptor (R) gene (LDL-R delta 5kb) confers familial hypercholesterolemia (FH). The FH phenotype is unexpected based on previous site-directed mutagenesis showing that deletion of exons 2 and 3 resulted in little or no defect in LDL-R activity. In the present study, we took unique advantage of the ability to distinguish the LDL-R delta 5kb from the normal receptor on the basis of size, in order to resolve this apparent discrepancy. Fibroblasts from heterozygotes for the LDL-R delta 5kb displayed 50% of normal capacity to bind LDL and beta-VLDL, apparently due to lower receptor number. Cellular mRNA for the delta 5kb allele was at least as abundant as that for the normal allele. Immunoblotting and cell binding assays with anti-LDL-R antibody IgG-4A4 demonstrated normal synthesis and transport of the delta 5kb receptor. Ligand blotting demonstrated that the delta 5kb receptor displayed minimal or no ability to bind LDL or beta-VLDL. Thus, in contrast to transfected cell lines, in human fibroblasts, the first two cysteine rich repeats of the LDL-R appear functionally necessary. These characteristics of the LDL-R delta 5kb in human fibroblasts explain the in vivo phenotype of carriers.     

Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.     

Alpha-crystallin, a molecular chaperone, forms a stable complex with carbonic anhydrase upon heat denaturation

Alpha-crystallin, a major eye lens protein of vertebrates has been characterized as a molecular chaperone based on its ability to inhibit the aggregation of proteins undergoing thermal denaturation (Horwitz, J., Proc. Natl. Acad. Sci. USA 1992, 89, 10449-10453). To understand the mechanisms underlying this chaperone-like activity, the present study addressed molecular interactions between alpha-crystallin and its target proteins. Using carbonic anhydrase as a model target protein, we demonstrate complex formation between the 2 proteins upon heating, as assessed by the criteria of agarose gel electrophoresis, immunoprecipitation, ultrafiltration and gel filtration chromatography. The complex of alpha-crystallin and carbonic anhydrase is stable, at room temperature and at 4 degrees C, for over 18 hours, and is non-covalent in nature. The results also indicate that alpha-crystallin binds the early non-native form of the target protein.