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F E Cohen

Publications and source records attributed to F E Cohen.

At least 73 records · Page 4Linked to original sources
PubMed1998

Eight prion strains have PrP(Sc) molecules with different conformations.

Variations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrP(Sc), are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrP(Sc) molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupies a unique position, indicative of a particular PrP(Sc) conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrP(Sc) found with each strain. Our findings indicate that each of the eight prion strains has a PrP(Sc) molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrP(Sc) and that the variation in incubation times is related to the relative protease sensitivity of PrP(Sc) in each strain.

Animals↗
PubMed1998

Pathologic conformations of prion proteins.

While many aspects of prion disease biology are unorthodox, perhaps the most fundamental paradox is posed by the coexistence of inherited, sporadic, and infectious forms of these diseases. Sensible molecular mechanisms for prion propagation must explain all three forms of prion diseases in a manner that is compatible with the formidable array of experimental data derived from histopathological, biochemical, biophysical, human genetic, and transgenetic studies. In this review, we explore prion disease pathogenesis initially from the perspective of an autosomal dominant inherited disease. Subsequently, we examine how an intrinsically inherited disease could present in sporadic and infectious forms. Finally, we explore the phenomenologic constraints on models of prion replication with a specific emphasis on biophysical studies of prion protein structures.

Humans↗
PubMed1997

Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice.

There is considerable concern that bovine prions from cattle with bovine spongiform encephalopathy (BSE) may have been passed to humans (Hu), resulting in a new form of Creutzfeldt-Jakob disease (CJD). We report here the transmission of bovine (Bo) prions to transgenic (Tg) mice expressing BoPrP; one Tg line exhibited incubation times of approximately 200 days. Like most cattle with BSE, vacuolation and astrocytic gliosis were confined in the brainstems of these Tg mice. Unexpectedly, mice expressing a chimeric Bo/Mo PrP transgene were resistant to BSE prions whereas mice expressing Hu or Hu/Mo PrP transgenes were susceptible to Hu prions. A comparison of differences in Mo, Bo, and Hu residues within the C terminus of PrP defines an epitope that modulates conversion of PrPC into PrPSc and, as such, controls prion transmission across species. Development of susceptible Tg(BoPrP) mice provides a means of measuring bovine prions that may prove critical in minimizing future human exposure.

Animals↗
PubMed1997

Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible.

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrPC to the infectious scrapie form PrPSc; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29-231 [PrP(29-231)] is investigated by multidimensional heteronuclear NMR. Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29-231) contains three helices and possibly one short beta-strand. Most striking is the random-coil nature of chemical shifts for residues 30-124 in the full-length PrP. Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121-231), the secondary structure boundaries of PrP(29-231) are different from those in mouse PrP(121-231) but similar to those found in the structure of Syrian hamster PrP(90-231). Comparison of resonance assignments of PrP(29-231) and PrP(90-231) indicates that there may be transient interactions between the additional residues and the structured core. Backbone dynamics studies done by using the heteronuclear [1H]-15N nuclear Overhauser effect indicate that almost half of PrP(29-231), residues 29-124, is highly flexible. This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of beta-structure.

Amino Acid Sequence↗
PubMed1997

Identification of functional surfaces of the zinc binding domains of intracellular receptors.

Transcriptional regulatory factor complexes assemble on genomic response elements to control gene expression. To gain insights on the surfaces that determine this assembly in the zinc binding domains from intracellular receptors, we systematically analyzed the variations in sequence and function of those domains in the context of their invariant fold. Taking the intracellular receptor superfamily as a whole revealed a hierarchy of amino acid residues along the DNA interface that correlated with response element binding specificity. When only steroid receptors were considered, two additional sites appeared: the known dimer interface, and a novel putative interface suitably located to contact regulatory factors bound to the free face of palindromic response elements commonly used by steroid receptors. Surprisingly, retinoic acid receptors, not known to bind palindromic response elements, contain both of these surfaces, implying that they may dimerize at palindromic elements under some circumstances. This work extends Evolutionary Trace analysis of functional surfaces to protein-DNA interactions, suggests how coordinated exchange of trace residues may predictably switch binding specificity, and demonstrates how to detect functional surfaces that are not apparent from sequence comparison alone.

Amino Acid Sequence↗
PubMed1997

A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform.

The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30.

Animals↗
PubMed1997

Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation.

Studies on the transmission of human (Hu) prions to transgenic (Tg) mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein (PrPSc). We report the identification of the site at which protein X binds to the cellular isoform of PrP (PrPC) using scrapie-infected mouse (Mo) neuroblastoma cells transfected with chimeric Hu/MoPrP genes even though protein X has not yet been isolated. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation. The side chains of these residues protrude from the same surface of the C-terminal alpha-helix and form a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at positions 167, 171, or 218 also prevented PrPSc formation: at a mechanistic level, these mutant PrPs appear to act as "dominant negatives" by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep and suggest therapeutic and prophylactic approaches to prion diseases.

Animals↗
PubMed1997

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform.

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Amino Acid Sequence↗
PubMed1997

Molecular properties of complexes formed between the prion protein and synthetic peptides.

Complexes of the Syrian hamster cellular prion protein (PrPC) and synthetic Syrian hamster PrP peptides were found to mimic many of the characteristics of the scrapie PrP isoform (PrPSc). Either PrPC expressed in chinese hamster ovary (CHO) cells or a C-terminal fragment of 142 residues of recombinant PrP protein (rPrP) produced in Escherichia coli was mixed with an excess of a synthetic 56 amino acid peptide, denoted PrP(90-145). Complex formation required PrPC or rPrP to be destabilized by guanidine hydrochloride (GdnHCl) or urea and PrP(90-145) to be in a coil conformation; it was enhanced by an acidic environment, salt and detergent. If PrP(90-145) was in a beta-sheet conformation, then no complexes were formed. While complex formation was rapid, acquisition of protease resistance was a slow process. Amorphous aggregates with a PrPC/PrP(90-145) ratio of 1:1 were formed in phosphate buffer, whereas fibrils with a diameter of approximately 10 nm and a PrPC/PrP(90-145) ratio of 1:5 were formed in Tris buffer. The complexes were stable only in the presence of excess peptide in either the coil or beta-sheet conformation; they dissociated rapidly after centrifugation and resuspension in buffer without peptide. Neither a peptide having a similar hydrophobicity profile/charge distribution to PrP(90-145) nor a scrambled version, denoted hPrP(90-145) and sPrP(90-145), respectively, were able to induce complex formation. Although hPrP(90-145) could stabilize the PrPC/PrP(90-145) complexes, sPrP(90-145) could not. Studies of PrPC/peptide complexes may provide insights into how PrPC interacts with PrPSc during the formation of a nascent PrPSc molecule and into the process by which PrPC is converted into PrPSc.

Amino Acid Sequence↗
PubMed1997

Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: a new homology modeling tool.

Modeling by homology is the most accurate computational method for translating an amino acid sequence into a protein structure. Homology modeling can be divided into two sub-problems, placing the polypeptide backbone and adding side-chains. We present a method for rapidly predicting the conformations of protein side-chains, starting from main-chain coordinates alone. The method involves using fewer than ten rotamers per residue from a backbone-dependent rotamer library and a search to remove steric conflicts. The method is initially tested on 299 high resolution crystal structures by rebuilding side-chains onto the experimentally determined backbone structures. A total of 77% of chi1 and 66% of chi(1 + 2) dihedral angles are predicted within 40 degrees of their crystal structure values. We then tested the method on the entire database of known structures in the Protein Data Bank. The predictive accuracy of the algorithm was strongly correlated with the resolution of the structures. In an effort to simulate a realistic homology modeling problem, 9424 homology models were created using three different modeling strategies. For prediction purposes, pairs of structures were identified which shared between 30% and 90% sequence identity. One strategy results in 82% of chi1 and 72% chi(1 + 2) dihedral angles predicted within 40 degrees of the target crystal structure values, suggesting that movements of the backbone associated with this degree of sequence identity are not large enough to disrupt the predictive ability of our method for non-native backbones. These results compared favorably with existing methods over a comprehensive data set.

Algorithms↗
PubMed1997

Physical studies of conformational plasticity in a recombinant prion protein.

PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.

Animals↗
PubMed1997

COOH-terminal sequence of the cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform.

Efficient formation of scrapie isoform of prion protein (PrP(Sc)) requires targeting PrP(Sc) by glycophosphatidyl inositol (GPI) anchors to caveolae-like domains (CLDs). Redirecting the cellular isoform of prion protein (PrP(C)) to clathrin-coated pits by creating chimeric PrP molecules with four different COOH-terminal transmembrane domains prevented the formation of PrP(Sc). To determine if these COOH-terminal transmembrane segments prevented PrP(C) from refolding into PrP(Sc) by altering the structure of the polypeptide, we fused the 28-aa COOH termini from the Qa protein. Two COOH-terminal Qa segments differing by a single residue direct the transmembrane protein to clathrin-coated pits or the GPI form to CLDs; PrP(Sc) was formed from GPI-anchored PrP(C) but not from transmembrane PrP(C). Our findings argue that PrP(Sc) formation is restricted to a specific subcellular compartment and as such, it is likely to involve auxiliary macromolecules found within CLDs.

Animals↗
PubMed1997

N-terminally tagged prion protein supports prion propagation in transgenic mice.

The eight amino acid sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, representing the FLAG peptide, was inserted after codons 22 or 88 of the mouse (Mo) prion protein (PrP) gene. Inclusion of the FLAG sequence at these locations interfered neither with the cellular processing of PrPC nor its conversion into PrPSc. Inclusion of the FLAG epitope at residue 22 but not at residue 88 facilitated immunodetection of tagged PrP by anti-FLAG monoclonal antibodies (mAbs). Inoculation of transgenic (Tg) mice expressing N-terminally tagged MoPrP with Mo prions resulted in abbreviated incubation times, indicating that the FLAG sequence was not deleterious to prion propagation. Immunopurification of FLAG-tagged MoPrPC in the brains of Tg mice was achieved using the calcium-dependent anti-FLAG M1 mAb and non-denaturing procedures. Although the function of PrPC remains unknown, our studies demonstrate that some modifications of PrPC do not inhibit the one biological activity that can be measured, i.e., conversion into PrPSc. Tagged PrP molecules may prove useful in the development of improved assays for prions as well as structural studies of the PrP isoforms.

Animals↗
PubMed1997

Bayesian statistical analysis of protein side-chain rotamer preferences.

We present a Bayesian statistical analysis of the conformations of side chains in proteins from the Protein Data Bank. This is an extension of the backbone-dependent rotamer library, and includes rotamer populations and average chi angles for a full range of phi, psi values. The Bayesian analysis used here provides a rigorous statistical method for taking account of varying amounts of data. Bayesian statistics requires the assumption of a prior distribution for parameters over their range of possible values. This prior distribution can be derived from previous data or from pooling some of the present data. The prior distribution is combined with the data to form the posterior distribution, which is a compromise between the prior distribution and the data. For the chi 2, chi 3, and chi 4 rotamer prior distributions, we assume that the probability of each rotamer type is dependent only on the previous chi rotamer in the chain. For the backbone-dependence of the chi 1 rotamers, we derive prior distributions from the product of the phi-dependent and psi-dependent probabilities. Molecular mechanics calculations with the CHARMM22 potential show a strong similarity with the experimental distributions, indicating that proteins attain their lowest energy rotamers with respect to local backbone-side-chain interactions. The new library is suitable for use in homology modeling, protein folding simulations, and the refinement of X-ray and NMR structures.

Bayes Theorem↗
PubMed1997

Leishmania major: molecular modeling of cysteine proteases and prediction of new nonpeptide inhibitors.

The crystal structures of papain, cruzain, and human liver cathepsin B were used to build homology-based enzyme models of a cathepsin L-like cysteine protease (cpL) and a cathepsin B-like cysteine protease (cpB) from the protozoan parasite Leishmania major. Although structurally a member of the cathepsin B subfamily, the L. major cpB is not able to cleave synthetic substrates having an arginine in position P2. This biochemical property correlates with the prediction of a glycine instead of a glutamic acid at position 205 (papain numbering). The modeled active sites of the L. major cpB and cpL were used to screen the Available Chemicals Directory (a database of about 150,000 commercially available compounds) for potential cysteine protease inhibitors, using DOCK3.5. Based on both steric and force field considerations, 69 compounds were selected. Of these, 18 showed IC50's between 50 and 100 microM and 3 had IC50's below 50 microM. A secondary library of compounds, originally derived from a structural screen against the homologous protease of Plasmodium falciparum (falcipain), and subsequently expanded by combinatorial chemistry, was also screened. Three inhibitors were identified which were not only effective against the L. major protease but also inhibited parasite growth at 5-50 microM.

Animals↗
PubMed1997

Entamoeba histolytica: computer-assisted modeling of phosphofructokinase for the prediction of broad-spectrum antiparasitic agents.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) is the rate-limiting glycolytic enzyme found in the pathogenic protists Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis, and Naegleria fowleri. The enzyme differs significantly from ATP-dependent phosphofructokinases found in humans and as such represents an important drug target. Current therapy for infections caused by these pathogens is inadequate, especially for children, pregnant women, and the immune compromised. The development of more selective, safer agents in imperative, as parasitic infections are currently a significant health threat worldwide and will likely become increasingly common agents of disease in the future. For the purpose of designing drugs to treat parasitic infections, we have constructed a model of PPi-PFK from E. histolytica based on the three-dimensional structure of the ATP-dependent PFK from Bacillus stearothermophilus. The model was used with the computer program Dock 3.5 (University of California, San Francisco) to predict the binding of pyrophosphate and selected bisphosphonates to the enzyme. The predicted drug-enzyme interactions suggested that two of these compounds would be competitive inhibitors of pyrophosphate. These drugs were tested against E. histolytica and inhibited the growth of amebae in vitro. This class of compounds may have broad-spectrum antiparasitic activity and, in the future, may facilitate the treatment of serious parasitic infections.

Amino Acid Sequence↗