Binding of bovine prion protein to heparin: a fluorescence polarization study

Glycosaminoglycans (GAGs) are believed to be associated with prion disease pathology and also with metabolism of the prion protein. Fluorescence polarization assay (FPA) of binding between bovine recombinant prion protein (brecPrP) and heparin labelled with AlexaFluor488 was used in model experiments to study glycosaminoglycan-prion protein interaction. Heparin binding to brecPrP was a rapid reversible event which occurred under defined conditions. The interaction of brecPrP with fluorophore-labelled heparin was inhibited by the presence of Cu(2+) ions and was sensitive to competition with heparin, heparan sulphate, and dextran. The dissociation constant of the heparin-brecPrP complex was 73.4+/-3.7 nM. Circular dichroism (CD) experiments indicated that the structure of brecPrP was less helical in the presence of heparin.     

Effect of metal ions on de novo aggregation of full-length prion protein

It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu(2+), Mn(2+), Ni(2+), Co(2+), and Zn(2+)) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn(2+) at low micromolar concentrations that could be blocked by nanomolar concentration of Cu(2+). These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo.     

Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful techniques to measure molecular interactions with high sensitivity in homogeneous solution and living cells. In this study, we developed methods for the detection of prion protein (PrP) using FCS and FCCS. A combination of a fluorescent-labeled Fab' fragment and another anti-PrP monoclonal antibody (mAb) enabled us to detect recombinant bovine PrP (rBoPrP) using FCS because there was a significant difference in the diffusion coefficients between the labeled Fab' fragment and the trimeric immune complex consisting of rBoPrP, labeled Fab' fragment, and another anti-PrP mAb. On the other hand, FCCS detected rBoPrP using two mAbs labeled with different fluorescence dyes. The detection limit for PrP in FCCS was approximately threefold higher than that in FCS. The sensitivity of FCCS in detection of abnormal isoform of PrP (PrP(Sc)) was comparable to that of enzyme-linked immunosorbent assay (ELISA). Because FCS and FCCS detect the PrP immune complex in homogeneous solution of only microliter samples with a single mixing step and without any washing steps, these features of measurement may facilitate automating bovine spongiform encephalopathy diagnosis.     

Rapid folding of the prion protein captured by pressure-jump

The conversion of the cellular form of the prion protein (PrP^C) to an altered disease state, generally denoted as scrapie isoform (PrP^Sc), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions. Equilibrium folding was studied by circular dichroism and fluorescence. Pressure-jump experiments were successfully applied to reveal rapid submillisecond folding events of PrP at temperatures not accessed before. D. C. Jenkins and D. S. Pearson contributed equally.     

A solid-phase assay for identification of modulators of prion protein interactions

The progression of the transmissible spongiform encephalopathies (TSEs) is characterized in part by accumulation of a proteinase K-resistant form of the prion protein, which has been converted from the endogenous, proteinase K-sensitive form. This conversion reaction provides a target for possible anti-TSE strategies. We have adapted a cell-free conversion reaction to a high-throughput, solid-phase format that can be used to screen possible therapeutic compounds for inhibitory activity or to illuminate inhibition and conversion mechanisms. The solid-phase assay was compatible with reactions performed under a variety of conditions. Using this assay, we report that phthalocyanine tetrasulfonate, a known modulator of conversion, inhibited conversion by interfering with binding between the protease-sensitive and the protease-resistant forms of the prion protein. A biotinylated form of the protease-sensitive prion protein was successfully converted to the protease-resistant isoform in the solid-phase assay, indicating that biotinylation provides a nonisotopic labeling strategy for large-scale screens.     

Conformational detection of p53’s oligomeric state by FlAsH Fluorescence

The p53 tumor suppressor protein is a critical checkpoint in prevention of tumor formation, and the function of p53 is dependent on proper formation of the active tetramer. In vitro studies have shown that p53 binds DNA most efficiently as a tetramer, though inactive p53 is predicted to be monomeric in vivo. We demonstrate that FlAsH binding can be used to distinguish between oligomeric states of p53, providing a potential tool to explore p53 oligomerization in vivo. The FlAsH tetra-cysteine binding motif has been incorporated along the dimer and tetramer interfaces in the p53 tetramerization domain to create reporters for the dimeric and tetrameric states of p53, though the geometry of the four cysteines is critical for efficient FlAsH binding. Furthermore, we demonstrate that FlAsH binding can be used to monitor tetramer formation in real-time. These results demonstrate the potential for using FlAsH fluorescence to monitor protein-protein interactions in vivo.     

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.     

Conformational preferences of non-polar amino acid residues: an additional factor in amyloid formation

Amyloid consists of β-sheet polymers and is associated with disease and with functional assemblies. Amyloid-forming proteins differ widely in native structures and sequences. We describe here how conformational preferences of non-polar amino acid residues can affect amyloid formation. The most non-polar residues promote either β-strands (Val, Ile, Phe, and Cys, VIFC) or α-helices (Leu, Ala, and Met, LAM), while the most polar residues promote only α-helices. For 12 proteins associated with disease, the localizations of the amyloid core regions are known. Eleven of these contain segments that are biased for VIFC, but essentially lack segments that are biased for LAM. For the amyloid β-peptide associated with Alzheimer’s disease and an amyloidogenic fragment of the prion protein, observed effects of mutations support that VIFC bias favors formation of β-sheet aggregates and amyloid, while LAM bias prevents it. VIFC and LAM profiles combine information on secondary structure propensities and polarity, and add a simple criterion to the prediction of amyloidogenic regions.     

Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line

An abnormal isoform of prion protein (PrP^Sc), which is composed of the same amino acids as cellular PrP (PrP^C) and has proteinase K (PK)-resistance, hypothetically converts PrP^C into PrP^Sc. To investigate the region important for PrP^Sc production, we examined the levels of PrP^Sc in PrP gene-deficient cells (HpL3-4) expressing PrP^C deleted of various regions including the octapeptide repeat region (OR) or hydrophobic region (HR). After Chandler or Obihiro prion infection, PrP^Sc was produced in HpL3-4 cells expressing wild-type PrP^C or PrP^C deleted of HR at an early stage and further reduced to below the detectable level, whereas cells expressing PrP^C deleted of OR showed no PrP^Sc production. The results suggest that OR of PrP^C is required for the early step of efficient PrP^Sc production.     

Molecular dynamics study of the fibril elongation of the prion protein fragment PrP106-126

The present paper aims at exploring the elongation of the PrP106-126 fibril under acid environments through molecular dynamics simulation. It shows that influenced by the edge strands of the fibril, single PrP106-126 peptide forms beta-sheet and becomes a new element of the fibril. Under acidic condition, single PrP106-126 fragment presents a much larger variety of conformations than it does under neural condition. However, acidic condition does not largely affect the stability of the PrP106-126 fibril. Consequently, the speed of the fibril elongation can be dramatically increased by lowering the pH value of the solution. The pH values are adjusted by either altering the protonation state of the residues or adding hydronium ions or hydroxyl ions.     

The fate of the prion protein in the prion/plasminogen complex

The cellular prion protein (PrP(c)) forms complexes with plasminogen. Here, we show that the PrP(c) in this complex is cleaved to yield fragments of PrP(c). The cleavage is accelerated by plasmin but does not appear to be dependent on it.     

The co-translational folding and interactions of nascent protein chains: a new approach using fluorescence resonance energy transfer

During protein biosynthesis, a nascent protein is exposed to multiple environments and proteins both inside and outside the ribosome that influence nascent chain folding and trafficking. Fluorescence resonance energy transfer between two dyes incorporated into a single nascent chain using aminoacyl-tRNA analogs can directly and selectively monitor changes in nascent chain conformation. This approach recently revealed the existence and functional ramifications of ribosome-mediated folding of nascent membrane proteins inside the ribosome and can be extended to characterize the effects of chaperones and other proteins and ligands on nascent protein folding, interactions, assembly, and avoidance of misfolding and degradation.     

Single particle analysis of manganese-induced prion protein aggregates

Prion diseases are characterized by the conversion of the cellular prion protein (PrP(C)) to a disease-specific aggregated isoform (PrP(Sc)). We have shown that Mn(2+) ions amplify aggregation, whereas Cu(2+) has an inhibitory effect. To characterize Mn(2+)-induced aggregates, we used cross-correlation analysis as well as scanning for intensely fluorescent targets in an SDS-dependent aggregation assay with fluorescently labeled PrP. We found that the effect of Mn(2+) was mainly due to the association of preformed PrP oligomers to larger aggregates, rapidly reversible by EDTA, and independent of the histidine-dependent copper-binding sites of PrP, suggesting that Mn(2+) induces reversible intermolecular binding. In contrast, the inhibitory effect of Cu(2+) required binding to histidine-containing binding sites, indicating that binding of copper affects the structure of PrP(C) which in turn modifies the susceptibility to manganese and the ability to aggregate. These findings suggest that copper and manganese may also affect prion propagation in vivo.     

THERPA: A small molecule database related to prion protein regulation and prion diseases progression

Prion diseases are fatal neurodegenerative disorders that affect humans and animals. Although various small molecules have been evaluated for application in the treatment of prion diseases, none have been shown to be efficacious. Expanding our knowledge of these molecules is important for understanding of the complex mechanisms of prion diseases. To improve access to the scattered information on small molecules related to prion diseases, we built a database of therapeutic molecules associated with prion diseases (THERPA, therpa.pythonanywhere.com). THERPA includes 119 small molecules and their 283 relationships with prion diseases. THERPA is an interactive visual database and useful for improving search efficiency which can help researchers identify intrinsic small molecules that can be used for developing therapeutics for prion diseases.     

Study on interaction between microtubule associated protein tau and prion protein

Microtubule-associated protein tau is considered to play roles in many neurodegenerative diseases including some transmissible spongiform encephalopathies. To address the possible molecular linkage of prion protein (PrP) and tau, a GST-fusion segment of human tau covering the three-repeat region and various PrP segments was used in the tests of GST pull-down and immunoprecipitation. We found tau protein interacted with various style prion proteins such as native prion protein (PrPC) or protease-resistant isoform (prpSc). Co-localization signals of tau and PrP were found in the CHO cell tranfected with both PrP and tau gene. The domain of interaction with tau was located at N-terminal of PrP (residues 23 to 91). The evidence of molecular interactions between PrP and tau protein highlights a potential role of tau in the biological function of PrP and the pathogenesis of TSEs.     

Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins

Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins by using prion protein (PrP) and amyloid precursor protein (APP) as models. In prion-infected cells, FlAsH-labeled tetracysteine-tagged PrP converted from the normal isoform (PrPsen) to the disease-associated isoform (PrPres), suggesting minimal steric effects of the tag. Pulse-chase analysis of PrP and APP by fluorescent gel imaging demonstrated the utility of IDEAL labeling in investigating protein metabolism by identifying an as-yet-unrecognized C-terminal fragment (C3) of PrPsen and by characterizing the kinetics of PrPres and APP metabolism. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor. Surprisingly, E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. To expand the versatility of tetracysteine tagging, we created new Alexa Fluor- and biotin-conjugated tetracysteine-binding molecules that were applied to imaging PrP endocytosis and ultrastructural localization. IDEAL-labeling extends the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.     

Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (6AP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as ’ribosomal folding modulators’ or RFMs) from both bacteria Escherichia coli and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.     

Conformational stability and folding mechanisms of dimeric proteins

The folding of multisubunit proteins is of tremendous biological significance since the large majority of proteins exist as protein-protein complexes. Extensive experimental and computational studies have provided fundamental insights into the principles of folding of small monomeric proteins. Recently, important advances have been made in extending folding studies to multisubunit proteins, in particular homodimeric proteins. This review summarizes the equilibrium and kinetic theory and models underlying the quantitative analysis of dimeric protein folding using chemical denaturation, as well as the experimental results that have been obtained. Although various principles identified for monomer folding also apply to the folding of dimeric proteins, the effects of subunit association can manifest in complex ways, and are frequently overlooked. Changes in molecularity typically give rise to very different overall folding behaviour than is observed for monomeric proteins. The results obtained for dimers have provided key insights pertinent to understanding biological assembly and regulation of multisubunit proteins. These advances have set the stage for future advances in folding involving protein-protein interactions for natural multisubunit proteins and unnatural assemblies involved in disease.