Molecular interactions between monoclonal oligomer-specific antibody 5E3 and its amyloid beta cognates.

Oligomeric amyloid ß (Aß) is currently considered the most neurotoxic form of the Aß peptide implicated in Alzheimer's disease (AD). The molecular structures of the oligomers have remained mostly unknown due to their transient nature. As a result, the molecular mechanisms of interactions between conformation-specific antibodies and their Aß oligomer (AßO) cognates are not well understood. A monoclonal conformation-specific antibody, m5E3, was raised against a structural epitope of Aß oligomers. m5E3 binds to AßOs with high affinity, but not to Aß monomers or fibrils. In this study, a computational model of the variable fragment (Fv) of the m5E3 antibody (Fv5E3) is introduced. We further employ docking and molecular dynamics simulations to determine the molecular details of the antibody-oligomer interactions, and to classify the AßOs as Fv5E3-positives and negatives, and to provide a rationale for the low affinity of Fv5E3 for fibrils. This information will help us to perform site-directed mutagenesis on the m5E3 antibody to improve its specificity and affinity toward oligomeric Aß species. We also provide evidence for the possible capability of the m5E3 antibody to disaggregate AßOs and to fragment protofilaments.

Preparation and characterization of a highly soluble Aß1-42 peptide variant.

Alzheimer's disease (AD) is a progressive neurological disease marked by the accumulation and deposition of misfolded amyloid beta or Abeta (Aß) peptide. Two species of Aß peptides are found in amyloid plaques, Aß1-40 and Aß1-42, with the latter being the more amyloidogenic of the two. Understanding how and why Aß peptides misfold, oligomerize and form amyloid plaques requires a detailed understanding of their structure and dynamics. The poor solubility and strong aggregation tendencies of Aß1-42 has made the isolation and characterization of its different structural isoforms (monomer, dimer, oligomer, amyloid) exceedingly difficult. Furthermore, while synthetic Aß1-42 peptides (Aß42syn) are readily available, the cost of isotopically labeled peptide is substantial, making their characterization by NMR spectroscopy cost prohibitive. Here we describe the design, cloning, high-level production, isotopic labeling and biophysical characterization of a modified (solubility-tagged) Aß1-42 variant that exhibits excellent water solubility and shares similar aggregation properties as wildtype Aß1-42. Specifically, we attached six lysines (6K) to the C-terminus of native Aß1-42 to create a more soluble, monomeric form of Aß1-42 called Aß42C6K. A gene for the Aß42C6K was designed, synthesized and cloned into Escherichia coli (E. coli) and the peptide was expressed at milligram levels. The Aß42C6K peptide was characterized using circular dichroism (CD), NMR, electron microscopy and thioflavin T fluorescence. Its ability to form stable monomers, oligomers and fibrils under different conditions was assessed. Our results indicate that Aß42C6K stays monomeric at high concentrations (unlike Aß1-42) and can be induced to oligomerize and form fibrils like Aß1-42. Our novel construct could be used to explore the structure and dynamics of Aß1-42 as well as the interaction of ligands with Aß1-42 via NMR.

A simple in vitro assay for assessing the efficacy, mechanisms and kinetics of anti-prion fibril compounds.

Prion diseases are caused by the conversion of normal cellular prion proteins (PrP) into lethal prion aggregates. These prion aggregates are composed of proteinase K (PK) resistant fibrils and comparatively PK-sensitive oligomers. Currently there are no anti-prion pharmaceuticals available to treat or prevent prion disease. Methods of discovering anti-prion molecules rely primarily on relatively complex cell-based, tissue slice or animal-model assays that measure the effects of small molecules on the formation of PK-resistant prion fibrils. These assays are difficult to perform and do not detect the compounds that directly inhibit oligomer formation or alter prion conversion kinetics. We have developed a simple cell-free method to characterize the impact of anti-prion fibril compounds on both the oligomer and fibril formation. In particular, this assay uses shaking-induced conversion (ShIC) of recombinant PrP in a 96-well format and resolution enhanced native acidic gel electrophoresis (RENAGE) to generate, assess and detect PrP fibrils in a high throughput fashion. The end-point PrP fibrils from this assay can be further characterized by PK analysis and negative stain transmission electron microscopy (TEM). This cell-free, gel-based assay generates metrics to assess anti-prion fibril efficacy and kinetics. To demonstrate its utility, we characterized the action of seven well-known anti-prion molecules: Congo red, curcumin, GN8, quinacrine, chloropromazine, tetracycline, and TUDCA (taurourspdeoxycholic acid), as well as four suspected anti-prion compounds: trans-resveratrol, rosmarinic acid, myricetin and ferulic acid. These findings suggest that this in vitro assay could be useful in identifying and comprehensively assessing novel anti-prion fibril compounds. Abbreviations: PrP, prion protein; PK, proteinase K; ShIC, shaking-induced conversion; RENAGE, resolution enhanced native acidic gel electrophoresis; TEM, transmission electron microscopy; TUDCA, taurourspdeoxycholic acid; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; CJD, Creutzfeldt Jakob disease; GSS, Gerstmann-Sträussler-Scheinker syndrome; FFI, fatal familial insomnia; PrPc, cellular prion protein; recPrPC, recombinant monomeric prion protein; PrPSc, infectious particle of misfolded prion protein; RT-QuIC, real-time quaking-induced conversion; PMCA, Protein Misfolding Cyclic Amplification; LPS, lipopolysaccharide; EGCG, epigallocatechin gallate; GN8, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide; DMSO, dimethyl sulfoxide; ScN2A, scrapie infected neuroblastoma cells; IC50, inhibitory concentration for 50% reduction; recMoPrP 23-231, recombinant full-length mouse prion protein residues 23-231; EDTA; PICUP, photo-induced cross-linking of unmodified protein; BSA, bovine serum albumin;; PMSF, phenylmethanesulfonyl fluoride.

Fluorescent Protein Visualization Immediately After Gel Electrophoresis Using an In-Gel Trichloroethanol Photoreaction with Tryptophan.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the essential techniques in molecular biology and biochemistry laboratories and requires rapid visualization methods for efficient sample analysis. Proteins on polyacrylamide gels can be visualized within 5 min via the photoreaction of tryptophan with trichloroethanol. This process does not require protein fixation, staining, or destaining. In this method polyacrylamide gels are prepared by adding trichloroethanol before polymerization. After electrophoresis, the gel is immediately activated on a standard UV transilluminator and the fluorescently labeled proteins are imaged. The reaction is based on the photoreaction of trichloroethanol with tryptophan residues within the protein. This generates a visible blue-green fluorescence (∼500 nm) that is accurately imaged. Here we describe the preparation of Tris-glycine and Tris-tricine SDS-polyacrylamide gels with trichloroethanol and the photoreaction and visualization of tryptophan containing proteins.

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation.

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of ß-sheet rich, insoluble and protease resistant protein deposits (called PrPSc) that occur throughout the brain. Formation of synthetic or in vitro PrPSc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrPSc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrPc (spanning residues 90-231). We then obtained two-dimensional solution NMR spectra of the PrPc monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (1H-15N) studies revealed that, in comparison to monomeric PrPc, the oligomer has intense amide peak signals in the N-terminal (residues 90-114) and C-terminal regions (residues 226-231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrPc monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.

Role of polysaccharide and lipid in lipopolysaccharide induced prion protein conversion.

Conversion of native cellular prion protein (PrPc) from an α-helical structure to a toxic and infectious ß-sheet structure (PrPSc) is a critical step in the development of prion disease. There are some indications that the formation of PrPSc is preceded by a ß-sheet rich PrP (PrPß) form which is non-infectious, but is an intermediate in the formation of infectious PrPSc. Furthermore the presence of lipid cofactors is thought to be critical in the formation of both intermediate-PrPß and lethal, infectious PrPSc. We previously discovered that the endotoxin, lipopolysaccharide (LPS), interacts with recombinant PrPc and induces rapid conformational change to a ß-sheet rich structure. This LPS induced PrPß structure exhibits PrPSc-like features including proteinase K (PK) resistance and the capacity to form large oligomers and rod-like fibrils. LPS is a large, complex molecule with lipid, polysaccharide, 2-keto-3-deoxyoctonate (Kdo) and glucosamine components. To learn more about which LPS chemical constituents are critical for binding PrPc and inducing ß-sheet conversion we systematically investigated which chemical components of LPS either bind or induce PrP conversion to PrPß. We analyzed this PrP conversion using resolution enhanced native acidic gel electrophoresis (RENAGE), tryptophan fluorescence, circular dichroism, electron microscopy and PK resistance. Our results indicate that a minimal version of LPS (called detoxified and partially de-acylated LPS or dLPS) containing a portion of the polysaccharide and a portion of the lipid component is sufficient for PrP conversion. Lipid components, alone, and saccharide components, alone, are insufficient for conversion.

Shaking alone induces de novo conversion of recombinant prion proteins to ß-sheet rich oligomers and fibrils.

The formation of ß-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into ß-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to ß-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of ß-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to ß-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable ß-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).