Soluble oligomers of A␤42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP C , on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128 -1132). Here we characterized the interaction between synthetic A␤42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met 129 and Val 129 polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with A␤ oligomers. It was also found that in addition to the previously identified ϳ95-110 segment, the second region of critical importance for the interaction with A␤42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for A␤42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well. Alzheimer disease (AD)2 is a devastating age-related neurodegenerative disorder leading to memory loss and progressive decline in cognitive ability (1-3). Pathologically, the disease is characterized by the formation of neurofibrillary tangles composed of hyperphosphorylated Tau and accumulation of extracellular amyloid plaques (1-6). The main component of these plagues is the 40 -42-amino acid residue amyloid-␤ (A␤) peptide, a product of proteolytic processing of a large membrane glycoprotein, amyloid precursor protein (APP).Although the etiology of AD remains poorly understood, the leading hypothesis is that the major causative agent is the aggregated form of A␤ (4, 7). Although in the past much attention has focused on mature ␤-sheet-rich amyloid fibrils, more recent evidence points to a critical role of smaller, soluble A␤ oligomers (8 -13). In contrast to poor quantitative correlation between the burden of insoluble fibrillar amyloid plagues and the degree of dementia, the severity of AD appears to correlate well with the concentration of soluble A␤ oligomers (9 -11). Furthermore, these soluble oligomers have been shown to be potent neurotoxins in vitro and in vivo; among other effects, they were reported to inhibit hippocampal long term potentiation, a widely used electrophysiological measure of synaptic plasticity related to learning and memory, and cause impairment of long term memory in rats (...

Background: Prion protein was found to interact with A␤, but the consequences of this interaction are largely unknown. Results: Prion protein and its N-terminal fragment inhibit A␤1-42 amyloidogenesis and cytotoxicity. Conclusion: Soluble prion protein is a potent inhibitor of A␤1-42 assembly into toxic oligomers. Significance: The results have important implications for understanding the pathogenesis of AD and for the development of novel therapeutic strategies.

The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity. Structural Overview of the c-Abl Kinase CoreThe kinase core of the c-Abl protein has a domain organization similar to that of the Src family kinases, with sequential Src homology (SH) 3 3 and SH2 domains, an SH2/kinase linker, and a bilobed kinase domain (Fig. 1). This core is flanked by an N-terminal "cap" (N-cap) region with a signal sequence for myristoylation, which serves dual roles in regulation of kinase activity and in membrane localization. C-terminal to the kinase domain is a long region of Ͼ600 amino acids encoded by a single exon, which controls interaction of Abl with other SH3-containing proteins and the actin cytoskeleton. This region also regulates nuclear-cytoplasmic shuttling of the kinase (1-4). These key structural and regulatory features are discussed in detail below. The Myristoylated N-cap Is Critical for Down-regulation of AblThe N-cap is ϳ80 amino acids in length and is myristoylated in the 1b splice variant of Abl (5). The first crystal structure of the Abl core (residues 1-531) revealed that this N-terminal myristic acid group binds a deep hydrophobic pocket in the C-terminal lobe (C-lobe) of the kinase domain ( Fig. 1) (6). Binding of the myristoyl group into this pocket induces a bend in C-lobe helix ␣I, allowing the SH2 domain to dock onto the C-lobe of the kinase domain (Fig. 2). Interaction of the myristoylated N-cap with the C-lobe is critical to maintenance of the autoinhibited state, as mutation of the myristoylation signal sequence results in a highly active kinase (7). Interestingly, small molecules that bind to this site also modulate kinase activity, supporting an allosteric connection between this regulatory pocket and the kinase active site (8 -11).In addition to binding the C-lobe of the kinase domain, the N-cap also influences kinase regulation via the SH3 and SH2 domains. Although the N-cap region was disordered in the first crystal structure of the c-Abl core, a more recent structure with a modified N-cap revealed that Ser 69 (numbered according to Protein Data Bank (PDB) code 2FO0) 4 is phosphorylated and contacts the short connector joining the SH3 and SH2 domains. Mutation of Ser 69 increased Abl activity, identifying this site as a potential input for regulatory kinases (12). Additional contacts were observed between N...

The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrPSc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrPSc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD), determined their conformational stability and replication tempo in vitro, as well as characterized structural organization using recently emerged approaches based on hydrogen/deuterium (H/D) exchange coupled with mass spectrometry. Our data clearly demonstrate that these phenotypically distant prions differ in a major way with regard to their structural organization, both at the level of the polypeptide backbone (as indicated by backbone amide H/D exchange data) as well as the quaternary packing arrangements (as indicated by H/D exchange kinetics for histidine side chains). Furthermore, these data indicate that, in contrast to previous observations on yeast and some murine prion strains, the replication rate of sCJD prions is primarily determined not by conformational stability but by specific structural features that control the growth rate of prion protein aggregates.

The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-LysHis-Met (MKHM) corresponding to human PrP-(109 -112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species-and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106 -110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the speciesand conformer-dependent preferences of 3F4 arise largely from the interactions between Met 112 (human PrP) or Val 115 (cervid PrP) and adjacent residues.

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

Background: Prion strains are believed to be enciphered by distinct conformations of misfolded prion protein (PrP). Results: Strains of PrP amyloid with different conformational stabilities were found to have identical ␤-sheet core regions but different steric zipper interfaces. Conclusion: Strain-specific differences in PrP amyloid stability are dictated by a packing polymorphism. Significance: These findings have implications for understanding the structural basis of prion strains.

Background: Abl kinases are regulated by noncatalytic domains that allosterically impact kinase domain structure and inhibitor sensitivity. Results: Enhanced SH3/linker interaction suppresses c-Abl core protein dynamics and sensitizes Bcr-Abl to kinase domain inhibitors. Conclusion: SH3/linker interaction influences kinase dynamics in the context of Bcr-Abl. Significance: Stabilizers of SH3/linker interaction may sensitize Bcr-Abl to kinase domain inhibitors, providing a new route to allosteric kinase control.