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Self association of the amyloid-β (Aβ42) peptide into oligomers, high molecular weight forms, fibrils and ultimately neuritic plaques, has been correlated with progressive cognitive decline in Alzheimer's disease. Thus, insights into the drivers of the aggregation pathway have the capacity to significantly contribute to our understanding of disease mechanism. Functional assays and a three-dimensional crystal structure of the P3 amyloidogenic region 18-41 of Aβ were used to identify residues important in self-association and to design novel non-aggregating variants of the peptide. Biophysical studies (gel filtration, SDS-PAGE, dynamic light scattering, thioflavin T assay, and electron microscopy) demonstrate that in contrast to wild type Aβ these targeted mutations lose the ability to self-associate. Loss of aggregation also correlates with reduced neuronal toxicity. Our results highlight residues and regions of the Aβ peptide important for future targeting agents aimed at the amelioration of Alzheimer's disease., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

The tendency of amyloid β (Aβ42) peptide to misfold and aggregate into insoluble amyloid fibrils in Alzheimer's disease (AD) has been well documented. Accumulation of Aβ42 fibrils has been correlated with abnormal apoptosis and unscheduled cell division which can also trigger the death of neuronal cells, while oligomers can also exhibit similar activities. While investigations using chemically-synthesized Aβ42 peptide have become common practice, there appear to be differences in outcomes from different preparations. In order to resolve this inconsistency, we report 2 separate methods of preparing chemically-synthesized Aβ42 and we examined their effects in yeast. Hexafluoroisopropanol pretreatment caused toxicity while, ammonium hydroxide treated Aβ42 induced cell proliferation in both C. glabrata and S. cerevisiae. The hexafluoroisopropanol prepared Aβ42 had greater tendency to form amyloid on yeast cells as determined by thioflavin T staining followed by flow cytometry and microscopy. Both quiescent and non-quiescent cells were analyzed by these methods of peptide preparation. Non-quiescent cells were susceptible to the toxicity of Aβ42 compared with quiescent cells (p < 0.005). These data explain the discrepancy in the previous publications about the effects of chemically-synthesized Aβ42 on yeast cells. The effect of Aβ42 on yeast cells was independent of the size of the peptide aggregates. However, the Aβ42 pretreatment determined whether the molecular conformation of peptide resulted in proliferation or toxicity in yeast based assays.

Extended X-ray absorption fine structure spectroscopy, mass spectrometry, dynamic light scattering and density functional theory are combined to derive structural models for the interaction of neurotoxicity-ablating platinum-based compounds with the amyloid-β peptide.

Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid-β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal-binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1-16 fused to the N-terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti-Aβ N-terminal antibody WO2. The structure demonstrates that Aβ residues 10-16, which are not in complex with the antibody, adopt a mixture of local polyproline II-helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10-16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13-metal-His14 coordination in the Aβ1-16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N-terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal-binding-induced neurotoxicity., (Copyright © 2013 Wiley Periodicals, Inc.)

Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction., (Copyright © 2011 Wiley Periodicals, Inc.)

Latrepirdine (Dimebon), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-β (Aβ)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Aβ yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aβ42. GFP-Aβ42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aβ42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aβ42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aβ42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aβ42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aβ42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aβ42.

This study has found that the Maltose binding protein Aβ42 fusion protein (MBP-Aβ42) forms soluble oligomers while the shorter MBP-Aβ16 fusion and control MBP did not. MBP-Aβ42, but neither MBP-Aβ16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aβ42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aβ42 characterization., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT.

The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-l-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.

Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.

Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 A. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 A.

Background: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT., Results: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 A. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent., Conclusions: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

A single-chain antibody fragment (scFv) of the humanised monoclonal antibody, hu3S193, that reacts specifically with Le(y) antigen expressed in numerous human epithelial carcinomas was constructed. A five-residue linker joined the C-terminus of the V(H) and the N-terminus of the V(L), which prevented V-domain association into a monomeric scFv and instead directed non-covalent association of two scFvs into a dimer or diabody. The diabody was secreted into the E. coli periplasm using a heat-inducible vector, pPOW3, and recovered as a soluble, correctly processed protein, following osmotic shock or solubilised with 4 M urea from the insoluble fraction. The diabody from both fractions was isolated by a rapid batch affinity chromatography procedure, using the FLAG affinity tag to minimise degradation and aggregation. The purified diabody has an Mr of approximately 54 kDa, was stable and demonstrated similar binding activity as the parent monoclonal antibody, as measured by FACS and BIAcore analyses. The radiolabelled diabody showed a rapid tumour uptake, with fast blood clearance, proving it to be an excellent potential candidate as a tumour-imaging agent.

The gene encoding phenylethanolamine N-methyltransferase (PNMT) has been amplified from a human adrenal medulla cDNA library. Following ligation of the gene into a pET3a-derived expression vector and transformation into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yielding about 10% of the soluble protein. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by ion-exchange chromatography and gel filtration. The Km for phenylethanolamine and S-adenosyl-L-methionine were determined to be 130 and 16 microM, respectively. The enzyme could be inhibited by reagents expected to modify cysteine, arginine, tyrosine, and histidine residues, but not by methyl acetimidate, a reagent expected to modify lysine residues.

p-Aminobenzoic acid (PABA) synthase catalyses the first step in folic acid biosynthesis, the conversion of chorismate to p-aminobenzoate. In general, difficulties in purification have permitted only limited investigation of this enzyme. However, in an attempt to identify possible active site residues, the E. coli enzyme has been incubated with a range of protein modifying agents. Results indicate that cysteine, histidine, arginine and tyrosine residues are important for enzyme activity. Attempts were made to determine the subunits upon which these residues were located.