Jose Varghese

Naturally occurring polyphenolic inhibitors of amyloid beta aggregation.

Alzheimers disease is the most common neurodegenerative disease and is one of the main causes of death in developed countries. Consumption of foods rich in polyphenolics is strongly correlated with reduced incidence of Alzheimers disease. Our study has investigated the biological activity of previously untested polyphenolic compounds in preventing amyloid β aggregation. The anti-aggregatory potential of these compounds was assessed using the Thioflavin-T assay, transmission electron microscopy, dynamic light scattering and size exclusion chromatography. Two structurally related compounds, luteolin and transilitin were identified as potent inhibitors of Aβ fibril formation. Computational docking studies with an X-ray derived oligomeric structure offer a rationale for the inhibitory activity observed and may facilitate development of improved inhibitors of Aβ aggregation and toxicity.

A New Method to Measure Cellular Toxicity of Non-Fibrillar and Fibrillar Alzheimer's Aβ Using Yeast

The 42 amino acid amyloid-β (Aβ) can exist in multiple physical states including oligomers and fibrils. This study shows that fibril formation is hastened by the biological buffers required to support the growth of mammalian cells, but is prevented if Aβ is maintained in water. Here we describe a method to produce Aβ in oligomeric form and the comparison of stable fibrillar and non-fibrillar forms in cell toxicity studies in water, achieved through the use of yeast. We show that extracellular, non-fibrillar Aβ causes a dose dependent loss of cell viability while fibrillar A β has low toxicity.

Sialidase inhibitors related to zanamivir. Further SAR studies of 4-amino-4H-pyran-2-carboxylic acid-6-propylamides

SAR investigations of the 4- and 5-positions of a series of 4-amino-4H-pyran-2-carboxylic acid 6-carboxamides are reported. Potent inhibitors of influenza A sialidase with marked selectivity over the influenza B enzyme were obtained when the basic 4-amino substituent was replaced by hydroxyl or even deleted. Modifications at the 5-position exhibited a tight steric requirement, with trifluoroacetamide being optimal.

Structural studies of the tethered N-terminus of the Alzheimer's disease amyloid-beta peptide.

Alzheimers 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 Alzheimers disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758.

Oligomerization and Toxicity of Aβ Fusion Proteins

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.

A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data

The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X‐ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low‐resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two‐dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low‐resolution phase information that can be refined further by the conventional methods of protein crystallography.

Computational studies on the stability of C-terminal beta-amyloid dimer and mutants

Background: Insoluble amyloid plaque deposits characterize the primary pathology of Alzheimer’s disease. The amyloid-beta (Ab) peptide, cleaved from the membrane-bound amyloid precursor protein (APP) by the action of b-secretase and g-secretase into 39-43 amino acid residue long fragments, is the major constituent of this plaque. It is now believed that a major source of the neurotoxicity in AD is due to the action of intermediate soluble Ab oligomers [1]. Recently, a deletion mutant at the Glu 22 position (D E22) of Ab was discovered in some Familial Alzheimer’s Disease (FAD) patients in Japan. This mutation, named the Osaka mutant, appears to cause the A b peptide to oligomerize and fibrillize much more rapidly compared to the wild type [2]. Methods: Streltsov et al. [3] have determined the x-ray crystal structure of Ab (18-41) within the framework of shark IgNAR (Ig New Antigen Receptor) single variable domain antibody CDR3 loop. The Ab portion of the crystal structure is observed to be a tetramer (or a dimer of dimers), and can provide valuable insight into the structure-function relationships of A b. In this work we perform in silico or computational studies in order to understand the stability and conformational dynamics of the Ab peptide and the Osaka mutant oligomers. Results: These simulations, employing molecular dynamics methodology in an explicit solvent environment, commence from the dimer present in the crystal structure. We observe the relative stability of the various secondary structure elements, inter-residue interactions and conformational transitions. Conclusions: The deletion mutation is located in an important turn region with formation and breaking of transient hydrogen bonds and ionic interactions contributing to the dynamical behaviour of the peptide. References: 1. Crouch, P.J., Harding, S.M., White, A.R., Camakaris, J., Bush, A.I., & Masters, C.L., (2008). International Journal of Biochemistry & Cell Biology, 40, 181198. 2. Inayathullah, M. & Teplow, D.P., (2011). Amyloid, 18(3), 98-107. 3. Streltsov, V.A., Varghese, J.N., Masters, C.L., & Nuttall, S.N. (2011). Journal of Neuroscience, 31, 1419 -1426.

Towards protein structure determination using two-dimensional crystals and powders

temperatures is thought to be from photoelectrons ejected from atoms following the absorption of X-rays. As the photoelectrons traverse the crystal, they lose energy through interactions with atoms in their path resulting in damage. If the X-ray beam is polarized, the photoelectrons are ejected preferentially along the polarization vector. Monte-Carlo simulations [Nave, C., and Hill, M. A. (2005). J Syn. Rad. 12, 299-303] suggest that, when the beam size is only a few microns, most photoelectrons escape the illuminated volume. This leads to the peculiar conclusion that the radiation damage due to photoelectrons may be significantly lower within the illuminated volume than in the volume immediately surrounding the irradiated spot. A second prediction of the calculations is that most of the photoelectron’s energy is abruptly dissipated within the last few microns of its trajectory. Recently, a long focal length Fresnel zone plate was used to provide a focused beam of ~1-micron cross section at the sample position, and high quality diffraction data was obtained from protein crystals. The 15.1 keV, 1-micron beam was used to probe the geometrical distribution and extent of radiation damage in protein crystals. These data confirm that radiation damage is greater along the polarization vector than in the perpendicular direction, radiation damage is maximal 3-4 microns from the center of the beam, and radiation damage does not extend beyond 6 microns.