Sajith Jayasinghe

MPtopo: A database of membrane protein topology

The reliability of the transmembrane (TM) sequence assignments for membrane proteins (MPs) in standard sequence databases is uncertain because the vast majority are based on hydropathy plots. A database of MPs with dependable assignments is necessary for developing new computational tools for the prediction of MP structure. We have therefore created MPtopo, a database of MPs whose topologies have been verified experimentally by means of crystallography, gene fusion, and other methods. Tests using MPtopo strongly validated four existing MP topology‐prediction algorithms. MPtopo is freely available over the internet and can be queried by means of an SQL‐based search engine.

Structure of α-Helical Membrane-bound Human Islet Amyloid Polypeptide and Its Implications for Membrane-mediated Misfolding

Human islet amyloid polypeptide (hIAPP) misfolding is thought to play an important role in the pathogenesis of type II diabetes mellitus. It has recently been shown that membranes can catalyze the misfolding of hIAPP via an α-helical intermediate of unknown structure. To better understand the mechanism of membrane-mediated misfolding, we used site-directed spin labeling and EPR spectroscopy to generate a three-dimensional structural model of this membrane-bound form. We find that hIAPP forms a single α-helix encompassing residues 9–22. The helix is flanked by N- and C-terminal regions that do not take up a clearly detectable secondary structure and are less ordered. Residues 21 and 22 are located in a transitional region between the α-helical structure and C terminus and exhibit significant mobility. The α-helical structure presented here has important implications for membrane-mediated aggregation. Anchoring hIAPP to the membrane not only increases the local concentration but also reduces the encounter between peptides to essentially a two-dimensional process. It is significant to note that the α-helical membrane-bound form leaves much of an important amyloidogenic region of hIAPP (residues 20–29) exposed for misfolding. Misfolding of this and other regions is likely further aided by the low dielectric environment near the membrane that is known to promote secondary structure formation. Based upon these considerations, a structural model for membrane-mediated aggregation is discussed.

Structural Features That Modulate the Transmembrane Migration of a Hydrophobic Peptide in Lipid Vesicles

Two approaches employing nuclear magnetic resonance (NMR) were used to investigate the transmembrane migration rate of the C-terminal end of native alamethicin and a more hydrophobic analog called L1. Native alamethicin exhibits a very slow transmembrane migration rate when bound to phosphatidylcholine vesicles, which is no greater than 1 x 10(-4) min(-1). This rate is much slower than expected, based on the hydrophobic partition energies of the amino acid side chains and the backbone of the exposed C-terminal end of alamethicin. The alamethicin analog L1 exhibits crossing rates that are at least 1000 times faster than that of native alamethicin. A comparison of the equilibrium positions of these two peptides shows that L1 sits approximately 3-4 A deeper in the membrane than does native alamethicin (Barranger-Mathys and Cafiso. 1996. Biochemistry. 35:489). The slow rate of alamethicin crossing can be explained if the peptide helix is irregular at its C-terminus and hydrogen bonded to solvent or lipid. We postulate that L1 does not experience as large a barrier to transport because its C-terminus is already buried within the membrane interface. This difference is most easily explained by conformational differences between L1 and alamethicin rather than differences in hydrophobicity. The results obtained here demonstrate that side-chain hydrophobicity alone cannot account for the energy barriers to peptide and protein transport across membranes.

Annexin A5 directly interacts with amyloidogenic proteins and reduces their toxicity.

Protein misfolding is a central mechanism for the development of neurodegenerative diseases and type 2 diabetes mellitus. The accumulation of misfolded alpha-synuclein protein inclusions in the Lewy bodies of Parkinsons disease is thought to play a key role in pathogenesis and disease progression. Similarly, the misfolding of the beta-cell hormone human islet amyloid polypeptide (h-IAPP) into toxic oligomers plays a central role in the induction of beta-cell apoptosis in the context of type 2 diabetes. In this study, we show that annexin A5 plays a role in interacting with and reducing the toxicity of the amyloidogenic proteins, h-IAPP and alpha-synuclein. We find that annexin A5 is coexpressed in human beta-cells and that exogenous annexin A5 reduces the level of h-IAPP-induced apoptosis in human islets by approximately 50% and in rodent beta-cells by approximately 90%. Experiments with transgenic expression of alpha-synuclein in Caenorhabditis elegans show that annexin A5 reduces alpha-synuclein inclusions in vivo. Using thioflavin T fluorescence, electron microscopy, and electron paramagnetic resonance, we provide evidence that substoichiometric amounts of annexin A5 inhibit h-IAPP and alpha-synuclein misfolding and fibril formation. We conclude that annexin A5 might act as a molecular safeguard against the formation of toxic amyloid aggregates.

Membrane Curvature-sensing and Curvature-inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption

Background: The mechanism behind diabetes-associated membrane damage by islet amyloid polypeptide (IAPP) is poorly understood. Results: IAPP induces and senses membrane curvature under conditions associated with membrane damage and binds to mitochondrial cristae in vivo. Conclusion: IAPP is a membrane-remodeling and curvature-sensing protein. Significance: Aberrant membrane remodeling could inform disruption of membrane integrity in diabetes and perhaps other amyloid diseases. Islet amyloid polypeptide (IAPP) is a 37-amino acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy, we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for human IAPP than for the less toxic rat IAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rat IAPP and human IAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well.

61 IDENTIFYING STRUCTURAL FEATURES OF ISLET AMYLOID POLYPEPTIDE USING SITE-DIRECTED SPIN LABELING.

Pancreatic amyloid deposits are found in over 90% of patients with type II diabetes mellitus. These deposits are primarily composed of a 37-residue islet amyloid polypeptide (IAPP). While evidence suggests an association between these amyloid plaques and pancreatic beta-cell dysfunction, elucidating the structure of these deposits can help further the understanding of its toxicity. We use electron paramagnetic resonance (EPR) spectroscopy to analyze spin-labeled derivatives of IAPP to determine structural features of the peptide in the fibrillar amyloid deposit form. A number of derivatives are made in which each residue of IAPP is spin-labeled one amino acid at a time and studied with EPR spectroscopy in order to obtain information about the local environment and structure in the region of the labeled site. While we are furthering our structural studies of the core amyloidgenic region, we have identified structural features of the amino and carboxy terminus. Analysis of the spin mobility and comparison to the amyloid found in Alzheimers patients (ABeta) indicates a high degree of order throughout the fibrillar peptide, but the N-terminal and C-terminal regions are less ordered and more mobile. Evidence suggests that these regions are not part of the ordered core region that gives rise to fibril formation. Based upon side-chain mobilities, we will present the structural features of different regions of IAPP. Combining these structural features can lead us to creating a 3-D model of IAPP and perhaps give way to methods of treatment or dissolution of the pancreatic deposit.