•Alzheimer’s disease (AD), the leading cause of dementia, affects ∼35 million people.•Effective, disease-modifying AD drugs are not available at present.•Better clinical trials and nonpharmacological ...therapies hold promise to stop or slow AD.•New drugs, surgical technologies, and healthy lifestyles may combine to halt AD.
Recent drug development efforts targeting Alzheimer’s disease (AD) have failed to produce effective disease-modifying agents for many reasons, including the substantial presymptomatic neuronal damage that is caused by the accumulation of the amyloid β (Aβ) peptide and tau protein abnormalities, deleterious adverse effects of drug candidates, and inadequate design of clinical trials. New molecular targets, biomarkers, and diagnostic techniques, as well as alternative nonpharmacological approaches, are sorely needed to detect and treat early pathological events. This article analyzes the successes and debacles of pharmaceutical endeavors to date, and highlights new technologies that may lead to the more effective diagnosis and treatment of the pathologies that underlie AD. The use of focused ultrasound, deep brain stimulation, stem cell therapy, and gene therapy, in parallel with pharmaceuticals and judicious lifestyle adjustments, holds promise for the deceleration, prevention, or cure of AD and other neurodegenerative disorders.
Fourier transform infrared (FTIR) spectroscopy has become one of the major techniques of structural characterization of proteins, peptides, and protein-membrane interactions. While the method does ...not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or intermolecular interactions. The sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as
C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between large protein complexes, interactions of proteins and peptides with lipid vesicles, or protein-nucleic acid interactions without light scattering problems often encountered in ultraviolet spectroscopy. Attenuated total reflection FTIR (ATR-FTIR) is a surface-sensitive version of infrared spectroscopy that has proved useful in studying membrane proteins and lipids, protein-membrane interactions, mechanisms of interfacial enzymes, the structural features of membrane pore forming proteins and peptides, and much more. The purpose of this chapter was to provide a practical guide to analyze protein structure and protein-membrane interactions by FTIR and ATR-FTIR techniques, including procedures of sample preparation, measurements, and data analysis. Basic background information on FTIR spectroscopy, as well as some relatively new developments in structural and functional characterization of proteins and peptides in lipid membranes, is also presented.
The insulin receptor (IR) is a (αβ)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αβ heterodimer consists of an extracellular ligand-binding α-subunit and a ...membrane-spanning β-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and β-subunits are linked via a single disulfide bridge, and the (αβ)2 tetramer is formed by disulfide bonds between the α-chains. Insulin binding induces conformational changes in IR that reach the intracellular β-subunit followed by a protein phosphorylation and activation cascade. Defects in this signaling process, including IR dysfunction caused by mutations, result in type 2 diabetes. Rational drug design aimed at treatment of diabetes relies on knowledge of the detailed structure of IR and the dynamic structural transformations during transmembrane signaling. Recent X-ray crystallographic studies have provided important clues about the mode of binding of insulin to IR, the resulting structural changes and their transmission to the TK domain, but a complete understanding of the structural basis underlying insulin signaling has not been achieved. This review presents a critical analysis of the current status of the structure–function relationship of IR, with a comparative assessment of the other IR family receptors, and discusses potential advancements that may provide insight into the molecular mechanism of insulin signaling.
Amyloid β (Aβ) peptide contributes to Alzheimer’s disease by a yet unidentified mechanism. In the brain tissue, Aβ occurs in various forms, including an undecapeptide Aβ25‑35, which exerts a ...neurotoxic effect through the mitochondrial dysfunction and/or Ca2+-permeable pore formation in cell membranes. This work was aimed at the biophysical characterization of membrane binding and pore formation by Aβ25‑35. Interaction of Aβ25‑35 with anionic and zwitterionic membranes was analyzed by microelectrophoresis. In pore formation experiments, Aβ25‑35 was incubated in aqueous buffer to form oligomers and added to Quin-2-loaded vesicles. Gradual increase in Quin-2 fluorescence was interpreted in terms of membrane pore formation by the peptide, Ca2+ influx, and binding to intravesicular Quin-2. The kinetics and magnitude of this process were used to evaluate the rate constant of pore formation, peptide–peptide association constants, and the oligomeric state of the pores. Decrease in membrane anionic charge and high ionic strength conditions significantly suppressed membrane binding and pore formation, indicating the importance of electrostatic interactions in these events. Circular dichroism spectroscopy showed that Aβ25‑35 forms the most efficient pores in β-sheet conformation. The data are consistent with an oligo-oligomeric pore model composed of up to eight peptide units, each containing 6–8 monomers.
The multiscale models for complex chemical systems constitute a powerful computational tool to describe biomolecular structure and dynamics, including enzymatic reactions. Here, the development of ...this method is presented as a miraculous chain of events, involving astoundingly lucky encounters of brilliant minds such as Planck, Schrödinger, Pauling, Karplus, Levitt, and Warshel.
Amyloid β (Aβ) peptide aggregation plays a central role in Alzheimer's disease (AD) etiology. AD drug candidates have included small molecules or peptides directed towards inhibition of Aβ ...fibrillogenesis. Although some Aβ-derived peptide fragments suppress Aβ fibril growth, comprehensive analysis of inhibitory potencies of peptide fragments along the whole Aβ sequence has not been reported. The aim of this work is (a) to identify the region(s) of Aβ with highest propensities for aggregation and (b) to use those fragments to inhibit Aβ fibrillogenesis. Structural and aggregation properties of the parent Aβ
peptide and seven overlapping peptide fragments have been studied, i.e. Aβ
(P1), Aβ
(P2), Aβ
(P3), Aβ
(P4), Aβ
(P5), Aβ
(P6), and Aβ
(P7). Structural transitions of the peptides in aqueous buffer have been monitored by circular dichroism and Fourier transform infrared spectroscopy. Aggregation and fibrillogenesis were analyzed by light scattering and thioflavin-T fluorescence. The mode of peptide-peptide interactions was characterized by fluorescence resonance energy transfer. Three peptide fragments, P3, P6, and P7, exhibited exceptionally high propensity for β-sheet formation and aggregation. Remarkably, only P3 and P6 exerted strong inhibitory effect on the aggregation of Aβ
, whereas P7 and P2 displayed moderate inhibitory potency. It is proposed that P3 and P6 intercalate between Aβ
molecules and thereby inhibit Aβ
aggregation. These findings may facilitate therapeutic strategies of inhibition of Aβ fibrillogenesis by Aβ-derived peptides.
Abstract
The amyloid β (Aβ) peptide and its shorter variants, including a highly cytotoxic Aβ
25–35
peptide, exert their neurotoxic effect during Alzheimer’s disease by various mechanisms, including ...cellular membrane permeabilization. The intrinsic polymorphism of Aβ has prevented the identification of the molecular basis of Aβ pore formation by direct structural methods, and computational studies have led to highly divergent pore models. Here, we have employed a set of biophysical techniques to directly monitor Ca
2+
-transporting Aβ
25–35
pores in lipid membranes, to quantitatively characterize pore formation, and to identify the key structural features of the pore. Moreover, the effect of membrane cholesterol on pore formation and the structure of Aβ
25–35
has been elucidated. The data suggest that the membrane-embedded peptide forms 6- or 8-stranded β-barrel like structures. The 8-stranded barrels may conduct Ca
2+
ions through an inner cavity, whereas the tightly packed 6-stranded barrels need to assemble into supramolecular structures to form a central pore. Cholesterol affects Aβ
25–35
pore formation by a dual mechanism, i.e., by direct interaction with the peptide and by affecting membrane structure. Collectively, our data illuminate the molecular basis of Aβ membrane pore formation, which should advance both basic and clinical research on Alzheimer’s disease and membrane-associated pathologies in general.
Amyloid β (Aβ) ion channels destabilize cellular ionic homeostasis, which contributes to neurotoxicity in Alzheimer's disease. The relative roles of various Aβ isoforms are poorly understood. We use ...bilayer electrophysiology, AFM imaging, circular dichroism, FTIR and fluorescence spectroscopy to characterize channel activities of four most prevalent Aβ peptides, Aβ
, Aβ
, and their pyroglutamylated forms (AβpE
, AβpE
) and correlate them with the peptides' structural features. Solvent-induced fluorescence splitting of tyrosine-10 is discovered and used to assess the sequestration from the solvent and membrane insertion. Aβ
effectively embeds in lipid membranes, contains large fraction of β-sheet in a β-barrel-like structure, forms multi-subunit pores in membranes, and displays well-defined ion channel features. In contrast, the other peptides are partially solvent-exposed, contain minimal β-sheet structure, form less-ordered assemblies, and produce irregular ionic currents. These findings illuminate the structural basis of Aβ neurotoxicity through membrane permeabilization and may help develop therapies that target Aβ-membrane interactions.
The amyloid β (Aβ) peptide and its shorter variants, including a highly cytotoxic Aβ
peptide, exert their neurotoxic effect during Alzheimer's disease by various mechanisms, including cellular ...membrane permeabilization. The intrinsic polymorphism of Aβ has prevented the identification of the molecular basis of Aβ pore formation by direct structural methods, and computational studies have led to highly divergent pore models. Here, we have employed a set of biophysical techniques to directly monitor Ca
-transporting Aβ
pores in lipid membranes, to quantitatively characterize pore formation, and to identify the key structural features of the pore. Moreover, the effect of membrane cholesterol on pore formation and the structure of Aβ
has been elucidated. The data suggest that the membrane-embedded peptide forms 6- or 8-stranded β-barrel like structures. The 8-stranded barrels may conduct Ca
ions through an inner cavity, whereas the tightly packed 6-stranded barrels need to assemble into supramolecular structures to form a central pore. Cholesterol affects Aβ
pore formation by a dual mechanism, i.e., by direct interaction with the peptide and by affecting membrane structure. Collectively, our data illuminate the molecular basis of Aβ membrane pore formation, which should advance both basic and clinical research on Alzheimer's disease and membrane-associated pathologies in general.
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