Biomedical subjects

Gal Bitan

Publications and source records attributed to Gal Bitan.

18 recordsLinked to original sources

PubMed2006

Dendrimeric Abeta1-15 is an effective immunogen in wildtype and APP-tg mice.

Immunization of humans and APP-tg mice with full-length beta-amyloid (Abeta) results in reduced cerebral Abeta levels. However, due to adverse events in the AN1792 trial, alternative vaccines are required. We investigated dendrimeric Abeta1-15 (dAbeta1-15), which is composed of 16 copies of Abeta1-15 peptide on a branched lysine core and thus, includes an Abeta-specific B cell epitope but lacks the reported T cell epitope. Immunization by subcutaneous, transcutaneous, and intranasal routes of B6D2F1 wildtype mice led to anti-Abeta antibody production. Antibody isotypes were mainly IgG1 for subcutaneous or transcutaneous immunization and IgG2b for intranasal immunization, suggestive of a Th2-biased response. All Abeta antibodies preferentially recognized an epitope in Abeta1-7. Intranasal immunization of J20 APP-tg mice resulted in a robust humoral immune response with a corresponding significant reduction in cerebral plaque burden. Splenocyte proliferation against Abeta peptide was minimal indicating the lack of an Abeta-specific cellular immune response. Anti-Abeta antibodies bound monomeric, oligomeric, and fibrillar Abeta. Our data suggest that dAbeta1-15 may be an effective and potentially safer immunogen for Alzheimer's disease (AD) vaccination.

Administration, Cutaneous↗

PubMed2006

Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.

Oligomers of amyloidogenic proteins are believed to be key effectors of cytotoxicity and cause a variety of amyloid-related diseases. Dissociation or inhibition of formation of the toxic oligomers is thus an attractive strategy for the prevention and treatment of these diseases. In order to develop reagents capable of inhibiting protein oligomerization, the structures and mechanisms of oligomer formation must be understood. However, structural studies of oligomers are difficult because of the metastable nature of the oligomers and their existence in mixtures with monomers and other assemblies. A useful method for characterization of oligomer size distributions in vitro is photo-induced cross-linking of unmodified proteins (PICUP) (Fancy and Kodadek, 1999). By providing "snapshots" of dynamic oligomer mixtures, PICUP enables quantitative analysis of the relations between primary and quaternary structures, offering insights into the molecular organization of the oligomers. This chapter discusses the photochemical mechanism; reviews the scope, usefulness, and limitations of PICUP for characterizing metastable protein assemblies; and provides detailed experimental instructions for performing PICUP experiments.

2,2'-Dipyridyl↗

PubMed2006

Elucidating amyloid beta-protein folding and assembly: A multidisciplinary approach.

Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer's and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils. A single methodological approach cannot elucidate the entire protein assembly process. An integrated multidisciplinary program is required. We discuss here the synergistic application of in hydro, in vacuo, and in silico methods to the study of the amyloid beta-protein, the key pathogenetic agent in Alzheimer's disease.

Amino Acid Sequence↗

PubMed2006

Amyloid beta-protein monomer structure: a computational and experimental study.

The structural properties of the Abeta42 peptide, a main constituent of the amyloid plaques formed in Alzheimer's disease, were investigated through a combination of ion-mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the -3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families. An analysis of secondary structure indicates the Abeta42 peptide conformations are dominated by loops and turns but show some helical structure in the C-terminal hydrophobic tail. A second calculation on Abeta42 in a solvent-free environment yields compact structures turned "inside out" from the solution structures (hydrophobic parts on the outside, polar parts on the inside). Ion mobility experiments on the Abeta42 -3 charge state electrosprayed from solution yield a bimodal arrival time distribution. This distribution can be quantitatively fit using cross-sections from dehydrated forms of the three families of calculated solution structures and the calculated solvent-free family of structures. Implications of the calculations on the early stages of aggregation of Abeta42 are discussed.

Amyloid beta-Peptides↗

PubMed2006

Early diagnostics and therapeutics for Alzheimer's disease--how early can we get there?

Alzheimer's disease (AD) is a major threat for the rapidly aging world population. AD is the leading cause of dementia and a major cause of death in developed countries. The disease puts a tremendous practical, emotional and financial burden on individuals and governments. Clinicians and researchers in the AD field face great challenges: the pathophysiological processes that cause AD are not well understood, definite diagnosis of AD requires autopsy, and therapeutic options are limited to treating the symptoms rather than the cause of the disease. Nevertheless, new insights into the earliest events that lead to development of AD increase hope that reliable diagnostics and efficacious therapies may emerge.

Alzheimer Disease↗

PubMed2005

En route to early diagnosis of Alzheimer's disease--are we there yet?

As therapeutics for Alzheimer's disease (AD) become available, it will become increasingly important to develop accurate and reliable tools for its early diagnosis. A recent paper by Georganopoulou et al. suggests that nanobiotechnology could help to overcome the limitations with the current diagnostic methods. The paper introduces a nanoparticle-based assay using antibodies that are specific for amyloid beta-protein (Abeta) oligomers with sub-femtomolar sensitivity. This assay appears to be more specific for AD than previous ELISA-based work and, if specificity of the assay for Abeta oligomers can be established, the method developed might provide a sensitive, reliable diagnostic tool for AD.

Alzheimer Disease↗

PubMed2005

Amyloid beta-protein: monomer structure and early aggregation states of Abeta42 and its Pro19 alloform.

The amyloid beta-protein (Abeta) is a seminal neuropathic agent in Alzheimer's disease (AD). Recent evidence points to soluble Abeta oligomers as the probable neurotoxic species. Among the naturally occurring Abeta peptides, the 42-residue form Abeta42 is linked particularly strongly with AD, even though it is produced at approximately 10% of the levels of the more abundant 40-residue form Abeta40. Here, we apply mass spectrometry and ion mobility to the study of Abeta42 and its Pro19 alloform. The Phe19 --> Pro19 substitution blocks fibril formation by [Pro19]Abeta42. Evidence indicates that solution-like structures of Abeta monomers are electrosprayed and characterized. Unfiltered solutions of Abeta42 produce only monomers and large oligomers, whereas [Pro19]Abeta42 solutions produce abundant monomers, dimers, trimers, and tetramers but no large oligomers. When passed through a 10,000 amu filter and immediately sampled, Abeta42 solutions produce monomers, dimers, tetramers, hexamers, and an aggregate of two hexamers that may be the first step in protofibril formation. These results are consistent with recently published photochemical cross-linking data and lend support to recent aggregation mechanisms proposed by Bitan, Teplow, and co-workers [J. Biol. Chem. 2003, 278, 34882-34889].

Amino Acid Sequence↗

PubMed2005

Neurotoxic protein oligomers--what you see is not always what you get.

An increasing body of evidence suggests that soluble assemblies of amyloid proteins are the predominant neurotoxic species in many amyloid-related diseases. Consequently, the focus of research on pathologic mechanisms underlying amyloidoses has shifted from amyloid fibrils to oligomers. Biophysical characterization of oligomers is difficult due to their metastable nature. The most popular experimental method for detection of oligomers has been SDS-PAGE. However, we provide experimental evidence that SDS-PAGE is not a reliable method for characterization of amyloid protein oligomers and discuss alternative approaches. In addition, we discuss how inconsistent nomenclature has obfuscated our understanding of the process and products of protein assembly. The goals of this paper are to identify pitfalls associated with the methods and language used to study protein oligomers and to provide alternatives, thereby facilitating successful elucidation of the mechanisms controlling amyloid protein oligomer assembly and toxicity.

Biopolymers↗

PubMed2005

Preparation of aggregate-free, low molecular weight amyloid-beta for assembly and toxicity assays.

More than 20 diseases have been identified which are caused by the deposition of amyloid. Natural and chemically synthesized amyloidogenic proteins are used widely to study the structure, assembly, and physiologic effects of both oligomeric and fibrillar forms of these proteins. In many cases, conflicting results arise in these studies, in part owing to difficulties in reproducibly preparing amyloidogenic proteins in a well-defined assembly state. To avoid these problems, several methods have been devised that provide reliable means of preparing amyloid-forming proteins for experimental use. Here, we discuss methods that have been used successfully to prepare one such protein, the amyloid beta-protein (Abeta), involved in Alzheimer's disease. Methods for reproducible preparation of Abeta in a well-defined assembly state include isolation of low molecular weight (LMW) Abeta by size exclusion chromatography, filtration through LMW cut-off filters, and solubilization/lyophilization in the presence of reagents which facilitate disassembly of Abeta. These reagents include strong bases and acids, and fluorinated alcohols. These methods, which were originally developed for Abeta, are generally applicable to amyloidogenic peptides and proteins. In this chapter, we describe the preparation of LMW Abeta using size exclusion chromatography and filtration. The advantages and disadvantages of each method are discussed.

Amyloid beta-Peptides↗

PubMed2005

Determination of Peptide oligomerization state using rapid photochemical crosslinking.

The assembly of the amyloid beta-protein (Abeta) into neurotoxic oligomers and fibrils is a seminal pathogenic process in Alzheimer's disease (AD). Understanding the mechanisms of Abeta assembly could prove useful in the identification of therapeutic targets. Owing to the metastable nature of Abeta oligomers, it is difficult to obtain interpretable data through application of classical methods, such as electrophoresis, chromatography, fluorescence, and light scattering. Here, we apply the method Photo-Induced Crosslinking of Unmodified Proteins (PICUP) to the study of Abeta oligomerization. This method directly produces covalent bonds among unmodified polypeptide chains through in situ generation of peptide free radicals. PICUP provides a snapshot of the native oligomerization state of proteins and can be used for assembly state analysis of a wide variety of peptides and proteins.

Amyloid beta-Peptides↗

PubMed2004

Rapid photochemical cross-linking--a new tool for studies of metastable, amyloidogenic protein assemblies.

Amyloidoses comprise a class of diseases characterized pathologically by the presence of deposits of fibrillar, aberrantly folded proteins, known as amyloids. Historically, these deposits were considered the key factors causing disease. However, recent evidence suggests that soluble protein oligomers, which are precursors for amyloid fibrils, are the primary toxic effectors responsible for the disease process. Understanding the mechanism by which these oligomers exert their toxicity requires knowledge of the structure, kinetics, and thermodynamics of their formation and conversion into larger assemblies. Such studies have been difficult due to the metastable nature of the oligomers. For the amyloid beta-protein (Abeta), a consensus about the size and relative abundance of small oligomers has not been achieved. We describe here the application of the method Photoinduced Cross-Linking of Unmodified Proteins (PICUP) to the study of Abeta oligomerization. This approach distinguishes oligomerization patterns of amyloidogenic and nonamyloidogenic proteins, allows quantification of each component in oligomer mixtures, and provides a means of correlating primary structure modifications with assembly characteristics. PICUP thus is a powerful tool for the investigation of small, metastable protein oligomers. The method provides essential insights into the factors that control the assembly of pathogenic protein oligomers, facilitating efforts toward the development of therapeutic agents.

Amyloid↗

PubMed2003

A molecular switch in amyloid assembly: Met35 and amyloid beta-protein oligomerization.

Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) forms neurotoxic oligomers. The predominant in vivo Abeta alloforms, Abeta40 and Abeta42, have distinct oligomerization pathways. Abeta42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. Abeta40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Abeta42 with AD. Here, we sought to determine the structural elements controlling paranucleus formation as a first step toward the development of strategies for treating AD. Because oxidation of Met(35) is associated with altered Abeta assembly, we examined the role of Met(35) in controlling Abeta oligomerization. Oxidation of Met(35) in Abeta42 blocked paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by Abeta40. Systematic structural alterations of the C(gamma)(35)-substituent group revealed that its electronic nature, rather than its size (van der Waals volume), was the factor controlling oligomerization pathway choice. Preventing assembly of toxic Abeta42 paranuclei through selective oxidation of Met(35) thus represents a potential therapeutic approach for AD.

Amyloid↗

PubMed2003

Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization.

Assembly of monomeric amyloid beta-protein (A beta) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight A beta 40 and A beta 42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight A beta 40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight A beta 42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant A beta alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in A beta 42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus self-association. A beta 40 oligomerization was particularly sensitive to substitutions of Glu22 or Asp23 and to truncation of the N terminus, but not to substitutions of Phe19 or Ala21. A beta 42 oligomerization, in contrast, was largely unaffected by substitutions at positions 22 or 23 or by N-terminal truncations, but was affected significantly by substitutions of Phe19 or Ala21. These results reveal how specific regions and residues control A beta oligomerization and show that these controlling elements differ between A beta 40 and A beta 42.

Alzheimer Disease↗

PubMed2003

Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease.

Alzheimer disease (AD) is characterized by the progressive deposition of the 42-residue amyloid beta protein (Abeta) in brain regions serving memory and cognition. In animal models of AD, immunization with Abeta results in the clearance of Abeta deposits from the brain. However, a trial of vaccination with synthetic human Abeta1-42 in AD resulted in the development of meningoencephalitis in some patients. We measured cellular immune responses to Abeta in middle-aged and elderly healthy subjects and in patients with AD. A significantly higher proportion of healthy elderly subjects and patients with AD had strong Abeta-reactive T cell responses than occurred in middle-aged adults. The immunodominant Abeta epitopes in humans resided in amino acids 16-33. Epitope mapping enabled the identification of MHC/T cell receptor (TCR) contact residues. The occurrence of intrinsic T cell reactivity to the self-antigen Abeta in humans has implications for the design of Abeta vaccines, may itself be linked to AD susceptibility and course, and appears to be associated with the aging process.

Adult↗

PubMed2002

Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways.

Amyloid beta-protein (Abeta) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of Abeta in AD is new evidence that oligomeric forms of Abeta are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Abeta, Abeta42, with the disease. Detailed knowledge of the structure and assembly dynamics of Abeta thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Abeta oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Abeta40 and Abeta42. Carefully prepared aggregate-free Abeta40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, Abeta42 preferentially formed pentamerhexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to Abeta40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that Abeta42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of Abeta42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

Alzheimer Disease↗

PubMed2002

Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by amyloid deposition in the cerebral neuropil and vasculature. These amyloid deposits comprise predominantly fragments and full-length (40 or 42 residue) forms of the amyloid beta-protein (Abeta) organized into fibrillar assemblies. Compelling evidence indicates that factors that increase overall Abeta production or the ratio of longer to shorter forms, or which facilitate deposition or inhibit elimination of amyloid deposits, cause AD or are risk factors for the disease. In vitro studies have demonstrated that fibrillar Abeta has potent neurotoxic effects on cultured neurons. In vivo experiments in non-human primates have demonstrated that Abeta fibrils directly cause pathologic changes, including tau hyperphosphorylation. In concert with histologic studies revealing a lack of tissue injury in areas of the neuropil in which non-fibrillar deposits were found, these data suggested that fibril assembly was a prerequisite for Abeta-mediated neurotoxicity in vivo. Recently, however, both in vitro and in vivo studies have revealed that soluble, oligomeric forms of Abeta also have potent neurotoxic activities, and in fact, may be the proximate effectors of the neuronal injury and death occurring in AD. A paradigm shift is thus emerging that necessitates the reevaluation of the relative importance of polymeric (fibrillar) vs. oligomeric assemblies in the pathobiology of AD. In addition to AD, an increasing number of neurodegenerative disorders, including Parkinson's disease, familial British dementia, familial amyloid polyneuropathy, amyotrophic lateral sclerosis, and prion diseases, are associated with abnormal protein assembly processes. The archetypal features of the assembly-dependent neuropathogenetic effects of Abeta may thus be of relevance not only to AD but to these other disorders as well.

Alzheimer Disease↗

PubMed2002

Polyglutamine repeat length-dependent proteolysis of huntingtin.

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than full-length huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeat-containing huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

Amino Acid Sequence↗

PubMed1997

Building Units for N-Backbone Cyclic Peptides. 3. Synthesis of Protected N(alpha)-(omega-Aminoalkyl)amino Acids and N(alpha)-(omega-Carboxyalkyl)amino Acids.

An improved synthesis of a family of amino acids that contain omega-aminoalkyl groups and of a new family containing omega-carboxyalkyl groups linked to the alpha-amine is described. The synthesis was performed by alkylation of suitably monoprotected alkylenediamines and protected omega-amino acids with triflates of alpha-hydroxy acid esters. The reaction proceeded with inversion of configuration yielding optically pure products. The N(alpha)-(omega-aminoalkyl)amino acids and N(alpha)-(omega-carboxyalkyl)amino acids were orthogonally protected to allow their incorporation into peptides by solid-phase peptide synthesis (SPPS) methodology.

Journal Article↗