James I. Elliott

Structural conversion of neurotoxic amyloid-[beta]1-42 oligomers to fibrils

The amyloid-β1–42 (Aβ42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimers disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the β-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Aβ42 oligomers to fibrils involves the association of these loosely aggregated strands into β-sheets whose individual β-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.

Detection of Alzheimer's amyloid in transgenic mice using magnetic resonance microimaging.

The presence of amyloid‐β (Aβ) plaques in the brain is a hallmark pathological feature of Alzheimers disease (AD). Transgenic mice overexpressing mutant amyloid precursor protein (APP), or both mutant APP and presenilin‐1 (APP/PS1), develop Aβ plaques similar to those in AD patients, and have been proposed as animal models in which to test experimental therapeutic approaches for the clearance of Aβ. However, at present there is no in vivo whole‐brain imaging method to detect Aβ plaques in mice or men. A novel method is presented to detect Aβ plaques in the brains of transgenic mice by magnetic resonance microimaging (μMRI). This method uses Aβ1‐40 peptide, known for its high binding affinity to Aβ, magnetically labeled with either gadolinium (Gd) or monocrystalline iron oxide nanoparticles (MION). Intraarterial injection of magnetically labeled Aβ1‐40, with mannitol to transiently open the blood–brain barrier (BBB), enabled the detection of many Aβ plaques. Furthermore, the numerical density of Aβ plaques detected by μMRI and by immunohistochemistry showed excellent correlation. This approach provides an in vivo method to detect Aβ in AD transgenic mice, and suggests that diagnostic MRI methods to detect Aβ in AD patients may ultimately be feasible. Magn Reson Med 50:293–302, 2003.

Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins.

The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90–104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp93 to Leu94 occurs through the β-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome cpeptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiological conditions. These post-translationally modified peptides may serve as an early immunologic stimulus in autoimmune disease.

Reversed-phase high-performance liquid chromatography for fractionation of enzymatic digests and chemical cleavage products of proteins.

Publisher Summary This chapter discusses reversed-phase high-performance liquid chromatography for fractionation of enzymatic digests and chemical cleavage products of proteins. The extremely high resolving power and speed of reversed-phase high-performance liquid chromatography (HPLC) make it the current method of choice for fractionating complex mixtures of peptides derived from the enzymatic and chemical cleavage of proteins. With its high peak capacity, reversed-phase HPLC can readily bring about a 100- to 125-fold purification of a typical tryptic peptide with a gradient time of only about 90 min. Although reversed-phase HPLC provides an invaluable tool for the protein chemist, its utility is enhanced even further by coupling it with mass spectrometry. A reversed-phase HPLC tryptic peptide/ fast atom bombardment (FAB) mass spectrometric approach provides an elegant and rapid means to accurately determine the molecular weights of the resulting peptides. This information can, in turn, be used to rapidly verify the primary structures of proteins that have been deduced from their DNA sequences.

pH-dependent fibrillogenesis of a VkappaIII Bence Jones protein.

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma‐cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light‐chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8–5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VκIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post‐mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N‐terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril‐formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro‐environments may contribute to the stabilization of amyloidogenic conformations.

Membrane-microdomain Localization of Amyloid β-Precursor Protein (APP) C-terminal Fragments is Regulated by Phosphorylation of the Cytoplasmic Thr668 Residue

Background: Phosphorylation of amyloid β-precursor protein (APP) at Thr668 alters the conformation of its cytoplasmic domain. Results: Phosphorylation of APP C-terminal fragments (pCTFs) at Thr668 decreases membrane lipid binding. Conclusion: Phosphorylation at Thr668 regulates the localization of pCTFs away from γ-secretase-containing, lipid raft-like membrane microdomains. Significance: Preservation of the phosphorylation of APP CTFs at Thr668 may be a useful treatment to lower amyloid β-protein generation. Amyloid β-precursor protein (APP) is primarily cleaved by α- or β-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr668 and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648–695)) with Thr(P)668 did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aβ generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aβ in Alzheimer disease.

In Vivo Magnetic Resonance of Amyloid Plaques in Alzheimer’s Disease Model Mice

A key feature of Alzheimer’s disease (AD) is the deposition of the amyloid β (Aβ) as neuritic plaques in the brain. Transgenic mice overexpressing mutant amyloid precursor protein (APP), or both mutant APP and presenilin-1 (APP-PS1), develop Aβ plaques similar to AD patients and are currently the most widely used models of AD. The definitive diagnosis of AD still requires post-mortem examination. We have developed a novel method for the detection of Aβ plaques in the brains of AD model transgenic mice using magnetic resonance micro-imaging (μMRI). Our method is dependent on ligands that bind to AD amyloid lesions, allowing their detection by μMRI. These ligands are Aβ1-40 peptides, magnetically labeled with either gadolinium (Gd) or monocrystalline iron oxide nanoparticles (MION). When these are systemically injected with mannitol to transiently open the blood-brain barrier, we are able to detect the majority of amyloid lesions. The number of lesions detected by μMRI showed a statistically significant correlation with the Aβ burden determined by histology. This approach, with additional development, may be used to detect amyloid lesions in humans. Similar methods may also be used to image other conformational neurodegenerative disorders.

Randomization of Amino Acid Sequence: An Important Control In Synthetic Peptide Analogue Studies of Nucleic Acid Binding Domains

Publisher Summary Over the past several years, improvements in automated peptide synthesis have helped to account for the increasing use of synthetic peptides for mapping epitopes, active site regions, and binding domains in proteins. This chapter discusses the randomization of amino acid sequence. By characterizing the nucleic acid binding properties of control peptides with the same composition as the actual peptide analog, but with randomized sequences, it is found that in at least one system the amino acid composition is more important than the amino acid sequence in determining the nucleic acid binding properties of the synthetic peptide. Data suggest that a randomized peptide that has the same composition but a different sequence than the correct peptide might serve as a better control than a completely unrelated peptide does for these studies. In this way, effects because of amino acid composition can begin to be differentiated from those resulting from amino acid sequence.