Conformational Switching in PolyGln Amyloid Fibrils Resulting from a Single Amino Acid Insertion

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of β-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-β structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10–20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated β-structure model for PolyQKd-33 and a model with two β-solenoid protofibrils for PolyQKd-32. These data provide evidence for β-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.     

Conformational Switching within Individual Amyloid Fibrils

A key structural component of amyloid fibrils is a highly ordered, crystalline-like cross-β-sheet core. Conformationally different amyloid structures can be formed within the same amino acid sequence. It is generally assumed that individual fibrils consist of conformationally uniform cross-β-structures. Using mammalian recombinant prion protein (PrP), we showed that, contrary to common perception, amyloid is capable of accommodating a significant conformational switching within individual fibrils. The conformational switch occurred when the amino acid sequence of a PrP variant used as a precursor substrate in a fibrillation reaction was not compatible with the strain-specific conformation of the fibrillar template. Despite the mismatch in amino acid sequences between the substrate and template, individual fibrils recruited the heterologous PrP variant; however, the fibril elongation proceeded through a conformational adaptation, resulting in a change in amyloid strain within individual fibrils. This study illustrates the high adaptation potential of amyloid structures and suggests that conformational switching within individual fibrils may account for adaptation of amyloid strains to a heterologous substrate. This work proposes a new mechanistic explanation for the phenomenon of strain conversion and illustrates the direction in evolution of amyloid structures. This study also provides a direct illustration that catalytic activity of self-replicating amyloid structures is not ultimately coupled with their templating effect.The ability to form amyloid structures is considered to be one of the most general properties of a polypeptide backbone (1). Regardless of the specific peptides or proteins involved in fibril formation, all types of amyloid fibrils share a common structural motif that consists of a cross-β-structure (2). Cross-β-structures are comprised of highly ordered, nearly anhydrous, crystalline-like β-sheets stabilized by hydrogen bonding and densely packed side chains (3, 4). Growing evidence indicates that multiple amyloid structures referred to as amyloid strains could be formed within the same amino acid sequence (5–7).Amyloids are capable of self-replicating (8). Self-replicating properties of amyloid fibrils are attributed to the unique arrangement of cross-β-strands that are assembled perpendicular to the fibrillar axis, where β-strands at the growing edge provide a template for recruiting and converting a monomeric precursor. The self-replicating property of the amyloid cross-β-structure consists of two activities: catalytic (i.e. the ability to convert a monomeric precursor into an amyloid state) and templating (i.e. the ability to accurately imprint the strain-specific conformation onto a newly recruited polypeptide). The templating activity is believed to be intimately coupled to the catalytic activity and accounts for the high fidelity of amyloid replication. High fidelity of replication requires identity or high homology between the amino acid sequences of a fibrillar template and a precursor substrate. The species specificity of a template-substrate interaction is believed to account for the species barrier in prion transmission and species specificity of in vitro cross-seeded fibrillation reactions. Local perturbations arising due to mismatches in packing of amino acid side chains within the crystalline-like cross-β-structures could prevent efficient replication of amyloid fibrils.It is generally assumed that individual fibrils are structurally uniform, i.e. maintain the same structure of a cross-β-core throughout the fibrillar length. In the current study, we showed that, contrary to the common perception, amyloid fibrils are capable of accommodating significant conformational switching within individual fibrils. The conformational switch occurred when the amino acid sequence of the precursor substrate was not compatible with the conformation of the template. Despite mismatched amino acid sequences, individual fibrils were able to recruit the heterologous recombinant prion protein (PrP)² variant; however, fibril elongation proceeded through switching to a new conformational state. The implications of these studies are multifold. First, our work illustrates the high adaptation potential of amyloid structures and suggests that the conformational switch accounts for adaptation of amyloid strains to the heterologous substrate. Second, the current studies propose a new molecular explanation for the phenomenon referred to as convergence of strains. Third, this work illustrates the directionality in evolution of amyloid structures, showing that the species-specific amyloid structures (i.e. structures that exist only within a single PrP sequence) can give rise to promiscuous or indiscriminative structures (structures compatible with several PrP variants), but not vice versa. Finally, our studies provide direct illustration that catalytic activity of self-replicating amyloid structures is not ultimately coupled with their templating effect.     

An Auto-Catalytic Surface for Conformational Replication of Amyloid Fibrils—Genesis of an Amyloid World?

Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65°C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.     

Amino Acid Replacements Resulting from Suppression and Missense Reversion of a Chain-Terminator Mutation in Neurospora

The Neurospora crassa super-suppressor mutation, ssu-1, suppresses the auxotrophic phenotype of the mutant am(17) by inserting tyrosine at residue 313 of NADP-specific glutamate dehydrogenase, a position occupied in the wild type by glutamate. Two classes of am(17) revertants due to further mutation within the am gene have, respectively, tyrosine and leucine at residue 313. These replacements are consistent with a chain-terminating codon in am(17) of either the amber (UAG) or the ochre type (UAA), but are inconsistent with UGA. The Leu³¹³ and Tyr³¹³ variants of the enzyme have effective activity but are grossly different from the wild type in Michaelis constants (especially for ammonium) and heat stabilities at two different pH values. They show smaller but significant differences in these respects from each other.     

Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ_1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ_1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ_1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.     

Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region

Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP^Sc. One key operational parameter used to define differences between strains has been conformational stability of PrP^Sc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP^Sc, especially because large strain-specific differences in PrP^Sc stability are often observed despite a similar size of the PrP^Sc core region.     

Imaging Amyloid Fibrils within Cells Using a Se-Labelling Strategy

The process of aggregation leading to amyloid formation by peptides and proteins is associated with diseases ranging from systemic amyloidoses to neurodegenerative disorders such as Alzheimer's disease. A key question in understanding the link between amyloid formation and its pathological consequences is the ultrastructural localisation and morphological form of amyloid species within the cellular environment. The acquisition of such information has proven to be challenging, but we report here a novel approach that enables amyloid fibrils to be visualised directly within a cell. First, fibrils are assembled from selenium analogues of the sulfur-containing cysteine peptides, and then, atomic number contrast transmission electron microscopy is used to detect the selenium doped species selectively within the carbon-rich background of the cell. We demonstrate the power of this approach by imaging human monocyte-derived macrophage cells that have been exposed to fibrils from an amyloidogenic fragment of the disease-associated protein transthyretin. The ready incorporation of seleno-cysteine and methionine instead of their natural sulfur-containing analogues, a feature that is already commonly used in X-ray diffraction studies of proteins, suggests that this method can be used as a general strategy to image specific peptides and proteins within the cellular environment using electron microscopy.     

Nanoscale Mechanical Characterisation of Amyloid Fibrils Discovered in a Natural Adhesive

Using the atomic force microscope, we have investigated the nanoscale mechanical response of the attachment adhesive of the terrestrial alga Prasiola linearis (Prasiolales, Chlorophyta). We were able to locate and extend highly ordered mechanical structures directly from the natural adhesive matrix of the living plant. The in vivo mechanical response of the structured biopolymer often displayed the repetitive sawtooth force-extension characteristics of a material exhibiting high mechanical strength at the molecular level. Mechanical and histological evidence leads us to propose a mechanism for mechanical strength in our sample based on amyloid fibrils. These proteinaceous, pleated β-sheet complexes are usually associated with neurodegenerative diseases. However, we now conclude that the amyloid protein quaternary structures detected in our material should be considered as a possible generic mechanism for mechanical strength in natural adhesives.     

AL Amyloid Imaging and Therapy with a Monoclonal Antibody to a Cryptic Epitope on Amyloid Fibrils

The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA) containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL) amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K_D of ∼10 nM. Binding was inhibited in the presence of the –Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas) as evidenced by single photon emission (SPECT) imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.     

Single Amino Acid Insertion in Loop 4 Confers Amphotropic Murine Leukemia Virus Receptor Function upon Murine Pit1

Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch of amino acids named region A was identified in Pit1 (residues 550 to 558 in loop 4) as critical for GALV and FeLV-B receptor function. We have here investigated the role of region A in A-MuLV and 10A1 entry. Insertion of a single amino acid in region A of mouse Pit1 resulted in a functional A-MuLV receptor, showing that region A plays a role in A-MuLV infection. Moreover, the downregulation of 10A1 receptor function by changes in region A of human Pit1 indicates that this region is also involved in 10A1 entry. Therefore, region A seems to play a role in infection by all viruses utilizing Pit1 and/or Pit2 as receptors.     

Analysis of Core Region from Egg White Lysozyme Forming Amyloid Fibrils

Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.     

Permeases Recycle Amino Acids Resulting from Autophagy

Autophagy is a major survival mechanism for eukaryotes to recycle cellular nutrients during stress conditions (such as nutrient limitation, or the accumulation of damaged organelles). We recently revealed a molecular mechanism by which Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Atg22 is not directly required for autophagic body breakdown, in contrast to previously reported data. Instead, we found that Atg22, Avt3 and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagy.

Addendum to:

Atg22 Recycles Amino Acids to Link the Degradative and Recycling Functions of Autophagy

Zhifen Yang, Ju Huang, Jiefei Geng, Usha Nair and Daniel J. Klionsky

Mol Biol Cell 2006; Oct. 4     

Identification of Single Amino Acid Substitutions in Proteogenomics

An important aim of proteogenomics, which combines data of high throughput nucleic acid and protein analysis, is to reliably identify single amino acid substitutions representing a main type of coding genome variants. Exact knowledge of deviations from the consensus genome can be utilized in several biomedical fields, such as studies of expression of mutated proteins in cancer, deciphering heterozygosity mechanisms, identification of neoantigens in anticancer vaccine production, search for RNA editing sites at the level of the proteome, etc. Generation of this new knowledge requires processing of large data arrays from high–resolution mass spectrometry, where information on single–point protein variation is often difficult to extract. Accordingly, a significant problem in proteogenomic analysis is the presence of high levels of false positive results for variant–containing peptides in the produced results. Here we review recently suggested approaches of high quality proteomics data processing that may provide more reliable identification of single amino acid substitutions, especially contrary to residue modifications occurring in vitro and in vivo. Optimized methods for assessment of false discovery rate save instrumental and computational time spent for validation of interesting findings of amino acid polymorphism by orthogonal methods.     

Approach to Minimize Damage Resulting from Microelectrode Insertion into Neuron

Biophysics - Penetration of a microelectrode into the cell soma is one of the most vulnerable stages of the method intracellular recording. Traumatic neuronal activity after piercing the membrane...     

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.     

Enhancement of Thermostability of Firefly Luciferase from Luciola lateralis by a Single Amino Acid Substitution

We constructed firefly luciferase mutants from Luciola lateralis in which Ala at position 217 was replaced by each of three hydrophobic amino acid residues (lie, Leu, and Val). These mutants were superior to the wild-type in thermostability. Especially, the purified Ala217Leu mutant still maintained over 70% of the initial activity after 60 min at 50°C. This mutant is the most thermostable firefly luciferase obtained.     

Insights into Stabilizing Forces in Amyloid Fibrils of Differing Sizes from Polarizable Molecular Dynamics Simulations

Pathological aggregation of amyloid-forming proteins is a hallmark of a number of human diseases, including Alzheimer's, type 2 diabetes, Parkinson's, and more. Despite having very different primary amino acid sequences, these amyloid proteins form similar supramolecular, fibril structures that are highly resilient to physical and chemical denaturation. To better understand the structural stability of disease-related amyloids and to gain a greater understanding of factors that stabilize functional amyloid assemblies, insights into tertiary and quaternary interactions are needed. We performed molecular dynamics simulations on human tau, amyloid-β, and islet amyloid polypeptide fibrils to determine key physicochemical properties that give rise to their unique characteristics and fibril structures. These simulations are the first of their kind in employing a polarizable force field to explore properties of local electric fields on dipole properties and other electrostatic forces that contribute to amyloid stability. Across these different amyloid fibrils, we focused on how the underlying forces stabilize fibrils to elucidate the driving forces behind the protein aggregation. The polarizable model allows for an investigation of how side-chain dipole moments, properties of structured water molecules in the fibril core, and the local environment around salt bridges contribute to the formation of interfaces essential for fibril stability. By systematically studying three amyloidogenic proteins of various fibril sizes for key structural properties and stabilizing forces, we shed light on properties of amyloid structures related to both diseased and functional states at the atomistic level.     

Death Resulting from Fatty Acid Starvation in Yeast

Mutants of Saccharomyces cerevisiae having the genotypes fas1 (fatty acid synthetase minus) and fas1, ole1 (fatty acid synthetase and fatty acid desaturase minus) were found to undergo logarithmic death when deprived of required fatty acids, whereas ole1 strains did not. During the first 2 to 3 h of fatty acid starvation, macromolecular synthesis occurred at apparently normal rates, although cell division stopped by the end of the 1st h. Cell death commenced at approximately the 2nd to the 3rd h, and within 24 h, depending upon conditions, 2 to 4 log orders of death had occurred. The loss of viability was accelerated by the addition of detergent, but could be largely prevented by the interruption of protein synthesis, either by amino acid starvation or by the use of cycloheximide. The possible significance of this phenomenon in terms of membrane biosynthesis is discussed.     

Amyloid Angiopathy of Alzheimer''s Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)