Fibrillogenic oligomers of human cystatin C are formed by propagated domain swapping

Cystatin C and the prion protein have been shown to form dimers via three-dimensional domain swapping, and this process has also been hypothesized to be involved in amyloidogenesis. Production of oligomers of other amyloidogenic proteins has been reported to precede fibril formation, suggesting oligomers as intermediates in fibrillogenesis. A variant of cystatin C, with a Leu68-->Gln substitution, is highly amyloidogenic, and carriers of this mutation suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adulthood. This work describes doughnut-shaped oligomers formed by wild type and L68Q cystatin C upon incubation of the monomeric proteins. Purified oligomers of cystatin C are shown to fibrillize faster and at a lower concentration than the monomeric protein, indicating a role of the oligomers as fibril-assembly intermediates. Moreover, the present work demonstrates that three-dimensional domain swapping is involved in the formation of the oligomers, because variants of monomeric cystatin C, stabilized against three-dimensional domain swapping by engineered disulfide bonds, do not produce oligomers upon incubation under non-reducing conditions. Redox experiments using wild type and stabilized cystatin C strongly suggest that the oligomers, and thus probably the fibrils as well, are formed by propagated domain swapping rather than by assembly of domain-swapped cystatin C dimers.     

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

The aggregation process of wild-type human lysozyme at pH 3.0 and 60 °C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the β-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.     

Characterization of oligomer formation of amyloid-beta peptide using a split-luciferase complementation assay

Amyloid-β peptide (Aβ) is the amyloid component of senile plaques in Alzheimer disease (AD) brains. Recently a soluble oliomeric form of Aβ in Aβ precursor protein transgenic mouse brains and AD brains was identified as a potential causative molecule for memory impairment, suggesting that soluble Aβ oligomers cause neurodegeneration in AD. Further characterization of this species has been hampered, however, because the concentrations are quite small and it is difficult to monitor Aβ oligomers specifically. Here we developed a novel method for monitoring Aβ oligomers using a split-luciferase complementation assay. In this assay, the N- and C-terminal fragments of Gaussia luciferase (Gluc) are fused separately to Aβ. We found that conditioned media from both N- and C-terminal fragments of Gluc-tagged Aβ1-42 doubly transfected HEK293 cells showed strong luminescence. We used gel filtration analyses to analyze the size of oligomers formed by the luciferase complementation assay, and found that it matched closely with oligomers formed by endogenous Aβ in Tg2576 neurons. Large oligomers (24-36-mers), 8-mers, trimers, and dimers predominate. In both systems, Aβ formed oligomers intracellularly, which then appear to be secreted as oligomers. We then evaluated several factors that might impact oligomer formation. The level of oligomerization of Aβ1-40 was similar to that of Aβ1-42. Homodimers formed more readily than heterodimers. The level of oligomerization of murine Aβ1-42 was similar to that of human Aβ1-42. As expected, the familial AD-linked Arctic mutation (E22G) significantly enhanced oligomer formation. These data suggest that Gluc-tagged Aβ enables the analysis of Aβ oligomers.     

The enzyme activity allosteric regulation model based on the composite nature of catalytic and regulatory sites concept

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure. The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.     

Remarks on the chemical conditions on the surface of the primitive earth and the probability of the evolution of life.

The inner planets were formed from smaller objects that had no gases associated with them. These objects contained relatively small amounts of water and carbon in a form similar to that found in carbonaceous chondrites. The first forms of life must have originated during the time when the water reacted with the carbon (and also with nitrides, phosphides, etc.), while the hydrogen formed by this reaction was continuously lost from the gravitational field of the Earth. About 10-44 atoms of carbon reacted with water during less than 10-17 s. The crucial question is whether some form of life will always develop under these conditions, or whether the origin of life is an improbable, perhaps an immensely improbable event. At present it is still impossible to answer this question.     

Internal correlations in minor groove profiles of experimental and computed B-DNA conformations.

It has been noticed that converged conformations of B-DNA oligomers obtained in MD calculations often have very small atom position rmsd values from the canonical B-DNA and all helical parameters close to the standard values, but their minor grooves tend to be somewhat narrower. This apparent bias disappears, however, when C5' rather than phosphorus atoms are used for measuring the groove width. At the origin of this effect is the specific orientation of phosphate groups in the canonical B-DNA model which maximizes their separation across the minor groove. When measured by C5' traces, minor groove profiles of experimental structures available in the Nucleic Acids Database show much less tendency to narrow below the canonical width. Correlation analysis reveals a high degree of correspondence in shapes of minor grooves of calculated and experimental single-crystal structures of B-DNA oligomers.     

The Enzyme Activity Allosteric Regulation Model Based on the Composite Nature of Catalytic and Regulatory Sites Concept

Abstract

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure.

The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric

The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.     

Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization.

Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on membranes. However, incorporation of the modified T250C mutant inhibited subsequent oligomerization, so that the hybrid oligomers were reduced in size. These appeared as typical arc lesions in the electron microscope. They formed pores that permitted passage of NaCl and calcein but restricted permeation of large dextran molecules. The data indicate that the SLO pore is formed gradually during oligomerization, implying that pores lined by protein on one side and an edge of free lipid on the other may be created in the plasma membrane. Intentional manipulation of the pore size may extend the utility of SLO as a tool in cell biological experiments.     

Ligand-Induced Reassembly of GroEL/ES Chaperone In Vitro: Visualization by Electron Microscopy

The products of the reassembly reaction of tetradecameric two-ring quaternary structure of GroEL chaperonin under the pressure of its heptameric co-chaperonin GroES have been visualized by electron microscopy. It has been shown that one-ring heptameric oligomers of GroEL have been formed at the beginning (after ~5 min) of the reaction, while at the final stage of the reaction (after ~70 min), both onering heptamers in complex with one GroES and two-rings tetradecamers in complexes with one (asymmetrical complex) or two (symmetrical complex) GroES heptamers are present. The relationship between the data of light scattering, native electrophoresis, and electron microscopy obtained earlier has been discussed.     

A molecular model of Alzheimer amyloid beta-peptide fibril formation

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.     

Chemical evolution XXIX. Pyrimidines from hydrogen cyanide.

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 8-9. Hydrolysis of these oligomers at pH 8.5 or with 6 N HCl yields 4,5-dihydroxypyrimidine, as the most abundant pyrimidine product along with orotic acid and 5-hydroxyuracil. These results, together with the earlier data, demonstrate that the three major nitrogen-containing classes of biomolecules could have originated from HCN on the primitive earth. The observation of the formation of orotic acid and 4-aminoimidazole-5-carboxamide by the hydrolysis of the HCN oligomers suggests that once the initially formed pyrimidines and purines were consumed, those life forms persisted which evolved enzymes for conversion of these intermediates to the pyrimidines and purines present in contemporary RNA.     

Heterogeneous triangular structures of human islet amyloid polypeptide (amylin) with internal hydrophobic cavity and external wrapping morphology reveal the polymorphic nature of amyloid fibrils

The misfolding and self-assembly of human islet amyloid polypeptide (hIAPP or amylin) into amyloid fibrils is pathologically linked to type II diabetes. The polymorphic nature of both hIAPP oligomers and fibrils has been implicated for the molecular origin of hIAPP toxicity to islet β-cells, but little is known about the polymorphic structure and dynamics of these hIAPP oligomers/fibrils at the atomic level. Here, we model the polymorphism of full length hIAPP(1-37) oligomers based on experimental data from solid-state NMR, mass per length, and electron microscopy using all-atom molecular dynamics simulation with explicit solvent. As an alternative to steric zipper structures mostly presented in the 2-fold symmetrical fibrils, the most striking structural feature of our proposed hIAPP oligomers is the presence of 3-fold symmetry along the fibril growth axis, in which three β-sheet-layers wind around a hydrophobic core with different periodicities. These 3-fold triangular hIAPP structures dramatically differ in the details of the β-layer assembly and core-forming sequence at the cross section, but all display a high structural stability with favorable layer-to-layer interactions. The 3-fold hIAPP structures can also serve as templates to present triple-stranded helical fibrils via peptide elongation, with different widths from 8.7 to 9.9 nm, twists from 2.8° to 11.8°, and pitches from 14.5 to 61.1 nm, in reasonable agreement with available biophysical data. Because similar 3-fold Aβ oligomers are also observed by both NMR experiments and our previous simulations, the 3-fold structure could be a general conformation to a broad range of amyloid oligomers and fibrils. Most importantly, unlike the conventional stacking sandwich model, the proposed wrapping-cord structures can readily accommodate more than three β-layers via a two dimension conformation search by rotating and translating the β-layers to adopt different favorable packings, which can greatly enrich the polymorphism of amyloid oligomers and fibrils.     

The instability of the autogen

The autogen theory has sought to provide a mechanism for the rapid origin of a self-replicating chemical system from short, random oligomers. The autogen is considered in terms of hypercycle theory, and its dynamic behavior is subjected to fixed point analysis. It is shown that the components of the autogen are incapable of stable coexistence.     

Studies on the biosynthesis of 16-membered macrolide antibiotics using carbon-13 nuclear magnetic resonance spectroscopy.

The origin of the skeletal carbons in the lactone ring of 16-membered macrolide antiobiotics has been studied. 13C-labeled antibiotics leucomycin and tylosin, have been obtained from the culture broth of Streptomyces kitasatoensis 66-14-3 and Streptomyces fradiae C-373, respectively in the presence of appropriate 13C-labeled precursors, and 13C NMR spectra of the antibiotics thus obtained have been measured. It was shown that the aglycone of leucomycin A3 is derived from five acetates, one propionate, one butyrate, and an unknown precursor corresponding to two carbons. The formyl carbon which is characteristic of the basic 16-membered macrolides orginates from C-4 butyrate. On the other hand, the aglycone of tylosin is formed from two acetates, five propionates and one butyrate. Butyric acid and ethylmalonic acid are metabolized to propionyl-CoA or methylmolonyl-CoA through a pathway involving methylmalonyl-CoA mutase, and subsequently incorporated into the lactone ring of tylosin.     

Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C): An RNA polymerase model

(Guanosine 5′-phosphor)-2-methylimidazolide (2-MeImpG), unlike guanosine 5′-phosphorimidazolide (ImpG), undergoes an efficient, buffer-independent, template-directed oligomerization in the presence of poly(C) at pH values above 7.6. The reaction occurs in a Watson-Crick double helix and yields predominantly 3′-5′-linked oligomers up to the 50-mer in above 80% yield. Synthesis proceeds in the 5′ → 3′ direction and has high fidelity in the sense that nucleotides other than G are not incorporated significantly into oligomers. Under some conditions, oligomers corresponding to approximately one and two turns of the helix are obtained in higher yield than somewhat longer or somewhat shorter oligomers.In the protonated triple-helical structure formed below pH 7, the efficiency of the oligomerization is much lower. Oligomers up to about the 10-mer are obtained. The most abundant products are “capped” at the 5′ terminus with a GppG pyrophosphate group.     

Racemization and the origin of optically active organic compounds in living organisms

The organic compounds synthesized in prebiotic experiments are racemic mixtures. A number of proposals have been offered to explain how asymmetric organic compounds formed on the Earth before life arose, with the influence of chiral weak nuclear interactions being the most frequent proposal. This and other proposed asymmetric syntheses give only slight enantiomeric excess and any slight excess will be degraded by racemization. This applies particularly to amino acids where half-lives of 10(5)-10(6) years are to be expected at temperatures characteristic of the Earth's surface. Since the generation of chiral molecules could not have been a significant process under geological conditions, the origins of this asymmetry must have occurred at the time of the origin of life or shortly thereafter. It is possible that the compounds in the first living organisms were prochiral rather than chiral; this is unlikely for amino acids, but it is possible for the monomers of RNA-like molecules.     

Structural transformations of oligomeric intermediates in the fibrillation of the immunoglobulin light chain LEN

LEN is a kappaIV immunoglobulin light chain variable domain from a patient suffering from multiple myeloma but with no evidence of amyloid fibrils. However, fibrils are formed when LEN solutions are agitated under mildly destabilizing conditions. Surprisingly, an inverse concentration dependence was observed on the kinetics of fibril formation because of the formation of off-pathway soluble oligomers at high protein concentration. Despite the fact that most of the protein is present in the off-pathway intermediates at relatively early times of aggregation, eventually all the protein forms fibrils. Thus, a structural rearrangement from the non fibril-prone off-pathway oligomers to a more fibril-prone species must occur. A variety of techniques were used to monitor changes in the size, secondary structure, solvent accessibility, and intrinsic stability of the oligomers, as a function of incubation time. The structural rearrangement was accompanied by a significant increase of disordered secondary structure, an increase in solvent accessibility, and a decrease in intrinsic stability of the soluble oligomeric species. We conclude that fibrils arise from the oligomers containing a less stable conformation of LEN, either directly or via dissociation. This is the first fibrillating system in which soluble off-pathway oligomeric intermediates have been shown to be the major transient species and in which fibrillation occurs from a relatively unfolded conformation present in these intermediates.     

The spectrin-actin complex and erythrocyte shape

The effects of phosphorylation of spectrin on the properties of the cytoskeletal network of the human erythrocyte have been studied. A suspension of the cytoskeletal residues obtained after extraction of the ghosts with the nonionic detergent Triton X-100 forms a gel on addition of membrane kinase and ATP. Phosphorylation has no effect on the association state of purified spectrin. No species higher than a tetramer of polypeptide chains is formed in vitro; in the absence of divalent cations, this tetramer is an entity liberated from and evidently present in the membrane. It has not so far proved possible to detect any F-actin in the cytoskeleton before or after phosphorylation. It is suggested that the consequence of phosphorylation is formation of additional interactions between spectrin and monomeric actin molecules. This view is supported by the formation, after phosphorylation of the Triton-extracted cytoskeleton, of an insoluble mass of protein on treatment with a cross-linking reagent. In the absence of divalent cations, a series of oligomeric species is progressively liberated from the cytoskeleton on extraction with solutions of low ionic strength. These oligomers contain actin as well as spectrin, and are thought to result from disruption of the network by random denaturation of the mono meric actin in the absence of divalent metal ions. A schematic view of the effects of phosphorylation on the structure of the cytoskeleton is presented.