Calcium and high molecular weight protein aggregates in bovine and human lens.
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Phenylhydrazine causes sulfhydryl oxidation and protein aggregation in hemoglobin-free human erythrocyte membranes.
Phenylhydrazine induces a remarkable decrease in total free sulfhydryls as well as spectrin and most other polypeptides in hemoglobin-free human erythrocyte ghosts. A parallel increase in coomassie blue stainable material that was retained on the gel tops was also observed. EDTA but not DM protected the membrane from phenylhydrazine-induced alterations, while antioxidants, catalase and SOD were ineffective. The aggregated material isolated from phenylhydrazine-treated membranes could be partially dissociated in presence of beta-mercaptoethanol to yield most of the membrane polypeptides.
Antioxidants↗
Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A.
Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.
Chromatography, High Pressure Liquid↗
Characterization of a phosphorylated form of the intermediate filament-aggregating protein filaggrin.
Filaggrin and a phosphorylated form of filaggrin, which has been shown by pulse-chase studies to be a precursor form of the protein [Dale, B. A., & Ling, S. Y. (1979) Biochemistry 18, 3539-3546], were compared for functional, biochemical, and physical properties. Filaggrin reacts with keratin filaments to form visible macrofibrils, unlike the precursor which does not. Biochemical and peptide-mapping studies suggest that the two proteins have similar, perhaps identical, amino acid sequences. The major differences between the two proteins are in molecular weight (precursor, 44 200 g/mol; filaggrin, 38 400 g/mol), the existence of oligomeric forms of the precursor, and the presence of phosphate in the precursor (15-20 mol/mol of protein). Phosphoserine was identified in the precursor, but neither phosphothreonine nor phosphotyrosine was observed. The results of proteolytic digests of [32P]phosphate-radiolabeled precursor show that the phosphate is unevenly distributed throughout the molecule and may be localized in approximately 30% of the precursor. A discrete localization of the phosphate in the precursor may block a specific keratin filament combining site and so prevent premature aggregation of these filaments during epidermal differentiation. It is suggested that a specific phosphatase is involved in the dephosphorylation, because several phosphatases of general specificity, including rat epidermal lysosomal acid phosphatase, did not catalyze this conversion.
Amino Acids↗
Electrostatically driven protein aggregation: beta-lactoglobulin at low ionic strength.
The aggregation of beta-lactoglobulin (BLG) at ambient temperature was studied using turbidimetry and dynamic light scattering in the range 3.8<pH<5.2 in 0.0045 M NaCl, and in the ionic strength range 0.0045-0.5 M at fixed pH=5.0. The initial rate of aggregation, taken as the initial slope of turbidity vs time, (dtau/dt)0, indicated maximum aggregation near pH 4.6 (below the isoelectric point of 5.2), but the dependence of the initial rate of aggregation on pH was highly asymmetric. At pH 5.0, (dtau/dt)0 strongly increased with a decrease in ionic strength I from 0.1 to 0.0045 M and was found to be nearly linear with 1/I. DLS measurements revealed an increase in particle size with time, with the appearance of bimodal distributions in which the fast and slow modes corresponded, respectively, to a BLG dimer and to larger aggregates in the 100-800 nm range. At conditions of slower aggregation, DLS revealed the consumption of dimers to form higher order aggregates with no intermediate species. Computer modeling (Delphi) was used to visualize the electrostatic potential around the dimer to elucidate the pH and ionic strength dependence of the initial aggregation rates. The aggregation process appears to comprise an initial fast consumption of the dimer, whose dependence on pH and I arises from the interaction of the positive and negative domains of interacting dimers, followed by the slow formation of much larger aggregates with relatively little sensitivity to pH and I. The open-ended nature of BLG aggregation is thought to arise from the asymmetry of the dimer charge distribution.
Hydrogen-Ion Concentration↗
Protein aggregation complicates the development of baculovirus-expressed African horsesickness virus serotype 5 VP2 subunit vaccines.
This paper describes the expression of a cloned African horsesickness virus (AHSV) serotype 5 VP2-gene by a baculovirus recombinant that was generated by the BAC-TO-BAC system. Immunization of horses with crude cell lysates containing recombinant baculovirus-expressed AHSV5 VP2 did induce neutralizing antibodies, but afforded only partial protection against virulent virus challenge. Further analysis of partially protective crude cell lysates revealed that baculovirus-expressed AHSV5 VP2 was predominantly present in the form of insoluble aggregates. Only approximately 10% of VP2 was present in a soluble form. Immunization of guinea-pigs with aggregated and soluble forms of AHSV5 VP2 established that only soluble VP2 was capable of inducing neutralizing antibodies. This finding adds a new dimension to the development of AHSV VP2s as subunit vaccines. Further investigation is needed to limit formation of insoluble aggregates and optimize conditions for producing VP2 in a form capable of inducing protective immunity.
African Horse Sickness Virus↗
A mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates.
BACKGROUND & AIMS: Wilson disease is a genetic disorder characterized by the accumulation of copper in the body as a result of a defect of copper excretion from hepatocytes. The intracellular localization of the Wilson disease gene product, ATP7B, was recently identified as the late endosomes. Various mutations have been documented in patients with Wilson disease. The clinical manifestations vary greatly among the patients; however, there is little information on the genotype-phenotype correlation. METHODS: We investigated the distribution of a common ATP7B mutant His1069Gln and a mutant Asp1270Ser by expressing the mutants tagged with green fluorescent protein in Huh7 and HEK293 cells. Intracellular organelles were visualized by fluorescence microscopy. RESULTS: Although the wild-type ATP7B and Asp1270Ser mutant localized in the late endosomes, His1069Gln mutant did not locate in the late endosomes and was degraded by the proteasomes in the cytoplasm. Furthermore, His1069Gln formed aggresomes composed of the degradates and intermediate filaments at the microtubule-organizing center. These aggresomes were similar to Mallory bodies on electron microscopy. CONCLUSIONS: The different protein properties of ATP7B mutants may explain the variety of clinical spectrums in patients with Wilson disease.
Acetylcysteine↗
Role of oxidative damage in protein aggregation associated with Parkinson's disease and related disorders.
Parkinson's disease, the most common movement disorder, is characterized by the loss of brainstem neurons, specifically dopaminergic neurons in the substantia nigra, as well as the accumulation of neuronal cytoplasmic filamentous proteinaceous inclusions comprised of polymerized alpha-synuclein. It was reported recently that alpha-synuclein can induce the formation of filamentous tau inclusions, which are characteristic of disorders like Alzheimer's disease and Lewy body variant of Alzheimer's disease, suggesting that a similar mechanism may exist between alpha-synuclein fibrillogenesis and tau polymerization. Pathological brain inclusions comprised of alpha-synuclein or tau proteins are associated with a spectrum of neurodegenerative disorders, and oxidative and nitrative injury has been implicated in all of these diseases. However, the role of oxidative damage in alpha-synuclein and tau polymerization and pathological inclusion formation is complex. Differences in the level, type, and temporal sequence of the oxidative alterations appear to result in both inhibitory and stimulatory effects on the fibrillogenesis of these proteins.
Animals↗
Anaphylactoid reactions due to non-immune complex serum protein aggregates.
The infusion of aggregate-containing i.v. human gamma-globulin as well as human serum albumin can lead to severe anaphylactoid reactions with decrease in mean arterial pressure, increase in pulmonary artery pressure and decrease in cardiac output in unsensitized dogs, while the deaggregated solutions are well tolerated. During these anaphylactoid reactions, no significant changes in the serum complement activity of the dogs were observed. In clinical human serum albumin incompatibility, stimulation with albumin aggregates led to a high response in the lymphocyte culture, whereas deaggregated albumin had no stimulatory effect. By deaggregation of horse anti-human lymphocyte globulin prior to clinical administration, the compatibility of ALG therapy was improved.
Anaphylaxis↗
Chain-length dependence of alpha-helix to beta-sheet transition in polylysine: model of protein aggregation studied by temperature-tuned FTIR spectroscopy.
The chain-length dependence of the alpha-helix to beta-sheet transition in poly(L-lysine) is studied by temperature-tuned FTIR spectroscopy. This study shows that heterogeneous samples of poly(L-lysine), comprising polypeptide chains with various lengths, undergo the alpha-beta transition at an intermediate temperature compared to homogeneous ingredients. This holds true as long as each individual fraction of the polypeptide is capable of adopting an antiparallel beta-sheet structure. The tendency is that the longer chain is, the lower the alpha-beta transition temperature is, which has been linked to the presence of distorted or solvated helices with turns or beta sheets in elongating chains of poly(L-lysine). As such helical structures are apparently conducive to the alpha-beta transition, this draws a comparison to the hypothesis of metastable protein conformational states being a common stage in amyloid-formation pathways. The antiparallel architecture of the beta sheet is likely to reflect the pretransition interhelical interactions in poly(L-lysine). Namely, the chains are arranged in an antiparallel manner because of energetically favored antiparallel pre-assembly of dipolar alpha helices.
Molecular Weight↗
X-ray small angle scattering of the human transferrin protein aggregates. A fractal study.
X-ray small angle scattering experiments, using a pin hole SAXS camera with Synchrotron radiation source, have been performed to study the conformational changes of lyophilized samples of Apo-, Mono-, and Diferric- human transferrin. We report the experimental evidence that the analysis of the scattered intensity through the fractal theory may give information on the particle size and its variation upon iron binding.
Apoproteins↗
Nanostructured dna-protein aggregates consisting of covalent oligonucleotide-streptavidin conjugates.
Covalent conjugates consisting of streptavidin and a 24-mer single-stranded DNA oligonucleotide have been oligomerized by cross-linking with a 5',5'-bis-biotinylated 169-base-pair double-stranded DNA (dsDNA) fragment. The oligomeric conjugates formed have been analyzed by nondenaturing gel electrophoresis and scanning-force microscopy (SFM). The comparison of analogous oligomers, prepared from native STV and the bis-biotinylated dsDNA fragment, revealed that the covalent STV-oligonucleotide hybrid conjugates self-assemble to generate oligomeric aggregates of significant smaller size, containing on average only about 2.5 times less dsDNA fragments per aggregate. Likely, this is a consequence of electrostatic or steric repulsion between the dsDNA and the single-stranded oligomer covalently attached to the hybrid, as indicated from control experiments. Nevertheless, the single-stranded oligonucleotide moiety within the oligomeric conjugates can be used as a selective molecular handle for further functionalization and manipulation. For instance, it was used for specific DNA-directed immobilization at a surface, previously functionalized with complementary capture oligonucleotides. Moreover, we demonstrate that macromolecules, such as STV and antibody molecules, which are tagged with the complementary oligonucleotide, specifically bind to the supramolecular DNA-STV oligomeric conjugates. This leads to a novel class of functional DNA-protein conjugates, suitable, for instance, as reagents in immuno-PCR or as building blocks in molecular nanotechnology.
Animals↗
Protein aggregation in C-phycocyanin. Studies at very low concentrations with the photoelectric scanner of the ultracentrifuge.
Solutions of C-phycocyanin of very low concentrations were examined by sedimentation-velocity studies in the Spinco model E ultracentrifuge equipped with a photoelectric scanning system and a monochromator. At sufficiently low concentrations complete disaggregation from the hexamer to the monomer was observed. The equilibrium constant of monomer to hexamer was estimated to be approx. 10(30). For studies of aggregation over the complete range of concentration, C-phycocyanins from Phormidium luridum and Lyngbya sp. were used. Sedimentation-velocity studies at high concentration with schlieren optics are reported for C-phycocyanins from Anabaena variabilis and Lyngbya sp. The pH-dependence of aggregation and the temperature-dependence of trimer-hexamer equilibrium for phycocyanins from these algae were found to be similar to those of other C-phycocyanins. The principal feature of the pH-dependence is the dominance of hexamers at the isoelectric point. Increasing temperature increased the amount of hexamer and decreased the amount of trimer.
Centrifugation, Density Gradient↗
Alterations in protein aggregation and degradation due to mild and severe missense mutations (A104D, R157N) in the human phenylalanine hydroxylase gene (PAH).
Phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine; its activity is the major determinant of phenylalanine disposal. Mutations in the corresponding human gene (PAH), which encodes the human hepatic PAH enzyme, result in hyperphenylalaninemia; the resulting phenotypes can range in severity from mild forms of hyperphenylalaninemia with benign outcome to the severe form, phenylketonuria with impaired cognitive development. This paper describes the detailed characterization of two inherited recessive missense mutations in PAH, c.311C-->A (A104D) and [c.470G-->A;c.471A-->C] (R157N), which are associated, respectively, in the homozygous or functionally hemizygous states, with mild and severe metabolic phenotypes. We used three different in vitro PAH expression systems (in Escherichia coli, cell-free rabbit reticulocyte lysates, and human embryonal kidney cells), as well as a unique assay for phenylalanine oxidation in vivo. In each system, we observed alterations of PAH function and physical properties, compared with wild-type enzyme, and differences in relative severity of effects between these two mutations. Pulse-chase experiments showed increased PAH degradation, probably related to observed aberrations in protein folding and altered oligomerization, as a basic mechanism underlying effects of these missense mutations.
Cell Line↗
Twisted protein aggregates and disease: the stability of sickle hemoglobin fibers.
We describe how twist could play an essential role in stabilizing 20 nm diameter sickle hemoglobin fibers. Our theory successfully reproduces the observed variation of helical pitch length with fiber diameter. With no remaining adjustable parameters it also yields a prediction for the torsional rigidity of sickle hemoglobin fibers that is in good agreement with experiment and hence retains the striking feature that such fibers can be highly mechanically anisotropic, even with a ratio of bending to torsional rigidity of about 50. We discuss how our study might be relevant to the development of treatment strategies.
Hemoglobin, Sickle↗
Familial amyloidotic polyneuropathy: protein aggregation in the peripheral nervous system.
The molecular pathology underlying transthyretin (TTR)-related amyloidosis is largely unknown. It is possible that a common factor in the amyloidogenesis process exists among the different forms; this common factor can involve changes produced by mutations in the three-dimensional structure of TTR, rendering it prone to deposition as amyloid. This amyloidogenic potential, together with other yet unidentified factors, contribute to amyloid deposition. The factors that trigger fibril formation and/or neurodegeneration in TTR-related amyloidosis present central questions for which there are still no available clues. We recently showed in vitro that TTR fibrils trigger NF-kappaB activation, and subsequent studies identified some inflammatory and apoptotic pathways opening perspectives to understand the neurodegeneration process in familial amyloidotic polyneuropathy (FAP). It is current opinion that the modified TTR represents an amyloidogenic intermediate, which integrates the fibril structure; analyses of FAP fibrils have proved that TTR in the fibrils maintains a beta-conformation and have suggested that the TTR monomer is the building block in fibrils. This concept has been questioned recently by investigators, and only future studies on native and synthetic TTR fibrils using high-resolution structural techniques will further elucidate fibril structure and the aggregation pathway. Modulators responsible for phenotypic diversity can be addressed by mice transgenic for different human TTR mutations. Different lines are now available; incomplete penetrance and environmental influence on the deposition of mutant TTR has been observed. Therefore, these animals constitute important tools to address modulators of phenotypic expression and pathophysiological consequences of amyloid deposition at cellular/molecular levels. They are pivotal for testing potential drugs for TTR amyloidosis as well.
Amyloid↗
Phenylalanyl-tRNA synthetase of Escherichia coli K10. Effects of zinc(II) on partial reactions of diadenosine 5',5"'-P1,P4-tetraphosphate synthesis, conformation, and protein aggregation.
The synthesis of diadenosine 5',5"'-P1-,P4-tetraphosphate (Ap4A) catalyzed by phenylalanyl-tRNA synthetase in the presence of Zn2+ involves the same partial reactions (synthesis of phenylalanyladenylate and transfer of the adenylate moiety to ATP) as occur in the absence of this metal ion. However, transfer is strongly stimulated while adenylate synthesis is depressed. Also inhibited are pyrophosphorolysis of phenylalanyladenylate and transfer of phenylalanine from the adenylate to cognate tRNA, because overall tRNA phenylalanylation is depressed [Mayaux, J.-F., & Blanquet, S. (1981) Biochemistry 20, 4647-4654], whereas binding of tRNA to the synthetase is not. At moderate concentrations of Zn2+, and in the presence of 5 microM phenylalanine and 0.5 mM ATP, transfer of AMP is rate limiting, while at higher concentrations of Zn2+ synthesis of adenylate is rate determining. The Zn2+ concentration optimum for stimulation depends on the concentration of phenylalanine and ATP. The effects of Zn2+ are mediated through two classes of binding site(s) on the synthetase, the half-saturations of which are 1-4 and 20-30 microM Zn2+, respectively. Binding of Zn2+ to the second class of site(s) causes inhibition of the synthetase, whereas binding to the first class is responsible for activation and inhibition, which may be caused by a conformational change. Evidence for the latter is the observed decrease in protein intrinsic fluorescence intensity and the decrease in fluorescence intensity of 6-(p-toluidinyl)naphthalene-2-sulfonate, which is used as a reporter group. The kinetics of the binding reaction show a saturation dependence on Zn2+, also suggesting that a conformational change occurs.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenine Nucleotides↗
A new point of view on the problem of G-protein aggregation. Assembly-disassembly of tubulin as an oscillation process.
Basic principles of nonlinear thermodynamics are used to develop a theory of tubulin assembly. The presence of GTP/GDP switch provides different ways for tubulin aggregation. The GTP-tubulin assembly results in determining the regime of limit cycle favoring growth of tubulin spiral with the formation of microtubules. The GDP-tubulin assembly, on the contrary, results in forming double rings. The difference between the GTP- and GDP-tubulin assemblies is that the former is a dynamical oscillation process while the latter is no more than a transition from a weakly nonequilibrium point to the equilibrium state along a thermodynamical path. Corresponding equations for the GTP- and GDP-tubulin assemblies are proposed.
GTP-Binding Proteins↗