Isoelectric point-amyloid formation of α-synuclein extends the generality of the solubility and supersaturation-limited mechanism.

Proteins in either a native or denatured conformation often aggregate at an isoelectric point (pI), a phenomenon known as pI precipitation. However, only a few studies have addressed the role of pI precipitation in amyloid formation, the crystal-like aggregation of denatured proteins. We found that α-synuclein, an intrinsically disordered protein of 140 amino acid residues associated with Parkinson's disease, formed amyloid fibrils at pI (= 4.7) under the low-sodium phosphate conditions. Although α-synuclein also formed amyloid fibrils at a wide pH range under high concentrations of sodium phosphate, the pI-amyloid formation was characterized by marked amyloid-specific thioflavin T fluorescence and clear fibrillar morphology, indicating highly ordered structures. Analysis by heteronuclear NMR in combination with principal component analysis suggested that amyloid formation under low and high phosphate conditions occurred by distinct mechanisms. The former was likely to be caused by the intermolecular attractive charge-charge interactions, where α-synuclein has +17 and -17 charges even with the zero net charge. On the other hand, the latter was caused by the phosphate-dependent salting-out effects. pI-amyloid formation may play a role in the membrane-dependent amyloid formation of α-synuclein, where the negatively charged membrane surface reduces the local pH to pI and the membrane hydrophobic environment enhances electrostatic interactions. The results extend the supersaturation-limited mechanism of amyloid formation: Amyloid fibrils are formed under a variety of conditions of decreased solubility of denatured proteins triggered by the breakdown of supersaturation.

Heating during agitation of ß2-microglobulin reveals that supersaturation breakdown is required for amyloid fibril formation at neutral pH.

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. ß2-Microglobulin (ß2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of ß2m in patients are observed at neutral pH, formation of ß2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that ß2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the ß2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.

Polyphosphates diminish solubility of a globular protein and thereby promote amyloid aggregation.

Structural changes of globular proteins and their resultant amyloid aggregation have been associated with various human diseases, such as lysozyme amyloidosis and light-chain amyloidosis. Because many globular proteins can convert into amyloid fibrils in vitro, the mechanisms of amyloid fibril formation have been studied in various experimental systems, but several questions remain unresolved. Here, using several approaches, such as turbidimetry, fluorescence and CD spectroscopy, EM, and isothermal titration calorimetry, we examined the binding of polyphosphates to hen egg-white lysozyme under acidic conditions and observed polyphosphate-induced structural changes of the protein promoting its aggregation. Our data indicate that negatively charged polyphosphates bind to protein molecules with a net positive charge. The polyphosphate-bound, structurally destabilized protein molecules then start assembling into insoluble amorphous aggregates once they pass the solubility limit. We further show that the polyphosphates decrease the solubility limit of the protein and near this limit, the protein molecules are in a labile state and highly prone to converting into amyloid fibrils. Our results explain how polyphosphates affect amorphous aggregation of proteins, how amyloid formation is induced in the presence of polyphosphates, and how polyphosphate chain length is an important factor in amyloid formation.

Possible mechanisms of polyphosphate-induced amyloid fibril formation of ß2-microglobulin.

Polyphosphate (polyP), which is found in various microorganisms and human cells, is an anionic biopolymer consisting of inorganic phosphates linked by high-energy phosphate bonds. Previous studies revealed that polyPs strongly promoted the amyloid formation of several amyloidogenic proteins; however, the mechanism of polyP-induced amyloid formation remains unclear. In the present study using ß2-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis, we investigated amyloid formation in the presence of various chain lengths of polyPs at different concentrations under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) conditions. We found that the amyloid formation of ß2m at acidic pH was significantly accelerated by the addition of polyPs at an optimal polyP concentration, which decreased with an increase in chain length. The results obtained indicated that electrostatic interactions between positively charged ß2m and negatively charged polyPs play a major role in amyloid formation. Under neutral pH conditions, long polyP with 60 to 70 phosphates induced the amyloid formation of ß2m at several micromoles per liter, a similar concentration range to that in vivo. Since ß2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with ß2m electrostatically. PolyPs appear to dehydrate water molecules around ß2m under the unfolded conformation, leading to the preferential stabilization of less water-exposed amyloid fibrils. These results not only revealed the pH-dependent mechanism of the amyloid formation of ß2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis.

Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces.

Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano-building block for further molecular assembly.

Aggregation-phase diagrams of ß2-microglobulin reveal temperature and salt effects on competitive formation of amyloids versus amorphous aggregates.

Several serious diseases are associated with crystal-like amyloid fibrils or glass-like amorphous aggregates of denatured proteins. However, protein aggregation involving both types of aggregates has not yet been elucidated in much detail. Using a protein associated with dialysis-related amyloidosis, ß2-microglobulin (ß2m), we previously demonstrated that amyloid fibrils and amorphous aggregates form competitively depending on salt (NaCl) concentration. To examine the generality of the underlying competitive mechanisms, we herein investigated the effects of heat on acid-denatured ß2m at pH 2. Using thioflavin fluorescence, CD, and light scattering analysis along with atomic force microscopy imaging, we found that the temperature-dependent aggregation of ß2m markedly depends on NaCl concentration. Stepwise transitions from monomers to amyloids and then back to monomers were observed at low NaCl concentrations. Amorphous aggregates formed rapidly at ambient temperatures at high NaCl concentrations, but the transition from amorphous aggregates to amyloids occurred only as the temperature increased. Combining the data from the temperature- and NaCl-dependent transitions, we constructed a unified phase diagram of conformational states, indicating a parabolic solubility curve with a minimum NaCl concentration at ambient temperatures. Although amyloid fibrils formed above this solubility boundary, amorphous aggregates dominated in regions distant from this boundary. Kinetic competition between supersaturation-limited slow amyloid fibrillation and supersaturation-unlimited fast amorphous aggregation deformed the phase diagram, with amyloid regions disappearing with fast titration rates. We conclude that phase diagrams combining thermodynamics and kinetics data provide a comprehensive view of ß2m aggregation exhibiting severe hysteresis depending on the heat- or salt-titration rates.

Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks.

The design of novel proteins that self-assemble into supramolecular complexes is important for development in nanobiotechnology and synthetic biology. Recently, we designed and created a protein nanobuilding block (PN-Block), WA20-foldon, by fusing an intermolecularly folded dimeric de novo WA20 protein and a trimeric foldon domain of T4 phage fibritin (Kobayashi et al., J. Am. Chem. Soc. 2015, 137, 11285). WA20-foldon formed several types of self-assembling nanoarchitectures in multiples of 6-mers, including a barrel-like hexamer and a tetrahedron-like dodecamer. In this study, to construct chain-like polymeric nanostructures, we designed de novo extender protein nanobuilding blocks (ePN-Blocks) by tandemly fusing two de novo binary-patterned WA20 proteins with various linkers. The ePN-Blocks with long helical linkers or flexible linkers were expressed in soluble fractions of Escherichia coli, and the purified ePN-Blocks were analyzed by native PAGE, size exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS), and transmission electron microscopy. These results suggest formation of various structural homo-oligomers. Subsequently, we reconstructed hetero-oligomeric complexes from extender and stopper PN-Blocks by denaturation and refolding. The present SEC-MALS and SAXS analyses show that extender and stopper PN-Block (esPN-Block) heterocomplexes formed different types of extended chain-like conformations depending on their linker types. Moreover, atomic force microscopy imaging in liquid suggests that the esPN-Block heterocomplexes with metal ions further self-assembled into supramolecular nanostructures on mica surfaces. Taken together, the present data demonstrate that the design and construction of self-assembling PN-Blocks using de novo proteins is a useful strategy for building polymeric nanoarchitectures of supramolecular protein complexes.

Membrane-mediated amyloid deposition of human islet amyloid polypeptide.

Amyloid deposition of human islet amyloid polypeptide (hIAPP) within the islet of Langerhans is closely associated with type II diabetes mellitus. Accumulating evidence indicates that the membrane-mediated aggregation and subsequent deposition of hIAPP are linked to the dysfunction and death of insulin-producing pancreatic ß-cells, but the molecular process of hIAPP deposition is poorly understood. In this review, I focus on recent in vitro studies utilizing model membranes to observe the membrane-mediated aggregation/deposition of hIAPP. Membrane surfaces can serve as templates for both hIAPP adsorption and aggregation. Using high-sensitivity surface analyzing/imaging techniques that can characterize the processes of hIAPP aggregation and deposition at the membrane surface, these studies provide valuable insights into the mechanism of membrane damage caused by amyloid deposition of the peptide.

Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation.

Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 °C. At concentrations lower than 20 µg/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.

Uptake of raft components into amyloid ß-peptide aggregates and membrane damage.

Amyloid aggregation and deposition of amyloid ß-peptide (Aß) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of Aß with membranes containing ganglioside GM1 (GM1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with Aß deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of Aß in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of Aß, uptake of the raft components into Aß aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into Aß deposits induces macroscopic heterogeneity of the membrane structure.

Amyloid aggregation and deposition of human islet amyloid polypeptide at membrane interfaces.

Amyloid deposition of human islet amyloid polypeptide (hIAPP) within the islets of Langerhans is a pathological feature of type 2 diabetes mellitus. Substantial evidence indicates that the membrane-mediated aggregation and subsequent deposition of hIAPP are linked to dysfunction and death of pancreatic ß-cells, but the molecular processes of hIAPP deposition are poorly understood. In this study, we examined the membrane-mediated aggregation and deposition of hIAPP at supported planar lipid bilayers with and without raft components (i.e. cholesterol and sphingomyelin) to provide insight into hIAPP-induced membrane dysfunction. The adsorption of hIAPP onto the bilayers was studied using a quartz crystal microbalance with dissipation monitoring, which showed enhanced accumulation of the peptide onto the bilayer containing raft components. Microscope observations demonstrated the growth of the aggregates formed from the membrane-adsorbed hIAPP. The examination of the membrane interfaces revealed that hIAPP aggregates retained the ability to associate with the membranes during the aggregation process, resulting in insertion of the aggregates into the bilayers. We also report the inhibitory effect of insulin on the hIAPP deposition. These findings demonstrate the aggregation of hIAPP at the membrane interfaces leading to amyloid deposits associated with the membrane and suggest a role for insulin in hIAPP deposition. A presumed mechanism regulating hIAPP deposition at the membrane interfaces is discussed.

Transcriptome profiling of brain edemas caused by influenza infection and lipopolysaccharide treatment.

Influenza A virus-associated encephalopathy triggered by influenza virus infection often occurs in children aged five and younger in Japan. However, the mechanisms behind Influenza A virus-associated encephalopathy are not yet well understood. This study developed an Influenza A virus-associated encephalopathy-like model using mice infected with Influenza A virus and given lipopolysaccharide treatment. The results showed that the mice used in the model suffered from brain edemas nearly three times more severe, as well as having higher cytokine levels in sera compared to those of the control groups. Using gene expression profiling, cytokine-related genes were found not to be up-regulated in the brain in situ, while protein coding genes, which are known to be involved in blood-brain barrier disruption, were up-regulated. Categorizing the functional groups using gene ontology revealed the terms "ion channels," "calcium oscillation," and "membrane transporter activities." The blood-brain barrier disruption found in this Influenza A virus-associated encephalopathy model can therefore be assumed to be due to a cellular electrolyte imbalance of the neuronal tissue, in addition to a cytokine storm.

Protease-dependent hemagglutinin cleavage contributes to alteration in chicken hemagglutination by the H3N2 influenza A virus.

The human influenza A virus (H3N2) has been the predominant influenza strain since 1992, and one property of this virus is non-agglutination of chicken erythrocytes [Ch(-) virus]. The Ch(-) virus in our study was able to acquire chicken hemagglutination [Ch(+)] by trypsin passage but not by chymotrypsin passage. Moreover, the trypsin-passaged Ch(+) viruses reacquired the Ch(-) property after a further chymotrypsin passage. In particular, genetic analysis showed no evidence of mutations in the hemagglutinin (HA) gene during either trypsin or chymotrypsin passages: the only differences found were in the HA cleavage sites between the trypsin-passaged virus and the chymotrypsin-passaged virus as determined by the N-terminal amino acid sequence. These results suggested that protease-dependent differences at the viral HA cleavage site, rather than genetic mutations, are likely to have a significant effect on the viral ability to produce chicken hemagglutination.

Effects of membrane interaction and aggregation of amyloid ß-peptide on lipid mobility and membrane domain structure.

Alzheimer's disease (AD) is the most prevalent age-dependent form of dementia, characterized by extracellular amyloid deposits comprising amyloid ß-peptide (Aß) in the cerebral cortex. Increasing evidence has indicated that ganglioside GM1 (GM1) in lipid rafts plays a pivotal role in amyloid deposition of Aß and the related cytotoxicity in AD. Despite recent efforts to characterize Aß-lipid interactions, the effect of Aß aggregation on dynamic properties and organization of lipid membranes is poorly understood. In this study, we examined the aggregation of Aß on supported lipid bilayers containing raft components (i.e., cholesterol, sphingomyelin, and GM1) and its effects on the membrane properties. We showed that the lateral fluidity of membranes was significantly affected by membrane binding and subsequent aggregation of Aß. Microscopic observations of the membrane surfaces demonstrated an enhancement in phase separation of lipids as a result of interactions between Aß and GM1 during induced aggregation of Aß. The uptake of GM1 into Aß aggregates and the attendant membrane damage were also observed under a microscope when the membrane-anchored aggregates were formed. On the basis of these observations, we propose that Aß aggregates formed in the presence of lipid membranes have a latent ability to trigger the uptake of raft components accompanied by phase separation of lipids.

Application and use of differential scanning calorimetry in studies of thermal fluctuation associated with amyloid fibril formation.

The aggregation of proteins into amyloid fibrils is a topic that has attracted great interest because the process is associated with the pathology of numerous human diseases. Despite considerable progress in the elucidation of the structure of amyloid fibrils and the kinetic mechanism of their formation, knowledge on the thermodynamic aspects underlying the formation and stability of amyloid fibrils is limited. In this review, we summarize recent calorimetric studies of amyloid fibril formation, with the goal of obtaining a better understanding of the causal factors that thermally induce proteins to aggregate into amyloid fibrils. Calorimetric data show that differential scanning calorimetry is a useful technique to study the causative factors that thermally trigger the conversion to the amyloid structure and highlight the physics related to the thermal fluctuation of proteins during this conversion.

Binding of islet amyloid polypeptide to supported lipid bilayers and amyloid aggregation at the membranes.

Amyloid deposition of human islet amyloid polypeptide (hIAPP) in the islets of Langerhans is closely associated with the pathogenesis of type II diabetes mellitus. Despite substantial evidence linking amyloidogenic hIAPP to loss of ß-cell mass and decreased pancreatic function, the molecular mechanism of hIAPP cytotoxicity is poorly understood. We here investigated the binding of hIAPP and nonamyloidogenic rat IAPP to substrate-supported planar bilayers and examined the membrane-mediated amyloid aggregation. The membrane binding of IAPP in soluble and fibrillar states was characterized using quartz crystal microbalance with dissipation monitoring, revealing significant differences in the binding abilities among different species and conformational states of IAPP. Patterned model membranes composed of polymerized and fluid lipid bilayer domains were used to microscopically observe the amyloid aggregation of hIAPP in its membrane-bound state. The results have important implications for lipid-mediated aggregation following the penetration of hIAPP into fluid membranes. Using the fluorescence recovery after photobleaching method, we show that the processes of membrane binding and subsequent amyloid aggregation are accompanied by substantial changes in membrane fluidity and morphology. Additionally, we show that the fibrillar hIAPP has a potential ability to perturb the membrane structure in experiments of the fibril-mediated aggregation of lipid vesicles. The results obtained in this study using model membranes reveal that membrane-bound hIAPP species display a pronounced membrane perturbation ability and suggest the potential involvement of the oligomeic forms of hAPP in membrane dysfunction.

Effect of lipid type on the binding of lipid vesicles to islet amyloid polypeptide amyloid fibrils.

Amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are observed near pancreatic beta-cells of type II diabetics, with their presence strongly correlating with a loss of beta-cell mass and decreased pancreatic function. Although beta-cell membranes have been implicated as the likely target of amyloidogenic IAPP toxicity, interactions between membranes and IAPP in the fibrillar state have not been well characterized. In this study, turbidity measurements were conducted to provide a detailed description of the binding reaction between IAPP fibrils and lipid vesicles made from phosphatidylcholine. The kinetic data representing the rate and extent of the fibril-vesicle binding reaction are described well by an empirical double-exponential equation. The extent of binding was found to increase with increasing amyloid fibril concentration. Modification of the vesicle composition significantly altered the observed binding reaction kinetics, with the change quantified using the parameters obtained from the double-exponential fitting analysis. When the vesicles contained a significant amount of the lipid phosphatidylglycerol, substantial sedimentation of the vesicles under gravity was detected following the initial binding reaction. To rationalize the observed kinetic binding data, we developed a mesoscopic simulation model based on a hard particle representation of the species involved. In light of the observed data and simulation predictions, the potential roles of IAPP amyloid fibrils in membrane binding are discussed.

Thermal response with exothermic effects of beta2-microglobulin amyloid fibrils and fibrillation.

Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of beta(2)-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid beta-peptides but also during the processes of the temperature-induced conversion of beta(2)-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules.

A comprehensive model for packing and hydration for amyloid fibrils of beta2-microglobulin.

Volume can provide informative structural descriptions of macromolecules such as proteins in solution because a final volumetric outcome accompanies the exquisite equipoise of packing effects between residues, and residues and waters inside and outside proteins. Here we performed systematic investigations on the volumetric nature of the amyloidogenic conformations of beta2-microglobulin (beta2-m) and its amyloidogenic core peptide, K3, using a high precision densitometer. The transition from the acid-denatured beta2-m to the mature amyloid fibrils was accompanied by a positive change in the partial specific volume, which was larger than that observed for the transition from the acid-denatured beta2-m to the native structure. The data imply that the mature amyloid fibrils are more voluminous than the native structure because of a sparse packing density of side chains. In contrast, the formation of the mature amyloid-like fibrils of the K3 from the random coil was followed by a considerable decrease in the partial specific volume, suggesting a highly compact core structure. Interestingly, the immature amyloid-like fibrils of beta2-m exhibited a volume intermediate between those of the mature fibrils of beta2-m and K3, because of the core structure at their center and the relatively noncompact region around the core with much hydration. These volumetric differences would result from the nature of main-chain-dominated fibrillogenesis. We suggest comprehensive models for these three types of fibrils illustrating packing and hydrational states.