Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The double W/F replacement renders apomyoglobin highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions. In this work we analyze the early stage of W7FW14F apomyoglobin aggregation following the time dependence of the process by far-UV CD, Fourier-transform infrared (FTIR) spectroscopy, and heme-binding properties. The results show that the aggregation of W7FW14F apomyoglobin starts from a native-like globin state able to bind the prosthetic group with spectroscopic properties similar to those observed for wild-type apoprotein. Nevertheless, it rapidly aggregates, forming amyloid fibrils. However, when the prosthetic group is added before the beginning of aggregation, amyloid fibrillization is inhibited, although the aggregation process is not prevented. Moreover, the apomyoglobin aggregates formed in these conditions are not cytotoxic differently from what is observed for all amyloidogenic proteins. These results open new insights into the relationship between the structure adopted by the protein into the aggregates and their ability to trigger the impairment of cell viability.     

Study of tetraphenylporphyrins as modifiers of insulin amyloid aggregation

Amyloid fibrils are rigid β‐pleated protein aggregates that are connected with series of harmful diseases and at the same time are promising as base for novel nanomaterials. Thus, design of compounds able to inhibit or redirect those aggregates formation is important both for the biomedical aims and for nanotechnology applications. Here, we studied the effect of tetraphenylporphyrins (metal free, their Cu and Pd complexes, and those functionalized by carboxy and amino groups on periphery) on insulin amyloid self‐assembling. The strongest impact on insulin aggregation was demonstrated by a metal‐free porphyrin bearing four carboxy groups. This compound strongly suppresses insulin aggregation (about 88% reduction in amyloid‐sensitive probe emission) inducing formation of fibrils with the length close to this of free insulin (1.7 ± 0.6 μm as compared with 1.4 ± 0.4 μm, respectively) with an essentially reduced tendency to lateral aggregation. Contrarily, the presence of tetraphenylporphyrin containing four amino groups only slightly affects fibrils' morphology and makes weaker impact on insulin aggregation yield (about 44% reduction). This is explained by the ability of aromatic carboxy groups of 5,10,15,20‐(tetra‐4‐carboxyphenyl)porphyrin to interact with complementary protein‐binding groups and thus stabilize the supramolecular complex. For 5,10,15,20‐(tetra‐4‐aminophenyl)porphyrin, full protonation takes place in acidic medium of protein aggregation reaction; this results in the high positive charge of TPPN4 (equal or close to +6) and hence higher contribution of coulombic repulsion to interaction of TPPN4 with insulin. One more possible mechanism of the lower inhibition effect of TPPN4 as compared with TPPC4 could be the more restricted possibility of the former as compared with the latter to form H bonds with insulin groups. It was also shown that metal‐free, Pd‐containing, and Cu‐containing tetraphenylporphyrins without peripheral substituents make almost the same impact on the protein self‐assembling. We suppose this to be due to coordination saturation of these metal atoms.     

Effects of copolypeptides on amyloid fibrillation of hen egg-white lysozyme

The fibrillation of hen egg-white lysozyme (HEWL) in the absence and presence of simple, unstructured D,L-lysine-co-glycine (D,L-Lys-co-gly) and D,L-lysine-co-L-phenylalanine (D,L-Lys-co-Phe) copolypeptides was studied by using a variety of analytical techniques. The attenuating and decelerating effects on fibrillation are significantly dependent on the polypeptide concentration and the composition ratios in the polypeptide chain. Interestingly, D,L-Lys-co-gly and D,L-Lys-co-Phe copolypeptides with the same composition ratio have comparable attenuating effects on fibrillation. The copolypeptide with highest molar fraction of glycine residue exhibits the strongest suppression of HEWL fibrillation. The copolypeptide has the highest hydrophobic interacting capacity due to the more molar ratio of apolar monomer in the polymer backbone. The major driving forces for the association of HEWL and copolypeptides are likely to be hydrogen bonding and hydrophobic interactions, and these interactions reduce the concentration of free protein in solution available to proceed to fibrillation, leading to the increase of lag time and attenuation of fibrillation. The results of this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and the amyloid proteins.     

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0–20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.     

Insights into the disparate action of osmolytes and macromolecular crowders on amyloid formation

It is widely recognized that amyloid formation sensitively responds to conditions set by myriad cellular solutes. These cosolutes include two important classes: macromolecular crowders and compatible osmolytes. We have recently found that addition of macromolecular PEG only slightly affects fibril formation of a model peptide in vitro. Polyol osmolytes, in contrast, lengthen the lag time for aggregation, and lead to larger fibril mass at equilibrium. To further hypothesize on the molecular underpinnings of the disparate effect of the two cosolute classes, we have further analyzed the experiments using an available kinetic mechanism describing fibril aggregation. Model calculations suggest that all cosolutes similarly lengthen the time required for nucleation, possibly due to their excluded volume effect. However, PEGs may in addition promote fibril fragmentation, leading to lag times that are overall almost unvaried. Moreover, polyols effectively slow the monomer-fibril detachment rates, thereby favoring additional fibril formation. Our analysis provides first hints that cosolutes act not only by changing association or dissociation rates, but potentially also by directing the formation of fibrils of varied morphologies with different mechanical properties. Although additional experiments are needed to unambiguously resolve the action of excluded cosolutes on amyloid formation, it is becoming clear that these compounds are important to consider in the search for ways to modulate fibril formation.     

Effects of salt concentration on association of the amyloid protofilaments of hen egg white lysozyme studied by time-resolved neutron scattering

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.     

Interaction of membrane-bound islet amyloid polypeptide with soluble and crystalline insulin

Islet amyloid polypeptide (IAPP, also known as amylin) is the major protein component of pancreatic amyloid fibers in type II diabetes and is normally cosecreted with insulin from the beta-cells of the pancreas. IAPP forms amyloid fibrils rapidly at concentrations well below those found in vivo, yet progression of type II diabetes occurs over many years. Insulin, a known inhibitor of IAPP fibrillogenesis, exists as a dense crystalline or near-crystalline core in the secretory vesicle, while IAPP localizes to the region between the crystal and the secretory vesicle membrane. In vitro, IAPP fibrillogenesis is both accelerated by lipid membranes and inhibited by monomeric insulin. In this work, we investigate insulin-IAPP-lipid interactions in vitro under conditions chosen to approximate native secretory vesicle physiology and the amyloid disease state. The effect of insulin on IAPP fibrillogenesis is investigated using fluorescence spectrometry. Additionally, interactions of IAPP and lipids with crystalline insulin are studied using fluorescence microscopy. We find that, while soluble states of insulin and IAPP do not interact significantly, large assemblies of either insulin (crystals) or IAPP (fibers) can lead to stable IAPP-insulin interactions. The results raise the possibility of multiple physiological interactions between these two beta-cell hormones.     

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.     

Measurements of residual dipolar couplings in peptide inhibitors weakly aligned by transient binding to peptide amyloid fibrils**

In this communication, we suggest that transferred residual dipolar couplings (trRDCs) can be employed to restrain the structure of peptide inhibitors transiently binding to beta-amyloid fibrils. The effect is based on the spontaneous alignment of amyloid fibrils with the fibril axis parallel to the magnetic field. This alignment is transferred to the transiently binding peptide inhibitor and is reflected in the size of the trRDCs. We find that the peptide inhibitor adopts a beta-sheet conformation with the backbone N-H and C-H dipolar vectors aligned preferentially parallel and perpendicular, respectively, to the fibril axis.     

Pathways and intermediates of amyloid fibril formation

The lack of understanding of amyloid fibril formation at the molecular level is a major obstacle in devising strategies to interfere with the pathologies linked to peptide or protein aggregation. In particular, little is known on the role of intermediates and fibril elongation pathways as well as their dependence on the intrinsic tendency of a polypeptide chain to self-assembly by β-sheet formation (β-aggregation propensity). Here, coarse-grained simulations of an amphipathic polypeptide show that a decrease in the β-aggregation propensity results in a larger heterogeneity of elongation pathways, despite the essentially identical structure of the final fibril. Protofibrillar intermediates that are thinner, shorter and less structured than the final fibril accumulate along some of these pathways. Moreover, the templated formation of an additional protofilament on the lateral surface of a protofibril is sometimes observed as a collective transition. Conversely, for a polypeptide model with a high β-aggregation propensity, elongation proceeds without protofibrillar intermediates. Therefore, changes in intrinsic β-aggregation propensity modulate the relative accessibility of parallel routes of aggregation.     

The mechanism of insulin action on islet amyloid polypeptide fiber formation

The pathology of type II diabetes includes the presence of cytotoxic amyloid deposits in the islets of Langerhans. The main component of these deposits, islet amyloid polypeptide (IAPP), is a hormone involved in glucose metabolism and is normally co-secreted with insulin by the beta-cells of the pancreas. Here, we perform in vitro IAPP fibrillogenesis experiments in the presence and in the absence of insulin to elucidate the mechanism by which insulin acts on fiber formation. We find that insulin is an exceptionally potent inhibitor. In contrast to the vast excess of insulin over IAPP in vivo, substoichiometric amounts of insulin inhibit seeded and unseeded reactions by more than tenfold in vitro. Unusually, the magnitude of the inhibitory effect is dependent on the concentration of insulin, yet independent of the concentration of IAPP. In addition, insulin appears to bind non-specifically to fiber surfaces, giving rise to altered morphology. IAPP fiber formation in vitro requires a minimum of three steps: fiber-independent nucleation, elongation, and fiber-dependent nucleation. Furthermore, these steps are attenuated by the presence of a dispersed-phase transition. We interpret these data in the context of the phase-mediated fibrillogenesis model (PMF) and conclude through experiment and kinetic simulation that the dominant effect of insulin is to act on the elongation portion of the reaction. These results suggest that amyloid formation in type II diabetes involves either an additional agent that acts as an accelerant, or a step that segregates IAPP from insulin.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

Rational design of a zinc phthalocyanine binding protein

Phthalocyanines have long been used as primary donor molecules in synthetic light-powered devices due to their superior properties when compared to natural light activated molecules such as chlorophylls. Their use in biological contexts, however, has been severely restricted due to their high degree of self-association, and its attendant photoquenching, in aqueous environments. To this end we report the rational redesign of a de novo four helix bundle di-heme binding protein into a heme and Zinc(II) phthalocyanine (ZnPc) dyad in which the ZnPc is electronically and photonically isolated. The redesign required transformation of the homodimeric protein into a single chain four helix bundle and the addition of a negatively charge sulfonate ion to the ZnPc macrocycle. To explore the role of topology on ZnPc binding two constructs were made and the resulting differences in affinity can be explained by steric interference of the newly added connecting loop. Singular binding of ZnPc was verified by absorption, fluorescence, and magnetic circular dichroism spectroscopy. The engineering guidelines determined here, which enable the simple insertion of a monomeric ZnPc binding site into an artificial helical bundle, are a robust starting point for the creation of functional photoactive nanodevices.     

The effects of shear flow on protein structure and function

Protein molecules are subjected to potentially denaturing fluid shear forces during processing and in circulation in the body. These complex molecules, involved in numerous biological functions and reactions, can be significantly impaired by molecular damage. There have been many studies on the effects of hydrodynamic shear forces on protein structure and function. These studies are reviewed and the implications to bioprocessing and pathophysiology of certain diseases are discussed.     

Role of the N and C-terminal strands of beta 2-microglobulin in amyloid formation at neutral pH

Beta 2-microglobulin (beta(2)m) is known to form amyloid fibrils de novo in vitro under acidic conditions (below pH 4.8). Fibril formation at neutral pH, however, has only been observed by deletion of the N-terminal six residues; by the addition of pre-assembled seeds; or in the presence of Cu(2+). Based on these observations, and other structural data, models for fibril formation of beta(2)m have been proposed that involve the fraying of the N and C-terminal beta-strands and the consequent loss of edge strand protective features. Here, we examine the role of the N and C-terminal strands in the initiation of fibrillogenesis of beta(2)m by creating point mutations in strands A and G and comparing the properties of the resulting proteins with variants containing similar mutations elsewhere in the protein. We show that truncation of buried hydrophobic side-chains in strands A and G promotes rapid fibril formation at neutral pH, even in unseeded reactions, and increases the rate of fibril formation under acidic conditions. By contrast, similar mutations created in the remaining seven beta-strands of the native protein have little effect on the rate or pH dependence of fibril formation. The data are consistent with the view that perturbation of the N and C-terminal edge strands is an important feature in the generation of assembly-competent states of beta(2)m.     

An examination of the binding behavior of histidine-containing peptides with immobilized metal complexes derived from the macrocyclic ligand, 1,4,7-triazacyclononane

In this study, two different experimental approaches have been employed to examine the binding behavior of histidine-containing peptides with metal ion complexes derived from the macrocyclic ligand 1,4,7-triazacyclononane (tacn). Firstly, a molecular modeling approach has been employed to derive the strain energies for test peptide sequences that have a predicted propensity to readily adopt an α-helical conformation. To this end, binuclear metal complexes were examined with peptides containing two histidine residues in different locations in a pair of peptides of the same composition but different sequence. These modeling results indicate that there are no energetic constraints for two-point binding to occur with dicopper(II) binuclear complexes when two histidine residues are appropriately placed in an α-helical conformation. Secondly, binding experiments were carried out to establish the effect of one or more histidine residues within a peptide sequence on the affinity of a peptide for these Cu(II)–tacn derived binuclear complexes when immobilized onto a chromatographic support material. The results confirm that for all chelating systems, higher affinity is achieved as the histidine number in the peptide structure increases, although the relative location of the histidine residues in these small peptides did not introduce a significant constraint to the conformation on interacting with the immobilized Cu(II) binuclear complexes.     

Mechanisms of ataxin-3 misfolding and fibril formation: kinetic analysis of a disease-associated polyglutamine protein

The polyglutamine diseases are a family of nine proteins where intracellular protein misfolding and amyloid-like fibril formation are intrinsically coupled to disease. Previously, we identified a complex two-step mechanism of fibril formation of pathologically expanded ataxin-3, the causative protein of spinocerebellar ataxia type-3 (Machado-Joseph disease). Strikingly, ataxin-3 lacking a polyglutamine tract also formed fibrils, although this occurred only via a single-step that was homologous to the first step of expanded ataxin-3 fibril formation. Here, we present the first kinetic analysis of a disease-associated polyglutamine repeat protein. We show that ataxin-3 forms amyloid-like fibrils by a nucleation-dependent polymerization mechanism. We kinetically model the nucleating event in ataxin-3 fibrillogenesis to the formation of a monomeric thermodynamic nucleus. Fibril elongation then proceeds by a mechanism of monomer addition. The presence of an expanded polyglutamine tract leads subsequently to rapid inter-fibril association and formation of large, highly stable amyloid-like fibrils. These results enhance our general understanding of polyglutamine fibrillogenesis and highlights the role of non-poly(Q) domains in modulating the kinetics of misfolding in this family.     

Structures of soluble amyloid oligomers from computer simulations

Alzheimer's, Parkinson's, and Creutzfeldt-Jakob's neurodegenerative diseases are all linked with the assembly of normally soluble proteins into amyloid fibrils. Because of experimental limitations, structural characterization of the soluble oligomers, which form early in the process of fibrillogenesis and are cytotoxic, remains to be determined. In this article, we study the aggregation paths of seven chains of the shortest amyloid-forming peptide, using an activitated method and a reduced atomic representation. Our simulations show that disordered KFFE monomers ultimately form three distinct topologies of similar energy: amorphous oligomers, incomplete rings with beta-barrel character, and cross-beta-sheet structures with the meridional but not the equatorial X-ray fiber reflections. The simulations also shed light on the pathways from misfolded aggregates to fibrillar-like structures. They also underline the multiplicity of building blocks that can lead to the formation of the critical nucleus from which rapid growth of the fibril occurs.     

Protective effect of extractive and biotechnological chondroitin in insulin amyloid and advanced glycation end product-induced toxicity

Glycosaminoglycans are extracellular matrix components related to several biological functions and diseases. Chondroitin sulfate is a sulphated glycosaminoglycan synthesized as part of proteoglycan molecules. They are frequently associated with amyloid deposits and possess an active role in amyloid fibril formation. Recently, a neuroprotective effect of extracellular matrix components against amyloid toxicity and oxidative stress has been reported. Advanced glycation end products (AGEs), the end products of the glycation reaction, have been linked to amyloid-based neurodegenerative disease as associated with oxidative stress and inflammation. In this study we have analyzed the effect of chondroitin sulfate isolated from different species, in comparison with a new biotechnological unsulfated chondroitin, in the amyloid aggregation process of insulin, as well as the ability to prevent the formation of AGEs and related toxicity. The results have showed a determining role of chondroitin sulfate groups in modulating insulin amyloid aggregation. In addition, both sulfated and unsulfated chondroitins have shown protective properties against amyloid and AGEs-induced toxicity. These data are very relevant as a protective effect of these glycosaminoglycans in the AGE-induced toxicity was never observed before. Moreover, considering the issues related to the purity and safety of chondroitin from natural sources, this study suggests a new potential application for the biotechnological chondroitin.