Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

A systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH

Beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How beta(2)m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on beta(2)m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (DeltaN6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that DeltaN6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of beta(2)m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type beta(2)m with that of DeltaN6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.     

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.     

Energetics of structural domains in alpha-lactalbumin.

alpha-Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in alpha-helix (the alpha-domain) and has Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds. The other domain is rich in beta-sheet (the beta-domain), has Cys 61-Cys 77 and Cys 73-Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine alpha-lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three-disulfide form, having a reduced Cys 6-Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact alpha-lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. the two-disulfide form of alpha-lactalbumin, having reduced Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds with carboxymethylated cysteines, retains about half the secondary and tertiary structure of the intact alpha-lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at lower temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, alpha-lactalbumin retains its calcium-binding beta-domain, whereas the alpha-domain is unfolded. It appears that the beta-domain does not require alpha-domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded alpha-domain.     

Amyloid fibril formation and disaggregation of fragment 1-29 of apomyoglobin: insights into the effect of pH on protein fibrillogenesis

The N-terminal fragment 1-29 of horse heart apomyoglobin (apoMb(1-29)) is highly prone to form amyloid-like fibrils at low pH. Fibrillogenesis at pH 2.0 occurs following a nucleation-dependent growth mechanism, as evidenced by the thioflavin T (ThT) assay. Transmission electron microscopy (TEM) confirms the presence of regular amyloid-like fibrils and far-UV circular dichroism (CD) spectra indicate the acquisition of a high content of beta-sheet structure. ThT assay, TEM and CD highlight fast and complete disaggregation of the fibrils, if the pH of a suspension of mature fibrils is increased to 8.3. It is of interest that amyloid-like fibrils form again if the pH of the solution is brought back to 2.0. While apoMb(1-29) fibrils obtained at pH 2.0 are resistant to proteolysis by pepsin, the disaggregated fibrils are easily cleaved at pH 8.3 by trypsin and V8 protease, and some of the resulting fragments aggregate very quickly in the proteolysis mixture, forming amyloid-like fibrils. We show that the increase of amyloidogenicity of apoMb(1-29) following acidification or proteolysis at pH 8.3 can be attributed to the decrease of the peptide net charge following these alterations. The results observed here for apoMb(1-29) provide an experimental basis for explaining the effect of charge and pH on amyloid fibril formation by both unfolded and folded protein systems.     

A common beta-sheet architecture underlies in vitro and in vivo beta2-microglobulin amyloid fibrils

Misfolding and aggregation of normally soluble proteins into amyloid fibrils and their deposition and accumulation underlies a variety of clinically significant diseases. Fibrillar aggregates with amyloid-like properties can also be generated in vitro from pure proteins and peptides, including those not known to be associated with amyloidosis. Whereas biophysical studies of amyloid-like fibrils formed in vitro have provided important insights into the molecular mechanisms of amyloid generation and the structural properties of the fibrils formed, amyloidogenic proteins are typically exposed to mild or more extreme denaturing conditions to induce rapid fibril formation in vitro. Whether the structure of the resulting assemblies is representative of their natural in vivo counterparts, thus, remains a fundamental unresolved issue. Here we show using Fourier transform infrared spectroscopy that amyloid-like fibrils formed in vitro from natively folded or unfolded beta(2)-microglobulin (the protein associated with dialysis-related amyloidosis) adopt an identical beta-sheet architecture. The same beta-strand signature is observed whether fibril formation in vitro occurs spontaneously or from seeded reactions. Comparison of these spectra with those of amyloid fibrils extracted from patients with dialysis-related amyloidosis revealed an identical amide I' absorbance maximum, suggestive of a characteristic and conserved amyloid fold. Our results endorse the relevance of biophysical studies for the investigation of the molecular mechanisms of beta(2)-microglobulin fibrillogenesis, knowledge about which may inform understanding of the pathobiology of this protein.     

Amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues via a liquid-crystalline intermediate phase

Chorion, the major component of silkmoth eggshell, consists of the A and B classes of low-molecular weight structural proteins. Chorion protects the oocyte and the developing embryo from environmental hazards and this is due to the extraordinary physical and chemical properties of its constituent proteins. We have shown previously [FEBS Lett. 479 (2000) 141; 499 (2001) 268] that peptide-analogues of the A and B classes of chorion proteins form amyloid fibrils under a variety of conditions, which led us to propose that silkmoth chorion is a natural, protective amyloid. In this work, we present data showing conclusively that, the first main step of amyloid-like fibrillogenesis of chorion peptides is the formation of nuclei of liquid crystalline nature, which is reminiscent of spider-silk formation. We show that these liquid-crystalline nuclei (spherulites) 'collapse'/deteriorate to form amyloid fibrils in a spectacular manner, important, it seems, for chorion morphogenesis and amyloid fibrillogenesis in general. The molecular 'switch' causing this spectacular transformation is, most probably, a conformational transition to the structure of chorion peptides, from a left-handed parallel beta-helix to an antiparallel beta-pleated sheet. Apparently, these peptides were suitably designed to play this role, after millions of years of molecular evolution.     

Identification of multiple amyloidogenic sequences in laminin-1

Amyloid fibril formation is associated with several pathologies, including Alzheimer's disease, Parkinson's disease, type II diabetes, and prion diseases. Recently, a relationship between basement membrane components and amyloid deposits has been reported. The basement membrane protein, laminin, may be involved in amyloid-related diseases, since laminin is present in amyloid plaques in Alzheimer's disease and binds to amyloid precursor protein. Recently, we showed that peptide A208 (AASIKVAVSADR), the IKVAV-containing peptide, formed amyloid-like fibrils. We previously identified 60 cell adhesive sequences in laminin-1 using a total of 673 12-mer synthetic peptides. Here, we screened for additional amyloidogenic sequences among 60 cell adhesive peptides derived from laminin-1. We first examined amyloid-like fibril formation by the 60 active peptides with Congo red, a histological dye binding to many amyloid-like proteins. Thirteen peptides were stained with Congo red. Four of the 13 peptides promoted cell attachment and neurite outgrowth like the IKVAV-containing peptide. The four peptides also showed amyloid-like fibril formation in both X-ray diffraction and electron microscopic analyses. The amyloidogenic peptides contain consensus amino acid components, including both basic and acidic amino acids and Ser and Ile residues. These results indicate that at least five laminin-derived peptides can form amyloid-like fibrils. We conclude that the laminin-derived amyloidogenic peptides have the potential to form amyloid-like fibrils in vivo, possibly when laminin-1 is degraded.     

Construction of a chemically and conformationally self-replicating system of amyloid-like fibrils

The amyloid-like fibril is considered to be a macromolecular self-assemblage with a highly-ordered quaternary structure, in which numerous beta-stranded polypeptide chains align regularly. Therefore, this kind of fibril has the potential to be engineered into proteinaceous materials, although conformational alteration of proteins from their native form to the amyloid form is a misfolding and undesirable process related to amyloid diseases. In this study, we have attempted to design an artificial system to explore applicability of using the amyloid-like fibril as a construct possessing self-recognition and self-catalytic abilities. A peptide self-replicating system based on the beta-structure of the amyloid-like fibril was designed and constructed. The beta-stranded peptide was self-replicated by the native chemical ligation reaction, and the newly generated peptide was self-assembled into amyloid-like fibrils. Thus, the constructed system was of both chemical and conformational self-replicating fibrils.     

Amyloid structure--one but not the same: the many levels of fibrillar polymorphism

Many proteins and peptides can form amyloid-like structures both in vivo and in vitro. Although strikingly similar fibrillar structures can be observed across a variety of amino acid sequences, the fibrils formed often exhibit a stunning wealth of polymorphisms at the level of electron or atomic force microscopy. This appears to violate the Anfinsen principle seen for globular proteins, where each protein sequence codes for just one well-defined fold. To a large extent, polymorphism reflects variable packing of a single protofilament structure in the mature fibrils. However, we and others have recently demonstrated that polymorphism can also reflect real structural differences in the molecular packing of the polypeptide chains leading to several possible protofilament structures and diverse mature fibrillar structures. Glucagon has been a particularly useful model system for studying the fibrillogenesis mechanisms that lead to the formation of structural polymorphism, thanks to its single tryptophan residue and the availability of large quantities at pharmaceutical-grade quality. Combinations of structural investigations and seed extension experiments have revealed the reproducible formation of at least five different self-propagating fibril types from subtle variations in growth conditions. These reflect the underlying complexity of the peptide conformational landscape and provide a link to natively disordered proteins, where structure is dictated by context in the form of different binding partners. Here we review some of the latest advances in the study of glucagon fibrillar polymorphism and their implications for mechanisms of fibril formation in general.     

EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils in solution. As this propensity of MSP2 to form fibrils in solution has the potential to impede its development as a vaccine candidate, finding an inhibitor that inhibits fibrillogenesis may enhance vaccine development. We have shown previously that EGCG inhibits the formation of MSP2 fibrils. Here we show that EGCG can alter the β-sheet-like structure of the fibril and disaggregate pre-formed fibrils of MSP2 into soluble oligomers. The fibril remodelling effects of EGCG and other flavonoids were characterised using Thioflavin T fluorescence assays, electron microscopy and other biophysical methods.     

Multifunctional peptide fibrils for biomedical materials

The Ile-Lys-Val-Ala-Val (IKVAV) containing peptide, A208 (AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108), was recently found to form amyloid-like fibrils. Fibril formation is critical for its biological activities, including promotion of cell adhesion and neurite outgrowth. In the present study, we designed multifunctional peptide fibrils using the A208 peptide and an Arg-Gly-Asp (RGD)-containing fibronectin active sequence for biomedical applications. The fibronectin active sequence GRGDS (FN) or a scrambled sequence RSGGD (SC) were conjugated to either A208 or to A208S (AASVVIAKSADR), a scrambled peptide of A208, with a glycine as a spacer. The FN-A208 and SC-A208 peptides formed a gel and were stained with Congo red similar to that of A208, but FN-A208S and SC-A208S did not form a gel. These results indicate that FN-A208 and SC-A208 form amyloid-like fibrils similar to A208. A208 and SC-A208 promoted cell attachment with filopodia formation, and this adhesion was inhibited by the IKVAV-containing peptide, but not by EDTA or a GRGDS peptide. FN-A208 promoted cell attachment with well-organized actin stress fibers, and this adhesion was partially inhibited by either EDTA, GRGDS, or IKVAV. These data suggest that A208 binds to only IKVAV receptor(s) while the FN-A208 interacts with both integrins and the IKVAV receptor(s). We conclude that multifunctional peptide fibrils can be designed by conjugation of active peptides on A208 and that this construct has potential to serve as a bioadhesive for tissue regeneration and engineering.     

Amphoterin includes a sequence motif which is homologous to the Alzheimer's beta-amyloid peptide (Abeta), forms amyloid fibrils in vitro, and binds avidly to Abeta

Many of the proteins associated with amyloidoses have been found to share structural and sequence similarities, which are believed to be responsible for their capability to form amyloid fibrils. Interestingly, some proteins seem to be able to form amyloid-like fibrils although they are not associated with amyloidoses. This indicates that the ability to form amyloid fibrils may be a general property of a greater number of proteins not associated with these diseases. In the present work, we have searched for amyloidogenic consensus sequences in two current protein/peptide databases and show that many proteins share structures which can be predicted to form amyloid. One of these potentially amyloidogenic proteins is amphoterin (also known as HMG-1), involved in neuronal development and a ligand for the receptor for advanced glycation end products (RAGE). It contains an amyloidogenic peptide fragment which is highly homologous to the Alzheimer's amyloid beta-peptide. If enzymatically released from the native protein, it forms amyloid-like fibrils which are visible in electron microscopy, exhibit apple green birefringence under polarized light after Congo red staining, and increases thioflavin T fluorescence. This fragment also shows high affinity to Abeta as a free peptide or while part of the native protein. Our results support the hypothesis that the potential to form amyloid is a common characteristic of a number of proteins, independent of their relation to amyloidoses, and that this potential can be predicted based on the physicochemical properties of these proteins.     

A partially structured region of a largely unstructured protein, Plasmodium falciparum merozoite surface protein 2 (MSP2), forms amyloid-like fibrils.

Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. It has been implicated in the process of erythrocyte invasion and is a leading vaccine candidate. MSP2 is an intrinsically unstructured protein (IUP), and recombinant MSP2 forms amyloid-like fibrils upon storage. We have examined synthetic peptides corresponding to sequences in the conserved N-terminal region of MSP2 for the presence of local structure and the ability to form fibrils related to those formed by full-length MSP2. In a 25-residue peptide corresponding to the entire N-terminal region of mature MSP2, structures calculated from NMR data show the presence of nascent helical and turn-like structures. An 8-residue peptide from the central region of the N-terminal domain (residues 8-15) also formed a turn-like structure. Both peptides formed fibrils that were similar but not identical to the amyloid-like fibrils formed by full-length MSP2. Notably, the fibrils formed by the peptides bound both Congo Red and Thioflavin T, whereas the fibrils formed by full-length MSP2 bound only Congo Red. The propensity of peptides from the N-terminal conserved region of MSP2 to form amyloid-like fibrils makes it likely that this region contributes to fibril formation by the full-length protein. Thus, in contrast to the more common pathway of amyloid formation by structured proteins, which proceeds via partially unfolded intermediates that then undergo beta-aggregation, MSP2 is an example of a largely unstructured protein with at least one small structured region that has an important role in fibril formation.     

Comparison of the denaturant-induced unfolding of the bovine and human alpha-lactalbumin molten globules.

Residue-specific information on the urea-induced unfolding of the molten globule state of bovine alpha-lactalbumin (BLA) has been obtained using NMR spectroscopy. In agreement with previous studies on human alpha-lactalbumin (HLA) the unfolding process for BLA has been found to be non-cooperative. Both the alpha and beta-domains of the protein are substantially collapsed in the absence of denaturant but in both proteins the majority of the structure in the beta-domain unfolds prior to that in the alpha-domain. However, in BLA the protein unfolds completely in 10 M urea at 50 degrees C, whilst in HLA a stable core region persists even under these extreme conditions. Previous studies on HLA have identified eight residues that are crucial for the stability of the molten globule. Of these residues, only three are conserved in the sequence of BLA. By taking into consideration the differences in inter-residue contacts between the four alpha-helices arising from these substitutions, and the relative hydrophobicity of the helices in the two proteins, we show that it is possible to rationalise the observed differences in the behaviour of the molten globule states of the two proteins. Taken together, these results suggest that there may be a number of ways of stabilising a given protein fold, and the specific manner that this is achieved for a particular protein is determined by details of its sequence.     

Amyloid-a state in many guises: survival of the fittest fibril fold

Under appropriate conditions, essentially all proteins are able to aggregate to form long, well-ordered and beta-sheet-rich arrays known as amyloid-like fibrils. These fibrils consist of varying numbers of intertwined protofibrils and can for any given protein exhibit a wealth of different forms at the ultrastructural level. Traditionally, this structural variability or polymorphism has been attributed to differences in the assembly of a common protofibril structure. However, recent work on glucagon, insulin, and the Abeta peptide suggests that this polymorphism can occur at the level of secondary structure. Simple variations in either solvent conditions such as temperature, protein concentration, and ionic strength or external mechanical influences such as agitation can lead to formation of fibrils with markedly different characteristics. In some cases, these characteristics can be passed on to new fibrils in a strain-specific manner, similar to what is known for prions. The preferred structure of fibrils formed can be explained in terms of selective pressure and survival of the fittest; the most populated types of fibrils we observe at the end of an experiment are those that had the fastest overall growth rate under the given conditions. Fibrillar polymorphism is probably a consequence of the lack of structural restraints on a nonfunctional conformational state.     

Triazavirine supramolecular complexes as modifiers of the peptide oligomeric structure

In this study, we present molecular dynamics simulations of the antiviral drug triazavirine, that affects formation of amyloid-like fibrils of the model peptide (SI). According to our simulations, triazavirine is able to form linear supramolecular structures which can act as shields and prevent interactions between SI monomers. This model, as validated by simulations, provides an adequate explanation of triazavirine’s mechanism of action as it pertains to SI peptide fibril formation.     

Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F

The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.     

Atomic force microscopy study of human amylin (20-29) fibrils

Here we present atomic force microscopy images of the fibrils formed by human amylin(20-29). This peptide is a fragment of the polypeptide amylin, the major proteinaceous component of amyloid deposits found in cases of type-II diabetes mellitus. Our results demonstrate that the amylin(20-29) peptide fragment forms amyloid-like fibrils that display polymorphic structures. Twisting along the axis of fibrils was often observed in fibrils aged for 6 hours but disappeared in mature fibrils aged for longer time periods.