Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis.     

Probing the mechanism of amyloidogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation

Aggregation of the SH3 domain of the PI3 kinase, both as a single domain and as a tandem repeat in which the C terminus of one domain is linked to the N terminus of another by a flexible linker of ten glycine/serine residues, has been studied under a range of conditions in order to investigate the mechanism of protein aggregation and amyloid formation. The tandem repeat was found to form amyloid fibrils much more readily than the single domain under the acidic conditions used here, and the fibrils themselves have higher morphological homogeneity. The folding-unfolding transition of the PI3-SH3 domain shows two-state behaviour and is pH dependent; at pH 3.6, which is near the pH mid-point for folding and only slightly below the isoelectric point of the protein, both the single domain and the tandem repeat spontaneously form broad distributions of soluble oligomers without requirement for nucleation. Under prolonged incubation under these conditions, the oligomers convert into thin, curly fibrils that interact with thioflavin-T, suggesting that they contain an organised beta-sheet structure. Under more acidic conditions (pH 2.0) where the proteins are fully denatured and carry a positive net charge, long, straight fibrils are formed in a process having a pronounced lag phase. The latter was found to be reduced dramatically by the addition of oligomers exceeding a critical size of approximately 20 molecules. The results suggest that the process of aggregation of these SH3 domains can take place by a variety of mechanisms, ranging from downhill formation of relatively amorphous species to nucleated formation of highly organised structures, the relative importance of which varies greatly with solution conditions. Comparison with the behaviour of other amyloidogenic systems suggests that the general mechanistic features outlined here are likely to be common to at least a wide variety of peptides and proteins.     

Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.     

Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6₅22 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P2₁2₁2₁, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.     

A single mutation induces amyloid aggregation in the alpha-spectrin SH3 domain: analysis of the early stages of fibril formation

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.     

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β₂-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.     

Human Immunoglobulin Light Chains λ Form Amyloid Fibrils and Granular Aggregates in Solution

Myeloma nephropathy is a disorder characterized by deposition of monoclonal immunoglobulin light chains in the kidneys. The chains deposited form either amyloid fibrils or granular (amorphous) aggregates. Distinct molecular mechanisms leading to the formation of different aggregate types in kidney of patients with multiple myeloma are poorly understood. Here we describe the self-association kinetics of human monoclonal immunoglobulin light chains lambda (GRY) isolated from urine of a patient with multiple myeloma. Under physiological conditions, the isolated light chain exists predominantly in a form of covalent dimer with apparent molecular mass of 50.1 kD. Spectral probe binding, analytical gel filtration, Western blot analysis, and electron microscopy indicate that GRY dimer aggregation occurs via two different pathways producing either amyloid fibrils or amorphous aggregates depending on microenvironment. Incubation of GRY (25 microM) for 4-14 days at 37 degrees C in phosphate buffered saline (PBS), pH 7.0, or in PBS containing urea (0.8 M), pH 6.5, leads to amyloid fibril formation. Under electron microscopy, the fibrils show unbranched thread-like structures, approximately 60-80 x 1000 A in size, which can bind thioflavin T and Congo Red. GRY maintained in acetate buffer, pH 3.5, forms granular aggregates. The structure of GRY oligomers formed during the early stage of amyloid fibril formation (1-4 days) has been examined by means of protein cross-linking with homobifunctional reagents. These oligomers are predominantly trimers and tetramers.     

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values ≤6, whereas the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥ 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.     

Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

Formation of soluble oligomers and amyloid fibrils with physical properties of the scrapie isoform of the prion protein from the C-terminal domain of recombinant murine prion protein mPrP-(121-231)

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP(C), into a misfolded, protease-resistant form, PrP(Sc). Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106-120 from the so-called amyloidogenic core of PrP (residues 106-126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 degrees C. Under partially denaturing conditions (65 degrees C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 degrees C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 degrees C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 degrees C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP(Sc) can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106-120 of PrP and does not require other cellular factors or a PrP(Sc) template.     

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.     

Aggregation properties of a short peptide that mediates amyloid fibril formation in model proteins unrelated to disease

Short peptides have been identified from amyloidogenic proteins that form amyloid fibrils in isolation. The hexapeptide stretch ²¹DIDLHL²⁶ has been shown to be important in the self-assembly of the Src homology 3 (SH3) domain of p85α subunit of bovine phosphatidylinositol-3-kinase (PI3-SH3). The SH3 domain of chicken brain α-spectrin, which is otherwise non-amyloidogenic, is rendered amyloidogenic if ²²EVTMKK²⁷ is replaced by DIDLHL. In this article, we describe the aggregation behaviour of DIDLHL-COOH and DIDLHL-CONH₂. Our results indicate that DIDLHL-COOH and DIDLHL-CONH₂ aggregate to form spherical structures at pH 5 and 6. At pH 5, in the presence of mica, DIDLHL-CONH₂ forms short fibrous structures. The presence of NaCl along with mica results in fibrillar structures. At pH 6, DIDLHL-CONH₂ forms largely spherical aggregates. Both the peptides are unstructured in solution but adopt β-conformation on drying. The aggregates formed by DIDLHL-COOH and DIDLHL-CONH₂ are formed during drying process and their structures are modulated by the presence of mica and salt. Our study suggests that a peptide need not have intrinsic amyloidogenic propensity to facilitate the self-assembly of the full-length protein. The propensity of peptides to form self-assembled structures that are non-amyloidogenic could be important in potentiating the self-assembly of full-length proteins into amyloid fibrils.     

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.     

NMR assignments of PI3-SH3 domain aided by protonless NMR spectroscopy

We report here the near complete assignments of native bovine PI3-SH3 domain, which has been a model system for protein folding, misfolding and amyloid fibril formation. The use of ¹³C-detected protonless NMR spectroscopy is instrumental in assigning the spin system of the proline residue at the C-terminus in addition to the missing resonances in proton-based NMR spectra due to rapid solvent exchange. It also helps assign the resonances of all three proline amine nitrogen nuclei, which are underrepresented in the database. Comparison of the backbone ¹³C resonances of PI3-SH3 in its native and amyloid fibril states shows that the aggregation of PI3-SH3 is accompanied by major conformational rearrangements.     

Immunoglobulin kappa light chain and its amyloidogenic mutants: a molecular dynamics study

AL amyloidosis and LCDD are pathological conditions caused by extracellural deposition of monoclonal Ig light chain variable domains. In the former case, deposits have a form of amyloid fibrils, in the latter, amorphous aggregates. 1REI kappa light chain variable domain and its two point mutants, R61N and D82I, were chosen for the analysis in this work. Wild 1REI does not create deposits in vitro, while R61N aggregates as amyloid fibrils and D82I creates amorphous aggregates. Both mutated residues create a conserved salt bridge; thus, substitution of any of them should decrease V(L) domain stability. For these three proteins, 5 ns MD simulations were conducted in temperatures of 300 K and 400 K, with protonated and unprotonated acidic residues, mimicking acidic and neutral experimental pH conditions (3 sets: N300, N400, and A400). The analysis of trajectories focused on characterization of changes in conformational behavior and stability of Ig kappa light chain variable domain caused by single aminoacid substitutions that were experimentally proved to enhance aggregation propensity, both in the form of amyloid and amorphous aggregates. Residue D82 turns out to be involved not only in R61-D82 but also in K45-D82 interaction, which was not observed in the X-ray structure, but frequently populated simulations of 1REI. The substitution D82I excludes both interactions, resulting in substantial destabilization (i.e., easier aggregation). Examination of behavior of edge regions of V(L) beta-sandwich reveals significant alterations in D82I mutant compared to wild 1REI, while relatively small changes occur in R61N. This suggests that mild and slow destabilization is the reason of the conversion of V(L) to partially folded amyloidogenic intermediate structure.     

Effects of succinylation on thermal induced amyloid formation in Concanavalin A

We have recently shown that upon slight thermal destabilization the legume lectin Concanavalin A may undergo two different aggregation processes, leading, respectively, to amyloid fibrils at high pH and amorphous aggregates at low pH. Here we present an experimental study on the amyloid aggregation of Succinyl Concanavalin A, which is a dimeric active variant of Concanavalin. The results show that, as for the native protein, the fibrillation process appears to be favoured by alkaline pH, far from the isoelectric point of the protein. Moreover, it strongly depends on temperature and requires large conformational changes both at secondary and tertiary structure level. With respect to the native protein, the succinyl derivative forms amyloid fibrils in considerably longer times and with a minor exposure of hydrophobic regions. At physiological conditions, Concanavalin A still displays a sizeable tendency to form amyloid fibril, while the succinyl variant does not. A close correlation was observed between the progress of amyloid formation and a narrowing of the tryptophans fluorescence emission band, indicating a reduction of protein conformational heterogeneity in amyloid fibrils. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Amyloid-like fibril formation by polyQ proteins: a critical balance between the polyQ length and the constraints imposed by the host protein

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.     

Effect of pH on the aggregate formation of a non-amyloid component (1-13).

The formation of aggregates including amyloid fibrils in the peptide fragment of non-amyloid-beta component (NAC(1-13)) was investigated under a variety of solution conditions. Two types of sample preparation method from neutral and acidic conditions were examined. Electron microscopy observation showed amorphous aggregates in the sample at pH 4.5 adjusted from the neutral condition. The CD and HPLC quantitative analyses indicated that the formation of the amorphous aggregate did not accompany a conformational conversion from a random coil in the sample solution. The analyses of pKa values determined by pH titration experiments in NMR spectroscopy indicated that the protonation of the carboxyl group of the N-terminal glutamic acid triggers the aggregation of NAC(1-13). On the other hand, electron microscopy observation showed that the samples at pH 2.2 and 4.5 adjusted from an initial pH of 2.2 form fibrils. A beta-structure was detected by CD spectroscopy in the 1 mM NAC(1-13) at pH 2.2 immediately after preparation. The CD analyses of samples at different concentrations and temperatures indicated that 1 mM NAC(1-13) immediately after preparation at pH 2.2 was oligomerized. The quantity of the beta-structure was increased depending on the Incubation time. The results strongly suggested that the beta-conformational oligomers play a critical role for the fibril nucleus.     

Structural studies reveal that the diverse morphology of beta(2)-microglobulin aggregates is a reflection of different molecular architectures

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of beta(2)-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the beta-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular beta-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular beta-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.