Hacking the Code of Amyloid Formation: The Amyloid Stretch Hypothesis

Many research efforts in the last years have been directed towards understanding the factors determining protein misfolding and amyloid formation. Protein stability and amino acid composition have been identified as the two major factors in vitro. The research of our group has been focused on understanding the relationship between amino acid sequence and amyloid formation. Our approach has been the design of simple model systems that reproduce the biophysical properties of natural amyloids. An amyloid sequence pattern was extracted that can be used to detect amyloidogenic hexapeptide stretches in proteins. We have added evidence supporting that these amyloidogenic stretches can trigger amyloid formation by nonamyloidogenic proteins. Some experimental results in other amyloid proteins will be analyzed under the conclusions obtained in these studies. Our conclusions together with evidences from other groups suggest that amyloid formation is the result of the interplay between a decrease of protein stability, and the presence of highly amyloidogenic regions in proteins. As many of these results have been obtained in vitro, the challenge for the next years will be to demonstrate their validity in in vivo systems.     

New strategy for the generation of specific D-peptide amyloid inhibitors

The conversion of a soluble protein into β-sheet-rich oligomeric structures and further fiber formation are critical steps in the pathogenesis of the group of human diseases known as amyloidoses. Drugs that interfere with this process may thus be able to prevent and/or cure these diseases. Recent results have shown that short amino acid stretches can provide most of the driving force needed to trigger amyloid formation of a protein. These evidence suggest that compounds that specifically bind to peptides synthesized upon the sequence of such amyloidogenic protein stretches might also be able to inhibit amyloid formation of the corresponding full-length protein and, likely, amyloid-induced cytotoxicity as well. Here we present a general strategy to obtain d-peptides that specifically interact with protein amyloid stretches. The screening of a d-peptide combinatorial library for inhibitors of an amyloidogenic peptide designed de novo has allowed us to extract a set of empirical rules for the design of d-peptide inhibitors of any six-residue amyloidogenic stretch. d-peptides generated on these bases prevent amyloid formation and disassemble preformed fibrils of different amyloid hexapeptides identified in human amyloid proteins. In addition, they are also specific for their target sequence. The d-peptide designed here for the Alzheimer's Aβ_1-42 peptide not only inhibits and disassembles amyloid material but also reduces Aβ_1-42 amyloid-induced cytotoxicity in cell culture.     

Is it possible to predict amyloidogenic regions from sequence alone?

Identification of potentially amyloidogenic regions in polypeptide chains is very important because the amyloid fibril formation can be induced in most normal proteins. In our work we suggest a new method to detect amyloidogenic regions in protein sequence. It is based on the assumption that packing is tight inside an amyloid and therefore regions which could potentially pack well would have a tendency to form amyloids. This means that the regions with strong expected packing of residues would be responsible for the amyloid formation. We use this property to identify potentially amyloidogenic regions in proteins basing on their amino acid sequences only. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Predictions of the regions which are responsible for the formation of amyloid fibrils in proteins unrelated to disease have been also done.     

A search for amyloidogenic regions in protein chain

We suggest a new method to detect amyloidogenic regions in a protein sequence. In the present work it is shown that regions enriched with amino acid residues which have a high expected packing density are responsible for the amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which positions of amyloidogenic regions have been revealed experimentally.     

Prediction of amyloidogenic and disordered regions in protein chains

The determination of factors that influence protein conformational changes is very important for the identification of potentially amyloidogenic and disordered regions in polypeptide chains. In our work we introduce a new parameter, mean packing density, to detect both amyloidogenic and disordered regions in a protein sequence. It has been shown that regions with strong expected packing density are responsible for amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for eight of 12 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Our findings support the concept that the mechanism of amyloid fibril formation is similar for different peptides and proteins. Moreover, we have demonstrated that regions with weak expected packing density are responsible for the appearance of disordered regions. Our method has been tested on datasets of globular proteins and long disordered protein segments, and it shows improved performance over other widely used methods. Thus, we demonstrate that the expected packing density is a useful value with which one can predict both intrinsically disordered and amyloidogenic regions of a protein based on sequence alone. Our results are important for understanding the structural characteristics of protein folding and misfolding.     

Cooperativity among short amyloid stretches in long amyloidogenic sequences

Amyloid fibrillar aggregates of polypeptides are associated with many neurodegenerative diseases. Short peptide segments in protein sequences may trigger aggregation. Identifying these stretches and examining their behavior in longer protein segments is critical for understanding these diseases and obtaining potential therapies. In this study, we combined machine learning and structure-based energy evaluation to examine and predict amyloidogenic segments. Our feature selection method discovered that windows consisting of long amino acid segments of ~30 residues, instead of the commonly used short hexapeptides, provided the highest accuracy. Weighted contributions of an amino acid at each position in a 27 residue window revealed three cooperative regions of short stretch, resemble the β-strand-turn-β-strand motif in A-βpeptide amyloid and β-solenoid structure of HET-s(218-289) prion (C). Using an in-house energy evaluation algorithm, the interaction energy between two short stretches in long segment is computed and incorporated as an additional feature. The algorithm successfully predicted and classified amyloid segments with an overall accuracy of 75%. Our study revealed that genome-wide amyloid segments are not only dependent on short high propensity stretches, but also on nearby residues.     

Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.     

Elongation in a beta-structure promotes amyloid-like fibril formation of human lysozyme

To understand the mechanism of amyloid fibril formation of a protein, we examined wild-type and three mutant human lysozymes containing both amyloidogenic and non-amyloidogenic proteins: I56T (amyloidogenic); EAEA, which has four additional residues (Glu-Ala-Glu-Ala-) at the N-terminus located on a beta-structure; and EAEA-I56T, which is an I56T mutant of EAEA. All formed amyloid-like fibrils through an in the increase contents of alpha-helix with increasing concentration of ethanol. The order of propensity for amyloid-like fibril formation in highly concentrated ethanol solution is EAEA-I56T > EAEA > I56T > wild-type. This order is almost the reverse of the order of conformational stability of these proteins, wild-type > EAEA > I56T > EAEA-I56T. The important views in this work are as follows. (i) Artificially modified proteins formed amyloid fibrils in vitro. This means that amyloid formation is a generic property of polypeptide chains. (ii) The amyloidogenic mutation Ile56 to Thr caused the destabilization and promoted fibril formation in the wild-type and EAEA human lysozymes, indicating that instability facilitates amyloid formation. (iii) The mutant protein EAEA human lysozyme had higher propensity for fibril formation than the amyloidogenic mutant protein, indicating that amyloid formation is controlled not only by stability but also by other factors. In this case, appending polypeptide chains to a beta-structure accelerated amyloid formation.     

Amino acid sequence determinants and molecular chaperones in amyloid fibril formation

Amyloid consists of cross-β-sheet fibrils and is associated with about 25 human diseases, including several neurodegenerative diseases, systemic and localized amyloidoses and type II diabetes mellitus. Amyloid-forming proteins differ in structures and sequences, and it is to a large extent unknown what makes them convert from their native conformations into amyloid. In this review, current understanding of amino acid sequence determinants and the effects of molecular chaperones on amyloid formation are discussed. Studies of the nonpolar, transmembrane surfactant protein C (SP-C) have revealed amino acid sequence features that determine its amyloid fibril formation, features that are also found in the amyloid β-peptide in Alzheimer’s disease and the prion protein. Moreover, a proprotein chaperone domain (CTC_Brichos) that prevents amyloid-like aggregation during proSP-C biosynthesis can prevent fibril formation also of other amyloidogenic proteins.     

Generic inhibition of amyloidogenic proteins by two naphthoquinone-tryptophan hybrid molecules

Amyloid formation is associated with several human diseases including Alzheimer's disease (AD), Parkinson's disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease-modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high-resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti- cancer, anti-viral and anti-bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone-tryptophan hybrids which are capable of inhibiting amyloid-beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α-synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti-amyloid drugs.     

Thermodynamic instability of human lambda 6 light chains: correlation with fibrillogenicity.

Certain types of human light chains have the propensity to deposit pathologically as amyloid fibrils as evidenced by the preferential association of monoclonal lambda 6 proteins with AL amyloidosis. However, the molecular features that render such proteins amyloidogenic have not been elucidated. Based upon the demonstrated relationship between the thermodynamic stability of light chains and their propensity to aggregate in vitro, we have initiated studies where the thermodynamic properties and fibrillogenic potential of two recombinant (r) V lambda 6 molecules were compared. The first protein was generated from cDNA cloned from marrow-derived plasma cells from a patient (Wil) who had AL amyloidosis and renal amyloid deposits; the second was from a patient (Jto) with multiple myeloma in whom the lambda 6 protein was deposited not as amyloid but in the form of renal tubular casts. The thermodynamic stabilities of rV lambda 6Wil and -Jto were determined from chaotropic and thermal denaturation studies. Based upon the Delta GH2O, Delta H, Delta G25 degrees C, Tm, and Cm values, the rV lambda 6Wil was less stable than its nonamyloidogenic counterpart, rV lambda 6Jto. Measurement of fibril formation using a novel in vitro fibril forming assay demonstrated that although both rV lambda 6 proteins formed fibrils in vitro, Wil had a shorter lag time and exhibited faster kinetics under physiologic conditions. Comparative amino acid sequence analyses of these two components and other lambda 6 amyloid-associated light chains revealed that the Jto protein had certain primary structural features that we posit contributed to its increased stability and thus rendered this protein nonamyloidogenic. Our studies provide the first evidence that stabilizing interactions within the V L domain can influence the kinetics of light chain fibrillogenicity.     

Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP)

The formation of amyloid fibrils is considered to be an important step in the aetiology of Alzheimer's disease and other amyloidoses. Fibril formation in vitro has been shown to depend on many different factors including modifications to the amino acid profile of fibrillogenic peptides and interactions with both large and small molecules of physiological significance. How these factors might contribute to amyloid fibril formation in vivo is not clear as very little is known about the promotion of fibril formation in undersaturated solutions of amyloidogenic peptides. We have used thioflavin T fluorescence and reverse phase high performance liquid chromatography to show that ATP, and in particular AlATP, promoted the formation of thioflavin T-reactive fibrils of beta amyloid and, an unrelated amyloidogenic peptide, amylin. Evidence is presented that induction of fibril formation followed the complexation of AIATP by one or more monomers of the respective peptide. However, the complex formed could not be identified directly and it is suggested that AlATP might be acting as a chaperone in the assembly of amyloid fibrils. The effect of AlATP was not mimicked by either AlADP or AlAMP. However, it was blocked by suramin, a P2 ATP receptor antagonist, and this has prompted us to speculate that the precursor proteins to beta amyloid and amylin may be substrates or receptors for ATP in vivo.     

Counteracting effects of renal solutes on amyloid fibril formation by immunoglobulin light chains

In primary (light chain-associated) amyloidosis, immunoglobulin light chains deposit as amyloid fibrils in vital organs, especially the kidney. Because the kidney contains high concentrations of urea that can destabilize light chains as well as solutes such as betaine and sorbitol that serve as protein stabilizers, we investigated the effects of these solutes on in vitro amyloid fibril formation and thermodynamic stability of light chains. Two recombinant light chain proteins, one amyloidogenic and the other nonamyloidogenic, were used as models. For both light chains, urea enhanced fibril formation by reducing the nucleation lag time and diminished protein thermodynamic stability. Conversely, betaine or sorbitol increased thermodynamic stability of the proteins and partially inhibited fibril formation. These solutes also counteracted urea-induced reduction in protein thermodynamic stability and accelerated fibril formation. Betaine was more effective than sorbitol. A model is presented to explain how the thermodynamic effects of the solutes on protein state equilibria can alter nucleation lag time and, hence, fibril formation kinetics. Our results provide evidence that renal solutes control thermodynamic and kinetic stability of light chains and thus may modulate amyloid fibril formation in the kidney.     

Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild‐type rats

Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation‐prone proteins such as αSN, which is central in the development of Parkinson''s disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThT‐positive aggregates that contain higher‐order β‐sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross‐seed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes.     

Determination of regions involved in amyloid fibril formation for Aβ(1-40) peptide

The studies of amyloid structures and the process of their formation are important problems of biophysics. One of the aspects of such studies is to determine the amyloidogenic regions of a protein chain that form the core of an amyloid fibril. We have theoretically predicted the amyloidogenic regions of the Aβ(1-40) peptide capable of forming an amyloid structure. These regions are from 16 to 21 and from 32 to 36 amino acid residues. In this work, we have attempted to identify these sites experimentally by the method of tandem mass spectrometry. As a result, we show that regions of the Aβ(1-40) peptide from 16 to 22 and from 28 to 40 amino acid residues are resistant to proteases, i.e. they are included in the core of amyloid fibrils. Our results correlate with the results of the theoretical prediction.     

Understanding and controlling amyloid aggregation with chirality

Amyloid aggregation and human disease are inextricably linked. Examples include Alzheimer disease, Parkinson disease, and type II diabetes. While seminal advances on the mechanistic understanding of these diseases have been made over the last decades, controlling amyloid fibril formation still represents a challenge, and it is a subject of active research. In this regard, chiral modifications have increasingly been proved to offer a particularly well-suited approach toward accessing to previously unknown aggregation pathways and to provide with novel insights on the biological mechanisms of action of amyloidogenic peptides and proteins. Here, we summarize recent advances on how the use of mirror-image peptides/proteins and d-amino acid incorporations have helped modulate amyloid aggregation, offered new mechanistic tools to study cellular interactions, and allowed us to identify key positions within the peptide/protein sequence that influence amyloid fibril growth and toxicity.     

Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein

Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity.     

Amyloid toxicity is independent of polypeptide sequence, length and chirality

By using an amyloid sequence pattern, here we have identified putative six-residue amyloidogenic stretches in several relevant amyloid proteins. Hexapeptides synthesized on the bases of the sequence stretches matching the pattern have been shown to form amyloid fibrils in vitro. As larger pathological peptides such as Aβ_1-42 do, these short amyloid peptides form heterogeneous mixtures of small aggregates that induce cell death in PC12 cells and primary hippocampal neurons. Toxic mixtures of small aggregates from these hexapeptides bind to cell membranes and can be further internalized, as also observed for natural amyloid proteins. In neurons, toxic aggregates obtained from the full length Aβ_1-42 amyloid peptide or their amyloid stretch Aβ_16-21 peptide preferentially localize in synapses, leading to the re-organization of the underlying actin cytoskeleton. This process does not involve stereospecific interactions between membrane and toxic species as D-sequences are as toxic as L ones, suggesting that is not receptor mediated. Based on these results, we propose here that regardless of polypeptide sequence, length and amino acid chirality, amyloid prefibrillar aggregates exert their cytotoxic effect through a common cell death mechanism related to a particular quaternary structure. The degree of toxicity of these species seems to depend, however, on cell membrane composition.     

Consensus prediction of amyloidogenic determinants in amyloid fibril-forming proteins

We combine the results of three prediction algorithms on a test set of 21 amyloidogenic proteins to predict amyloidogenic determinants. Two prediction algorithms are recently developed prediction algorithms of amyloidogenic stretches in protein sequences, whereas the third is a secondary structure prediction algorithm capable of identifying 'conformational switches' (regions that have both the propensity for alpha-helix and beta-sheet). Surprisingly, the results of prediction agree well and also agree with experimentally investigated amyloidogenic regions. Furthermore, they suggest several previously not identified amino acid stretches as potential amyloidogenic determinants. Most predicted (and experimentally observed) amyloidogenic determinants reside on the protein surface of relevant solved crystal structures. It appears that a consensus prediction algorithm is more objective than individual prediction methods alone.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.