As a proof of principle our approach was first tested in the molecule from which the experimental amino acid aggregation propensities were derived. Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the patient's memory loss and impairment of cognitive abilities. The extracellular amyloid is found in the brain and is widely believed to be involved in the progression of the disease [15]. The principal component of the lesions is the hydrophobic polypeptide Aβ. The most abundant forms found in amyloid plaques are a 40-mer (Aβ40) and a 42-mer (Aβ42). Although less abundant, Aβ42 is more amyloidogenic than Aβ40 and is the major component of neuritic plaques [16]. Two main regions with high aggregation propensity can be distinguished in the aggregation profile for this polypeptide (Fig. 1). The second region arises from the contribution of two sequence stretches comprising residues 30–36 and 38–42, respectively. The predicted aggregation-prone regions are in excellent agreement with the experimental data in the literature. Residues 16–21 overlap with the CHC sequence comprising residues 17–21, a particular region recognized to play a key role in Aβ aggregation and that is defined as specially relevant for the amyloidogenesis of the Aβ40 and Aβ42 peptides by two recent proline-scanning-mutagenesis studies [17,18]. In addition, structural studies using solid state-NMR [19] and site-directed spin labeling [20] have revealed that residues 16–21 are located in the core of Aβ fibrils. Accordingly, a short 7 residues fragment comprising residues 16–22 is able to form ordered amyloid fibrils [21] and, more interestingly, 16-LVAFF-20 and derived peptides have been shown to bind to Aβ42 and act as potent inhibitors of amyloid formation [22]. The region 30–42, including both 30–36 and 38–42 stretches, has been also implicated in Aβ aggregation. Proline-scanning-mutagenesis revealed that the region 31–36 is sensitive to proline replacement and likely to include a β-sheet portion of the Aβ fibrils [17,18]. The contribution of the C-terminal region 38–42 to Aβ amyloidogenesis becomes clear from the observation that, although Aβ40 is produced in greater abundance in vivo, the prevalence of the full-length 42-mer in plaques is much higher [16]. Experiments with truncated synthetic Aβ peptides have confirmed that Aβ39 and Aβ40 are kinetically soluble for several days, whereas Aβ42 immediately aggregates into amyloid fibrils [23]. The relevance of the predicted 30–42 region is confirmed by structural studies that demonstrate that residues 30–40 are located in the core of the Aβ fibrils [20].

A set of mutations in the CHC and adjacent positions of Aβ42 is intimately associated to early-onset familial Alzheimer diseases (FAD). The substitutions include A21G (Flemish), E22Q (Dutch) and E22G (Arctic) [24]. Aβ42 congeners bearing these mutations display distinct aggregation kinetics. The rate of fibril formation by the Flemish mutant is decreased relative to WT Aβ42, whereas the Dutch mutant peptide aggregates substantially faster. The Arctic peptide does not shows an overall change in the rate of fibrillogenesis relative to WT Aβ42, but rather accelerated protofibril formation. To assess whether the effect of such mutations could be predicted by the present approach we calculated ΔAP for the different sequences. The results obtained describe accurately the effects documented in the literature (Table 2).

Adding to the mutations present in the population, a large set of mutations has been artificially introduced on Aβ that result in changes in its aggregation propensity. ΔAP values were also calculated for several of them and the results compared with the experimental data (Table 2). The calculated changes in aggregation propensity are in excellent agreement with the trends reported in the literature. Briefly, we predict the changes in aggregation of F19 mutants, those of I31 and I32 in the 30–36 region and those of I41 and A42 in the C-terminal region, as well as the effects of deletions both in the N and the C ends. Finally, we also predict the high solubility of Aβ versions generated by random mutagenesis [25].

Type II diabetes is associated with progressive beta-cell failure manifested as a decline in insulin secretion and increasing hyperglycemia. A growing body of evidence suggests that beta-cell failure in type II diabetes correlates with the formation of pancreatic islet amyloid. Islet amyloid polypeptide (IAPP, amylin), the major component of islet amyloid, is co-secreted with insulin from beta-cells. In type II diabetes, this peptide aggregates to form amyloid fibrils that are toxic to beta-cells [26]. IAPP is an unstructured peptide hormone of 37 amino acid residues. Two "hot spots" of aggregation comprising residues 12–18 and 22–28 are detected for this peptide (Fig. 1). Interestingly enough, a 8–37 IAPP-fragment including both "hot spots", has been shown to form amyloid fibrils under physiological conditions [27]. The two aggregation prone regions sharply coincide with those protected in the core of the fibrils in a recently described structural model of IAPP aggregates [28]. In this study, residues 12–17 and 22–27 are proposed to form the inner β-sheets in the fibril protofilament structure. According to this hypothesis, peptides corresponding to residues 8–20, 10–19, 20–29 of human IAPP, which include one of the "hot spots" described here, all form amyloid [29-31]. Smaller peptides derived from these regions have also been shown to form amyloid, and a recent investigation suggests that the minimal amyloid forming fragment of IAPP consists of residues 22–27. This hexapeptide fragment, NFGAIL, forms β-sheet-containing fibrils that coil around each other in typical amyloid fibril morphology [32].

The analysis also explains the available mutational data on IAPP. Diabetes-associated IAPP amyloid occurs in primates and cats but not in rodents [33]. Consistently, the sequences of peptides 20–29 of rodents display reduced average aggregation propensity relative to that of cat and human (Table 3). We also predict the slightly increased aggregation propensities of single or multiple mutations of rat IAPP to the corresponding residues of human IAPP [33]: R18H, L23F or V26I, as well as the results from alanine-scanning-mutagenesis in a peptide encompassing residues 22–27 [32] (Table 3). It has been found that a substitution at position 20 (S20G) in the IAPP molecule in a reduced subpopulation of Japanese people with type II diabetes is associated with an earlier onset and more severe form of disease [34]. In this case, our approach does no predict an increased but a slightly reduced aggregation propensity in the mutant, suggesting that the pathological symptoms in this variant may arise from non-intrinsic factors. In fact, it has been suggested that the accelerated aggregation of the S20G variant could be related to structural reasons, resulting from a better packing of the turns connecting the β-sheets in the final protofilament structure [28] that cannot be predicted by the present approach.

Several mechanisms have been proposed for IAPP fibril formation in type II diabetes. One widely accepted mechanism is that in type II diabetes, increased production and secretion of IAPP associated with increased demand for insulin might result in accumulation and aggregation of IAPP [35]. A second view considers that impaired processing of the IAPP precursor molecule, proIAPP, by islet beta-cells may lead to hypersecretion of unprocessed or partially processed forms of proIAPP that may have a higher tendency for aggregation compared to mature IAPP [35]. Our calculated average aggregation propensities for proIAPP and processed IAPP support this view (Table 3).

Parkinson disease is the most common neurodegenerative movement disorder and is pathologically characterized by the presence of neuronal intracytoplasmatic deposits of aggregated protein called Lewy bodies [36]. Lewy bodies also occur in other cognitive disorders, globally known as α-synucleinopathies. α-Synuclein is the major component of the fibrils that form the Lewy bodies [36]. It is a small (137 residues), natively unfolded, soluble, presynaptic and highly conserved protein without a well-defined function. The aggregation profile for this polypeptide is shown in Fig. 1. Several large aggregation-prone stretches were predicted for the α-Synuclein sequence: region 1–18, region 27–56 and specially region 61–94. Again, our predictions are in complete agreement with the experimental data in the literature, as many studies suggest that the central region of the protein, known as the non-Aβ component of amyloid plaques (NAC, amino acids 61–95), is the responsible for its aggregation process [37]. A peptide comprising residues 68–78 of α-synuclein has been shown to be the minimum fragment that, like α-synuclein itself, forms amyloid fibrils and exhibits toxicity towards cells in culture [38]. This fragment is included in the region 62–80 which we predict as the sequence stretch with the highest aggregation propensity. All the α-synucleinopathies are characterized by the accumulation of the 35 residues NAC fragment in the insoluble deposits [37]. Accordingly, this central region is predicted to have a much higher average aggregation propensity than its soluble precursor (ΔAP = +38.47). The importance of this hydrophobic stretch is further supported by its absence in β-synuclein, a homologue of α-synuclein, with strongly reduced propensity for fibril formation. It has been shown that the deletion of amino acids 71–82 within the hydrophobic region abrogated the ability of human α-synuclein to polymerize into fibrils [39]. Protease digestion studies suggest that the core region of α-synuclein in the fibrils could be longer, since a 7-kDa fragment (comprising residues 31–109) was shown to be protected from proteinase K digestion [40]. This region contains the putative 12-residue core domain, as well as the NAC region and includes the second and third "hot spots" in our profile. A structural study on the organization of α-synuclein in the fibrilar state using site-directed spin labelling confirms that the 34–101 residues region constitutes the core of the fibrils forming a parallel in-register β-sheet structure whereas the N terminus is structurally more heterogeneous and the C terminus (40 amino acids) is completely unfolded [41].

Several α-Synuclein mutations appear associated with familial early-onset Parkinson Disease: A30P, A53T and E46K. All they map into our predicted second "hot spot". The rates of fibril assembly of the E46K and A53T mutants have been shown to be greater than those of the wild type and A30P proteins [42]. We predict a similar average aggregation propensity for the wild-type and the A30P mutant and an slightly increased aggregation propensity for the E46K mutant, but fail to foresee the effect of the A53T mutation in promoting the formation of protofibrils. Obviously, other functional factors apart from the intrinsic aggregation propensities can strongly influence the aggregation tendency of unfolded polypeptide chains within the cell. In fact the effects of α-synuclein mutations have been associated either to an impaired degradation inside lysosomes or to a reduced axonal transport of the variants [43,44]. Both situations may result in increased concentrations of the protein in certain regions of the neuron that may favor the nucleation step of amyloid formation. According to this, α-synuclein gene triplication identified in two independent families [45] has been shown to accelerate the development of Parkinson disease. Thus, an increase in the amount of cellular α-synuclein appears to be important for the pathogenesis of Parkinson disease, suggesting that the effects of the different α-synuclein mutations on protein aggregation could be quantitative, in terms of local concentration, rather that qualitative. Thus, experimental deviations from the theoretical predictions in natively unfolded proteins, in addition to reflect limitations of the approach, might also contain relevant information, prompting to find alternative structural, as in the case of amylin, or functional, as in the case of α-synuclein, explanations for the observed behavior.

β2-Microglobulin-related amyloidosis is a common and serious complication in patients on longterm hemodialysis [46]. Intact β2-microglobulin is a major structural component of the amyloid fibrils. β2-Microglobulin (β2-m) is a small (99 residues) non-glycosilated protein with an immunoglobulin-like fold consisting in two antiparallel pleated β-sheets linked by a disulfide bond (Fig. 2). β2-m has been shown to form amyloid fibrils in vitro under different conditions, but in all cases β2-m populates unfolded non-native states as precursors to fibril assembly [47]. Under these conditions aggregation-prone regions, if present, may promote and drive the aggregation event. According to the analysis of the aggregation profile, shown in Fig. 3, this protein displays four "hot spots" encompassing residues 21–31, 56–69 and 79–85, and 87–91. These regions sharply coincide with four different secondary structure elements in β2-m: β-strand 2, formed by residues 21–31; β-strand 6, formed by residues 61–71; β-strand 7; formed by residues 77–85 and β-strand 8, formed by residues 86–95 (Fig. 2). In agreement with our prediction a peptide comprising residues 21–41 has been shown to form fibrils in isolation [48]. In addition, a N-terminal fragment of this short peptide corresponding exactly to our first "hot spot" [21-31] is also able to self-assemble into fibrillar structures [49]. Interestingly enough, the peptides 23–31 and 21–29 exhibited reduced amyloidogenesis [49]. Thus, in this particular "hot spot" the prediction delimits not only the overall region important for aggregation but also its precise size. The amino acid stretches 59–79 and its shorter version 59–71 which overlap with the predicted second aggregation-prone region of β2-m have been also shown to form fibrils [50]. The C-terminal fragment 72–99 of β2-m has been also reported to form amyloid [51]. This 29 residues sequence includes our third and fourth "hot spots" of aggregation. The peptide 91–99 does not aggregate, indicating that the last 9 residues of β2-m are not relevant for amyloidogenesis as predicted here [49]. The N-terminal region, for which no aggregation propensity is predicted, is probably not involved inthe aggregation process as evidenced by the fact that the fragment 6–12 does not form fibrils [49]. This observation could be physiologically relevant since the N-terminus of β2-m is truncated in 30% of the molecules extracted from ex vivo fibrils [52].

In contrast to the human protein, mouse β2-m does not form fibrils even at high concentration [53]. Based on this observation a seven residues region corresponding to residues 83–89 of human β2-m has been suggested to be particularly important for aggregation, since it corresponds to the sequence with the highest divergence between both species. This hypothesis has been tested experimentally, since a heptapeptide bearing the human sequence is able to self-assemble whereas the mouse version is not [53]. The complete mouse sequence is predicted to have a strongly reduced aggregation propensity (ΔAP = -47.86).

Overall, our predictions on the presence and location of "hot spots" in β2-m are extremely accurate and overlap with the experimentally found relevant regions (Fig. 2). The observation that short peptides including the aggregation-prone regions described here form amyloids implies that exposure of previously hidden short segments can nucleate native proteins into the amyloid state and reinforces the hypothesis that fibril formation is sequence specific.

One of the most urgent issues in the study of amyloid fibrils is to reproduce the formation of fibrils under physiological conditions. Recently, it has been found that low concentrations of SDS around the critical micelle concentration induce the extensive growth of β2-m amyloid fibrils at physiological pH, probably through the SDS-induced conformational change of β2-m monomers [54]. Contrarily to what was expected, the presence of low concentration of SDS had little effect on the stability of the protein and did not promote global protein unfolding. Our results strongly suggest that in β2-m the parts of the molecule involved in aggregation are located in pre-formed β-strands. Therefore, it is possible that local unfolding events may allow anomalous intermolecular interaction between this preformed elements leading to the formation of an aggregated β-sheet structure. This would explain the formation of amyloid deposits in hemodyalisis patients in which no major unfolding of the protein is expected to occur, as well as the effect of seeds, which may have exposed aggregation prone β-strands, in strongly accelerating the aggregation process of β2-m under physiological conditions [55].

Human lysozyme has been shown to form amyloid fibrils in individuals suffering from nonneuropathic systemic amyloidosis. The disease is always associated to point mutations in the lysozyme gene and fibrils are deposited widely in tissues [56]. The properties of two amyloidogenic lysozyme mutants (I56T and D67H) have been studied in detail and, when compared to those of the wild-type protein, the mutants were found to have reduced structural stability allowing unfolding to take place at least partially at physiologically relevant temperatures [57,58]. Thus, the formation of amyloid fibrils by human lysozyme is likely to occur by the exposure of aggregation-prone region previously hidden in the native structure. The aggregation profile of lysozyme identifies three main "hot spots" corresponding to residues 20–34, 50–62 and 73–104 (Fig. 3). The last large aggregation-prone region includes several local maxima. The first "hot spot" maps in helix B, the second in a β-hairpin of the β-domain and the third includes helix C and a large flanking unstructured region at its N-terminus (Fig. 2). Although there is no experimental characterization of amyloidogenic regions in human lysozyme in the literature, this information is available for the homologous hen lysozyme molecule, which displays an almost identical 3D-structure. The aggregation profile for the hen protein is very similar to that of the human one despite the fact that our input consists solely on the sequence and the identity between both molecules is only of 40%. The equivalent "hot spots" in hen lysozyme comprise residues 24–34, 50–62 and 76–98. Experimental data suggests that the sequence of the β-domain could be of particular relevance for lysozyme aggregation since it unfolds prior to the α-domain [58]. Two peptides encompassing the β-domain of native lysozyme displayed very different behavior: peptide 61–82 appeared to be predominantly unstructured whereas peptide 41–60 showed a high tendency to aggregate and form extended β-sheet structures [59]. The first peptide coincides with a region of very low aggregation propensity in the aggregation profiles, whereas the second one covers the region with the highest aggregation propensity in the profile (residues 50–64). Interestingly enough, a peptide spanning residues 49–64 has been shown to form fibrils with the typical structure of amyloid showing that the first residues of the 41–60 peptide are not relevant for aggregation, as predicted by our approach [60]. Another study has reported that the major fragment incorporated in the core of the fibril structure, as monitored using proteolysis, encompasses the chain region 49–101 [61]. These lysozyme fragments contain helix C and two of the three β-strands of the β-domain of the native protein structure and coincide with the limits of the second and third regions in our predictions (Fig. 2 and 3). This observation could be biologically relevant, since the β-domain and C-helix of the human lysozyme have been shown to unfold locally in the amyloidogenic variant D67H, which is associated with the familial cases of systemic amyloidosis linked to lysozyme deposition [58]. The C-helix is the α-helix with the lowest helical propensity of hen lysozyme according to both theoretical and peptide based studies [59]. This low propensity might be related to the ability of this region to be incorporated into the β-sheet rich fibrillar structured as have been reported for other protein systems [62]. Limited proteolysis of hen lysozyme renders fragments 57–107 and 1–38/108–129 [61]. In the 1–38/108–129 fragment the N-terminal and C-terminal ends of the molecule are joined by a disulfide bond. Only fragment 57–107, but not fragment 1–38/108–129, is able to generate well defined amyloid [61]. Whereas the behavior of the 57–107 fragment is expected from the analysis, one should also expect the fragment 1–38 to have a high tendency to aggregate. Two explanations are possible to account for this discordance. First, it could occur that the helical structure of this region prevents its conversion to β-sheet conformation, since the A-helix displays the highest helical propensity out of all lysozyme helices [59]. The second possibility is that, being joined to the 108–129 region, predicted to have lower aggregation propensity, steric hindrances limit self-assembly or alternatively the average aggregation tendency of this peptide becomes reduced. The analysis supports this last hypothesis reporting a decrease in aggregation propensity (ΔAP = -5.34) in the joined peptide respect the 1–38 peptide alone.

Transthyretin (TTR) is a homotetramer of 127-amino acid subunits. TTR is found in human plasma and cerebral spinal fluid, the plasma form being the amyloidogenetic precursor. TTR constitutes the fibrillar protein found in familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA) [63]. In the case of FAP, the amyloid is associated with a point mutation in the TTR gene. To date, 100 different TTR mutations have been reported, many of which are amyloidogenic [64]. The FAP-associated variants characterized thus far although tetrameric, are destabilized [65]. This destabilization allows tetramer dissociation to the amyloidogenic monomeric intermediate to occur under the influence of mild denaturing denaturation conditions. More than 10 FAP-related variants crystal structures have been solved, revealing that the tertiary and quaternary structures are essentially identical to the wild type form [65]. This observation suggests that the partial denaturation of TTR is a requirement for amyloidogenesis. In this state, the presence of "hot spots" of aggregation could play an especially important role in promoting/driving amyloid formation. According to the analysis of the aggregation profile shown in Fig. 3, the TTR monomer displays three main "hot spots" encompassing residues 10–20, 23–33 and 105–118. Also in this case, aggregation-prone sequences appear to be located in preformed β-sheet structures: A β-strand (11–19), part of the B β-strand (28–36) and G and beginning of H β-strands (104–123) (Fig. 3). Most of these secondary structure elements are involved in the formation of the tetrameric structure: H strands mediate the dimerization whereas A and G provide the contacts for the tetramerization of two preformed dimmers. This explains the protective role played by the TTR quaternary structure against aggregation, since it hides or blocks most of the aggregation prone regions. Dissociation of the tetramer has been reported as a prerequisite for amyloidosis and according to our results might be associated to the exposure of previously hidden amyloidogenic sequences. We detect several short peaks exhibiting high aggregation propensities in the central region (63–94) of TTR. These result from the presence of almost regularly placed residues with low aggregation propensity (Asp, Glu, Arg, Lys, Gly) in this rather hydrophobic sequence, which probably act as disrupters, significantly lowering the aggregation tendency of this particular region, a strategy suggested to be used by nature to avoid edge-to-edge aggregation [66].

To date two different fragments of TTR have been shown to form amyloid fibrils. The peptide 105–115 can be assembled into homogeneous amyloid fibrils with favorable spectroscopic properties [67]. This has allowed to solve its fibrillar structure at high-resolution, showing that it adopts an antiparallel extended beta-strand conformation in the amyloid fibrils [68]. This peptide coincides with the region with the highest aggregation propensity in the profile. Also in excellent agreement with the prediction, the peptide 10–20 is the only other fragment of TTR reported to form amyloid fibrils [69]. No data are available on the region 23–33 but the success of the present method in predicting relevant regions in TTR suggests that it is worth to characterize its in vitro aggregation capabilities.

Misfolded isoforms of the naturally occurring prion protein (PrP) have been shown to be the causative agents in many mammalian neurodegenerative disorders, including Cruetzfeldt-Jakob disease (CJD) in human, scrapie in sheep, and bovine spongiform encephalopathy in cows. Prion infectivity is unique in that the pathogenic prion form (PrP^Sc) is involved in the conversion of the endogenous conformation (PrP^C) into transformed PrP^Sc. The "protein-only" hypothesis [70] asserts further that no extraneous agents are necessary to explain the unusual behavior of prions. Prion diseases can have infectious, familial, and sporadic origins. The basic infectious mechanism is thought to be a conformational change of the normal prion protein (PrPC) into the pathogenic PrP^Sc catalyzed by PrP^Sc itself.

The normal prion protein (PrP^C) is a GPI-anchored glycoprotein constitutively expressed on the surface of primarily neuronal cells. It consists of two structurally different parts; a C-terminal, globular part mainly α-helical in nature (Fig. 2) and an unstructured, N-terminal part [71]. Misfolding of PrP^C into PrP^Sc occurs posttranslationally and results in increased β-sheet content and gain of protease-resistance. Fig. 3 shows the predicted "hot spots" in the aggregation profile of the full-length human prion protein. They are located at the N-terminus (1–32), in the central region (105–146) and the C-terminus (208–252), respectively.

The role of the detected aggregation-prone sequence at the N-terminus is uncertain since it is out of the protease resistant core of PrP^Sc. Little information exits about the role of this region, although it appears to be unnecessary both for prion transmission and aggregation. The predicted C-terminal "hot spot" includes almost all the C-terminal α-helix, named C, from the globular domain (Fig. 2). Interestingly, some of the human mutations linked to Creutzfeldt-Jakob disease occur in this region of the prion protein and it has been related to the conversion of PrP^C into the toxic PrP^Sc. Moreover, some strains of PrP resistant to conversion to PrP^Sc have been found to bear mutations in helix C, and positions 214 and 218 have been shown to modulate PrP^Sc formation [72]. It is also important to note that the main structural differences between prion proteins from different species have been found at the end of helix C [71].

The central region of PrPC linking the unstructured N-terminal part with the globular C-terminal domain is believed to play a pivotal role in the PrP^C conformational changes. Extensive studies on the secondary structure and fibrillogenic properties of synthetic peptides of PrP have established that the continuous segment of the prion protein spanning residues 106–147, coincident with the second "hot spot" predicted using our approach, is important for the fibrillogenic properties of the protein [73]. One of the synthetic peptides, that named PrP106-126 within the central region of PrP and near the N-terminal of the protease resistant core of PrP^Sc, shares many properties with the infectious form as it readily forms amyloid fibrils with a high β-sheet content, shows partial proteinase K resistance and is neurotoxic in vivo [74]. The neurotoxicity of PrP106-126 depends on the expression of endogenous PrP^C which makes PrP106-126 a relevant model for PrP^Sc neurotoxicity [74]. Also another prion derived peptide – PrP118-135 – has been found to cause neuronal death via induction of apoptosis [75]. The toxicity of PrP118-135 is, however, independent of endogenous PrP^C expression. Both peptides map in our predicted central aggregation-prone region of PrP^C.