Sequence specificity of amylin-insulin interaction: a fragment-based insulin fibrillation inhibition study

Subcutaneous insulin delivery serves as the major treatment for the ever-increasing spread of type II diabetes worldwide. However, long-term exposure to insulin results in local aggregates at the site of injection. This therapeutic concern accentuates the need to develop newer effective excipients to stabilize the insulin in pharmaceutical formulations. The fact that in normal physiological conditions, insulin interacts with the amylin hormone co-secreted from the pancreas, we targeted a peptide-mimetic approach based on the amylin sequence. The amylin-fibrillating core (NL6- N²²FGAIL²⁷ from the human Islet Amyloid Poly-Peptide) and its derivative NFGAXL (NL6X, X = 2-aminobenzoic acid) were used as potential inhibitory peptides against insulin amyloidogenesis. The fibrillation kinetics in the presence of the inhibitors was studied using an array of biophysical and microscopic techniques. High-resolution NMR spectroscopy enabled probing of the inhibitory interaction at an atomic resolution. Our results highlight the potential of using the naturally evolved NL6 peptide as an effective inhibitor against insulin fibrillation.     

Effect of sodium tetrathionate on amyloid fibril: Insight into the role of disulfide bond in amyloid progression

Tissue deposition of fibrillar protein aggregates called amyloid is the root cause of several degenerative diseases. Thus identification of compounds which can prevent or reduce protein aggregation can serve as a potential therapeutic target. In the present study we have shown inhibitory effect of sodium tetrathionate toward Hen egg white lysozyme (HEWL) amyloidogenesis at pH 2.0. Our study reveals that without sulfonation, sodium tetrathionate prevents amyloid fibril progression. Moreover, it shows that formation of disulfide bonds rather than exposure of hydrophobic surface in protein plays a critical role in initiating fibrillation process. Inhibitory effect of reducing agent β-mercaptoethanol toward fibrillation process also confirms the involvement of disulfide bond in initiating HEWL amyloidogenesis. These results provide important information toward understanding key interactions that guide amyloidogenesis, which may facilitate development of potential therapeutics.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

Identification of regions responsible for heparin-induced amyloidogenesis of human serum amyloid A using its fragment peptides

Human serum amyloid A (SAA) is a precursor protein of amyloid fibrils. Although several studies have been performed, a detailed understanding of the molecular mechanism for SAA fibrillation remains elusive. Glycosaminoglycans such as heparin are suggested to serve as scaffolds in amyloid fibril formation in some cases. In the present study, amyloidogenic properties of synthetic fragment peptides corresponding to the N-terminal (residues 1-27), central (residues 43-63), and C-terminal (residues 77-104) regions of SAA molecule induced by heparin were examined using fluorescence, circular dichroism (CD), and electron microscopy. Fluorescence and CD measurements demonstrated that SAA (1-27) peptide is evidently involved in heparin-induced amyloidogenesis. Correspondingly, relatively minor changes in fluorescence and a quite different pattern in the CD spectrum were observed in SAA (43-63) peptide. In contrast, SAA (77-104) peptide did not show any changes induced by heparin. Transmission electron microscopy indicated that SAA (1-27) peptide forms short and straight fibrils, whereas SAA (43-63) peptide forms much longer and seemingly elastic fibrils. These results suggest that the N-terminal region plays a crucial role as a rigid core and the central region facilitates the elongation of fibrils in heparin-induced amyloidogenesis of SAA molecule.     

Impact of membrane curvature on amyloid aggregation

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs.We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-β peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic β cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed.     

Inhibition of amyloid fibril formation and disassembly of pre-formed fibrils by natural polyphenol rottlerin

Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aβ_1–40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of β-sheets. The apparent IC₅₀ and DC₅₀ values were calculated in the range of 1.3–36.4 μM and 15.6–25.8 μM, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aβ_1–40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50 μM. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation.     

IAPP in type II diabetes: Basic research on structure,molecular interactions,and disease mechanisms suggests potential intervention strategies

Islet amyloid polypeptide (a.k.a. IAPP, amylin) is a 37 amino acid hormone that has long been associated with the progression of type II diabetes mellitus (TIIDM) disease. The endocrine peptide hormone aggregatively misfolds to form amyloid deposits in and around the pancreatic islet β-cells that synthesize both insulin and IAPP, leading to a decrease in β-cell mass in patients with the disease. Extracellular IAPP amyloids induce β-cell death through the formation of reactive oxygen species, mitochondrial dysfunction, chromatin condensation, and apoptotic mechanisms, although the precise roles of IAPP in TIIDM are yet to be established. Here we review aspects of the normal physiological function of IAPP in glucose regulation together with insulin, and its misfolding which contributes to TIIDM, and may also play roles in other pathologies such as Alzheimer's and heart disease. We summarize information on expression of the IAPP gene, the regulation of the hormone by post-translational modifications, the structural properties of the peptide in various states, the kinetics of misfolding to amyloid fibrils, and the interactions of the peptide with insulin, membranes, glycosaminoglycans, and nanoparticles. Finally, we describe how basic research is starting to have a positive impact on the development of approaches to circumvent IAPP amyloidogenesis. These include therapeutic strategies aimed at stabilizing non-amyloidogenic states, inhibition of amyloid growth or disruption of amyloid fibrils, antibodies directed towards amyloid structures, and inhibition of interactions with cofactors that facilitate aggregation or stabilize amyloids.     

Acceleration of alpha-synuclein aggregation by homologous peptides

alpha-Synuclein (alpha-Syn), amyloid beta-protein and prion protein are among the amyloidogenic proteins that are associated with the neurodegenerative diseases. These three proteins share a homologous region with a consensus sequence mainly consisting of glycine, alanine and valine residues (accordingly named as the GAV motif), which was proposed to be the critical core for the fibrillization and cytotoxicity. To understand the role of the GAV motif in protein amyloidogenesis, we studied the effects of the homologous peptides corresponding to the sequence of GAV motif region (residues 66-74) on alpha-Syn aggregation. The result shows that these peptides can promote fibrillization of wild-type alpha-Syn and induce that of the charge-incorporated mutants but not the GAV-deficient alpha-Syn mutant. The acceleration of alpha-Syn aggregation by the homologous peptides is under a sequence-specific manner. The interplay between the GAV peptide and the core regions in alpha-Syn may accelerate the aggregation process and stabilize the fibrils. This finding provides clues for developing peptide mimics that could promote transforming the toxic oligomers or protofibrils into the inert mature fibrils.     

Heparin Oligosaccharides that Pass the Blood-Brain Barrier Inhibit β-Amyloid Precursor Protein Secretion and Heparin Binding to β-Amyloid Peptide

Abstract: We have previously demonstrated that full-length heparin stimulates the synthesis and secretion of β-amyloid precursor protein (APP) through an amyloidogenic pathway in neuroblastoma cells. In the present study, heparin was chemically depolymerized, and the effect of low-molecular-weight (LMW) heparin on APP secretion was investigated. In contrast to full-length heparin, LMW heparin had no significant effect on APP secretion. However, LMW heparin fragments, especially heparin disaccharides, were able to inhibit efficiently the stimulatory effect of heparin on APP secretion. LMW heparin derivatives also inhibit the binding of heparin to the β-amyloid peptide (1–28). Using an in vitro model, we further demonstrated the passage of LMW heparin derivatives through the blood-brain barrier. This study suggests that LMW heparin derivatives or analogues may be effective as therapeutic agents to prevent or slow the process of amyloidogenesis in Alzheimer's disease.     

Inhibition of insulin fibrillogenesis with targeted peptides

Under conditions of acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Aggregation of insulin leads to loss of activity and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin; these compounds generally suppress aggregation only at relatively high inhibitor concentrations. For example, effective inhibition of aggregation of 0.5 mM insulin required arginine concentrations of > or =100 mM. Here, we investigate a targeted approach toward inhibiting insulin aggregation. VEALYL, corresponding to residues B12-17 of full-length insulin, was identified as a short peptide that interacts with full-length insulin. A hybrid peptide was synthesized that contained this binding domain and hexameric arginine; this peptide significantly reduced the rate of insulin aggregation at near-equimolar concentrations. An effective binding domain and N-terminal placement of the arginine hexamer were necessary for inhibitory activity. The data were analyzed using a simple two-step model of aggregation kinetics. These results are useful not only in identifying an insulin aggregation inhibitor but also in extending a targeted protein strategy for modifying aggregation of amyloidogenic proteins.     

How do amino acid substitutions affect the amyloidogenic properties and seeding efficiency of prion peptides

The amino acid sequences in the amyloidogenic region (amino acids 108–144) of several mammalian prion proteins were compared and variations were found to occur at residues 109 (M or L), 112 (M or V), 129 (M, V, or L), 135 (N or S), 138 (M, L, or I), 139 (M or I), and 143 (N or S). Using the bovine PrP peptide (residues 108–144 based on the numbering of the human prion protein sequence) as a control peptide, several peptides with one amino acid differing from that of the bovine PrP peptide at residues 109, 112, 135, 138, 139, or 143 and several mammalian PrP peptides were synthesized, and the effects of these amino acid substitutions on the amyloidogenic properties of these peptides were compared and discussed on the basis of the chemical and structural properties of amino acids. Our results showed that the V112M substitution accelerated nucleation of amyloidogenesis, while the N143S and I139M substitutions retarded nucleation. These effects tended to cancel each other out when two substitutions with opposite effects were present on the same peptide. Moreover, acceleration or inhibition of nucleation was not necessarily correlated with effect on seeding efficiency. Using amyloid fibrils prepared from the bovine PrP peptide as seeds, the seeding efficiency for the monomer peptides with the M129L, S135N, N143S, or I139M substitution was decreased compared to that for bPrP peptide. Of all the mammalian peptides used in this study, the dog, mule deer, and pig PrP peptides had the lowest seeding efficiencies.     

Tuning amyloidogenic conformations through cosolvents and hydrostatic pressure: when the soft matter becomes even softer

Compact packing, burial of hydrophobic side-chains, and low free energy levels of folded conformations contribute to stability of native proteins. Essentially, the same factors are implicated in an even higher stability of mature amyloid fibrils. Although both native insulin and insulin amyloid are resistant to high pressure and influence of cosolvents, intermediate aggregation-prone conformations are susceptible to either condition. Consequently, insulin fibrillation may be tuned under hydrostatic pressure or-- through cosolvents and cosolutes-- by preferential exclusion or binding. Paradoxically, under high pressure, which generally disfavors aggregation of insulin, an alternative "low-volume" aggregation pathway, which leads to unique circular amyloid is permitted. Likewise, cosolvents are capable of preventing, or altering amyloidogenesis of insulin. As a result of cosolvent-induced perturbation, distinct conformational variants of fibrils are formed. Such variants, when used as templates for seeding daughter generations, reproduce initial folding patterns regardless of environmental biases. By the close analogy, this suggests that the "prion strains" phenomenon may mirror a generic, common feature in amyloids. The susceptibility of amyloidogenic conformations to pressure and cosolvents is likely to arise from their "frustration", as unfolding results in less-densely packed side-chains, void volumes, and exposure of hydrophobic groups. The effects of cosolvents and pressure are discussed in the context of studies on other amyloidogenic protein models, amyloid polymorphism, and "strains".     

(−)-Epigallocatechin-3-Gallate (EGCG) Maintains κ-Casein in Its Pre-Fibrillar State without Redirecting Its Aggregation Pathway

The polyphenol (−)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-β, α-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. Whether this anti-fibril activity is specific to these disease-related target proteins or is more generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMκ-CN (reduced and carboxymethylated κ-casein) and thereby protect pheochromocytoma-12 cells from RCMκ-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMκ-CN [the IC₅₀ for 50 μM RCMκ-CN is 13 ± 1 μM]. Biophysical studies reveal that EGCG prevents RCMκ-CN fibril formation by stabilising RCMκ-CN in its native-like state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMκ-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent π-π stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated.     

Flavonoid-mediated presenilin-1 phosphorylation reduces Alzheimer's disease beta-amyloid production

Glycogen synthase kinase 3 (GSK-3) dysregulation is implicated in the two Alzheimer's disease (AD) pathological hallmarks: β-amyloid plaques and neurofibrillary tangles. GSK-3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic γ-secretase cleavage of amyloid precursor protein (APP). Here, we report that the citrus bioflavonoid luteolin reduces amyloid-β (Aβ) peptide generation in both human 'Swedish' mutant APP transgene-bearing neuron-like cells and primary neurons. We also find that luteolin induces changes consistent with GSK-3 inhibition that ( i ) decrease amyloidogenic γ-secretase APP processing, and ( ii ) promote presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Importantly, we find GSK-3α activity is essential for both PS1 CTF phosphorylation and PS1-APP interaction. As validation of these findings in vivo , we find that luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Aβ levels, reduces GSK-3 activity, and disrupts PS1-APP association. In addition, we find that Tg2576 mice treated with diosmin, a glycoside of a flavonoid structurally similar to luteolin, display significantly reduced Aβ pathology. We suggest that GSK-3 inhibition is a viable therapeutic approach for AD by impacting PS1 phosphorylation-dependent regulation of amyloidogenesis.     

Exploring critical determinants of protein amyloidogenesis: a review

The transformation of polypeptide chains from their globular native structure to fibrillar aggregates has been a matter of great concern because of the involvement of these aggregates in the onset of several degenerative diseases. These aggregates exhibit highly ordered cross β sheet structures and are known as ‘amyloids’. Formation of amyloids in the body is associated with cytotoxicity due to direct interaction of the aggregated species with the cell membrane leading to cellular permeability or due to loss of functionality of the proteins involved in the amyloid formation. The preference of polypeptide chains to remain in their native conformation or to aggregate into amyloids is guided by several factors such as its conformation at specific condition, concentration, physicochemical properties of the amino acid sequence and so on. In the current review, we have reviewed the different factors that guide the transition of proteins from their natively folded state to the amyloidogenic state. Understanding the critical determinants of amyloidogenesis is vital towards deciphering the molecular mechanism of amyloidogenesis and for the development of effective therapeutics against amyloidosis. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Beta-hairpin conformation of fibrillogenic peptides: structure and alpha-beta transition mechanism revealed by molecular dynamics simulations

Understanding the conformational transitions that trigger the aggregation and amyloidogenesis of otherwise soluble peptides at atomic resolution is of fundamental relevance for the design of effective therapeutic agents against amyloid-related disorders. In the present study the transition from ideal alpha-helical to beta-hairpin conformations is revealed by long timescale molecular dynamics simulations in explicit water solvent, for two well-known amyloidogenic peptides: the H1 peptide from prion protein and the Abeta(12-28) fragment from the Abeta(1-42) peptide responsible for Alzheimer's disease. The simulations highlight the unfolding of alpha-helices, followed by the formation of bent conformations and a final convergence to ordered in register beta-hairpin conformations. The beta-hairpins observed, despite different sequences, exhibit a common dynamic behavior and the presence of a peculiar pattern of the hydrophobic side-chains, in particular in the region of the turns. These observations hint at a possible common aggregation mechanism for the onset of different amyloid diseases and a common mechanism in the transition to the beta-hairpin structures. Furthermore the simulations presented herein evidence the stabilization of the alpha-helical conformations induced by the presence of an organic fluorinated cosolvent. The results of MD simulation in 2,2,2-trifluoroethanol (TFE)/water mixture provide further evidence that the peptide coating effect of TFE molecules is responsible for the stabilization of the soluble helical conformation.     

BIGH3 (TGFBI) Arg124 mutations influence the amyloid conversion of related peptides in vitro.

Amyloid deposits with Arg124 mutated TGFBI protein have been identified in autosomal dominant blinding corneal dystrophies. We assessed in vitro the mechanisms determining TGFBI protein amyloid transformation involving mutations of Arg124. Eight peptides synthesized following the TGFBI protein sequence, centered on codon Arg124 holding the previously reported amyloidogenic mutations and the respective controls were studied. Cys124 and His124 mutated peptide preparations contained significantly higher amounts of amyloid than the native peptide. Blocking the SH group of Cys124 and deleting the first four NH2-terminal amino acids including Val112-Val113 resulted in a decrease in amyloid fibril formation while deletion of the nine CONH2-terminal residues increased amyloid fibril concentration. Fourrier transformed-infrared spectroscopy analysis of the different peptide solutions showed an increase in beta-pleated sheet structures in those with enhanced amyloid yielding. We designed a peptide (BB1) likely to counteract the role of Val112-Val113 in amyloid fibril formation. Incubation of Cys124 peptide with BB1 indeed resulted in a 35% inhibition of amyloid fibril formation. Our results are in keeping with the clinical observations of Arg124 mutation-linked amyloidosis and show the importance of Val112-Val113, disulfide and hydrogen bonding in increasing the beta-pleated conformation and amyloid formation. These findings shed new light on the molecular mechanisms of TGFBI protein amyloidogenesis and encourage further research on the use of specifically designed peptides as putative therapeutic agents for these disabling diseases.     

Proinsulin C-peptide interferes with insulin fibril formation

Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrils formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic β-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.     

Structural insights into the role of mutations in amyloidogenesis

Mechanisms of amyloidogenesis are not well understood, including potential structural contributions of mutations in the process. Our previous research indicated that the dimer interface of amyloidogenic immunoglobulin light chain protein AL-09 is twisted 90 degrees relative to the protein from its germline sequence, kappaI O18/O8. Here we report a systematic restoration of AL-09 to its germline sequence by mutating the non-conservative somatic mutations located in the light chain dimer interface. Among these mutants, we find a correlation between increased thermodynamic stability and an increase in the lag time for fibril formation. The restorative mutant AL-09 H87Y completes the trifecta and restores the dimer interface observed in kappaI O18/O8, emphasizing the potential importance of the structural integrity of these proteins to protect against amyloidogenicity. We also find that adding amyloidogenic mutations into the germline protein illustrates mutational cooperativity in promoting amyloidogenesis.     

Transthyretin amyloidosis: a tale of weak interactions.

Over 70 transthyretin (TTR) mutations have been associated with hereditary amyloidoses, which are all autosomal dominant disorders with adult age of onset. TTR is the main constituent of amyloid that deposits preferentially in peripheral nerve giving rise to familial amyloid polyneuropathy (FAP), or in the heart leading to familial amyloid cardiomyopathy. Since the beginning of this decade the central question of these types of amyloidoses has been why TTR is an amyloidogenic protein with clinically heterogeneous pathogenic consequences. As a result of amino acid substitutions, conformational changes occur in the molecule, leading to weaker subunit interactions of the tetrameric structure as revealed by X-ray studies of some amyloidogenic mutants. Modified soluble tetramers exposing cryptic epitopes seem to circulate in FAP patients as evidenced by antibody probes recognizing specifically TTR amyloid fibrils, but what triggers dissociation into monomeric and oligomeric intermediates of amyloid fibrils is largely unknown. Avoiding tetramer dissociation and disrupting amyloid fibrils are possible avenues of therapeutic intervention based on current molecular knowledge of TTR amyloidogenesis and fibril structure.