Elongation dynamics of amyloid fibrils: a rugged energy landscape picture

Protein amyloid fibrils are a form of linear protein aggregates that are implicated in many neurodegenerative diseases. Here, we study the dynamics of amyloid fibril elongation by performing Langevin dynamic simulations on a coarse-grained model of peptides. Our simulation results suggest that the elongation process is dominated by a series of local minimum due to frustration in monomer-fibril interactions. This rugged energy landscape picture indicates that the amount of recycling of monomers at the fibrils' ends before being fibrilized is substantially reduced in comparison to the conventional two-step elongation model. This picture, along with other predictions discussed, can be tested with current experimental techniques

Reverse immunodynamics : a new method for identifying targets of protective immunity

Despite a dramatic increase in our ability to catalogue variation among pathogen genomes, we have made far fewer advances in using this information to identify targets of protective immunity. Epidemiological models predict that strong immune selection can cause antigenic variants to structure into genetically discordant sets of antigenic types (e.g. serotypes). A corollary of this theory is that targets of immunity may be identified by searching for non-overlapping associations of amino acids among co-circulating antigenic variants. We propose a novel population genetics methodology that combines such predictions with phylogenetic analyses to identify genetic loci (epitopes) under strong immune selection. We apply this concept to the AMA-1 protein of the malaria parasite Plasmodium falciparum and find evidence of epitopes among certain regions of low variability which could render them ideal vaccine candidates. The proposed method can be applied to a myriad of multi-strain pathogens for which vast amounts of genetic data has been collected in recent years

Structural Elements Regulating Amyloidogenesis: A Cholinesterase Model System

Polymerization into amyloid fibrils is a crucial step in the pathogenesis of neurodegenerative syndromes. Amyloid assembly is governed by properties of the sequence backbone and specific side-chain interactions, since fibrils from unrelated sequences possess similar structures and morphologies. Therefore, characterization of the structural determinants driving amyloid aggregation is of fundamental importance. We investigated the forces involved in the amyloid assembly of a model peptide derived from the oligomerization domain of acetylcholinesterase (AChE), AChE586-599, through the effect of single point mutations on β-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE586-599 was chosen due to its fibrilization tractability and AChE involvement in Alzheimer's disease. The results revealed how specific regions and residues can control AChE586-599 assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high β-strand propensity, for the conformational transition to β-sheet, and for the first stage of aggregation. We also demonstrated that positively charged side-chains might be involved in electrostatic interactions, which could control the transition to β-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE586-599 surfactant activity and that amyloid assembly might preferentially occur at an air-water interface. Consistently with the experimental observations and assembly models for other amyloid systems, we propose a model for AChE586-599 assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-π, SH-aromatic, metal chelation and polar-polar) would maintain the β-sheets together. We also propose that the stacking between the strands in the β-sheets along the fiber axis could be stabilized through π-π interactions and metal chelation. The dissection of the specific molecular recognition driving AChE586-599 amyloid assembly has provided further knowledge on such poorly understood and complicated process, which could be applied to protein folding and the targeting of amyloid diseases

Heterologous Amyloid Seeding: Revisiting the Role of Acetylcholinesterase in Alzheimer's Disease

Neurodegenerative diseases associated with abnormal protein folding and ordered aggregation require an initial trigger which may be infectious, inherited, post-inflammatory or idiopathic. Proteolytic cleavage to generate vulnerable precursors, such as amyloid-β peptide (Aβ) production via β and γ secretases in Alzheimer's Disease (AD), is one such trigger, but the proteolytic removal of these fragments is also aetiologically important. The levels of Aβ in the central nervous system are regulated by several catabolic proteases, including insulysin (IDE) and neprilysin (NEP). The known association of human acetylcholinesterase (hAChE) with pathological aggregates in AD together with its ability to increase Aβ fibrilization prompted us to search for proteolytic triggers that could enhance this process. The hAChE C-terminal domain (T40, AChE575-614) is an exposed amphiphilic α-helix involved in enzyme oligomerisation, but it also contains a conformational switch region (CSR) with high propensity for conversion to non-native (hidden) β-strand, a property associated with amyloidogenicity. A synthetic peptide (AChE586-599) encompassing the CSR region shares homology with Aβ and forms β-sheet amyloid fibrils. We investigated the influence of IDE and NEP proteolysis on the formation and degradation of relevant hAChE β-sheet species. By combining reverse-phase HPLC and mass spectrometry, we established that the enzyme digestion profiles on T40 versus AChE586-599, or versus Aβ, differed. Moreover, IDE digestion of T40 triggered the conformational switch from α- to β-structures, resulting in surfactant CSR species that self-assembled into amyloid fibril precursors (oligomers). Crucially, these CSR species significantly increased Aβ fibril formation both by seeding the energetically unfavorable formation of amyloid nuclei and by enhancing the rate of amyloid elongation. Hence, these results may offer an explanation for observations that implicate hAChE in the extent of Aβ deposition in the brain. Furthermore, this process of heterologous amyloid seeding by a proteolytic fragment from another protein may represent a previously underestimated pathological trigger, implying that the abundance of the major amyloidogenic species (Aβ in AD, for example) may not be the only important factor in neurodegeneration

Unique insertions within Plasmodium falciparum subtilisin-like protease-1 are crucial for enzyme maturation and activity.

Parasite serine proteases play essential roles in the asexual erythrocytic life cycle of the malaria parasite. The timing and location of expression of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) are consistent with a role in erythrocyte invasion. Maturation of PfSUB-1 involves two autocatalytic processing events in which an 82 kDa precursor is converted to a 54 kDa form, followed by further cleavage to produce a 47 kDa form. Here we have compared PfSUB-1 with a number of Plasmodium orthologues and the most closely related bacterial subtilase sequences and find that, like many malarial proteins, PfSUB-1 possesses both low and high complexity insertions. The latter take the form of six surface-associated strands or loops which are conserved in all SUB-1 orthologues but not present in any other subtilase. Several mutants of PfSUB-1 with deletions of all, or part, of each of the six loop insertions were produced in an insect cell expression system. Aside from loop III, which was dispensable, individual deletion of the loop insertions revealed a role in protein maturation and/or stability. Specific substitutions within loop II inhibited maturation and enzyme activity. Mutations in loops V and VI specifically inhibited the second step of autocatalytic maturation providing evidence that the two processing steps have distinct structural requirements and that conversion to p47 is not a prerequisite for proteolytic activity in trans

Structure of Pb²⁺/dCMP and Pb²⁺/CMP complexes as characterized by tandem mass spectrometry and IRMPD spectroscopy

International audienceThe structure of Pb2+/cytidine-5′-monophosphate (CMP) and Pb2+/deoxycytidine-5′-monophosphate (dCMP) complexes were probed in the gas phase by combining electrospray ionization, tandem mass spectrometry and mid-infrared multiple-photon dissociation (IRMPD) spectroscopy. The fragment ions detected upon collision suggest that the metal interacts with both the phosphate group and the cytosine. This finding is further confirmed by IRMPD spectroscopy. The IRMPD spectrum of the ESI-generated [Pb(dCMP)−H]+ and [Pb(CMP)−H]+ complexes is indeed in very good agreement with DFT-computed infrared absorption spectra of macrochelate forms, in which the Pb2+ ion not only interacts with the phosphate group but also with the carbonyl group of the nucleobase moiety, by folding of the mononucleotides. The structures thus characterized therefore differ from those proposed in solution and deduced from potentiometric studies. Our data also suggest that losing the nucleobase residue under CID conditions does not necessarily mean a lack of interaction between the metal and the nucleobase moiety, as commonly mentioned in the literature for large oligonucleotides

Dynamics of the formation of a hydrogel by a pathogenic amyloid peptide: islet amyloid polypeptide

Many chronic degenerative diseases result from aggregation of misfolded polypeptides to form amyloids. Many amyloidogenic polypeptides are surfactants and their assembly can be catalysed by hydrophobic-hydrophilic interfaces (an air-water interface in-vitro or membranes in-vivo). We recently demonstrated the specificity of surface-induced amyloidogenesis but the mechanisms of amyloidogenesis and more specifically of adsorption at hydrophobic-hydrophilic interfaces remain poorly understood. Thus, it is critical to determine how amyloidogenic polypeptides behave at interfaces. Here we used surface tensiometry, rheology and electron microscopy to demonstrate the complex dynamics of gelation by full-length human islet amyloid polypeptide (involved in type II diabetes) both in the bulk solution and at hydrophobic-hydrophilic interfaces (air-water interface and phospholipids). We show that the hydrogel consists of a 3D supramolecular network of fibrils. We also assessed the role of solvation and dissected the evolution over time of the assembly processes. Amyloid gelation could have important pathological consequences for membrane integrity and cellular functions

Formation of amyloid oligomers by AChE_586-599 and AChE_586-599 mutants.

<p>Oligomers of AChE_586-599 and AChE_586-599 mutants (12 µM) were cross-linked by photo-induced cross-linking. Cross-linked products were resolved (16.5% Tris-Tricine SDS-PAGE), electro-blotted onto nitrocellulose and probed with Mab 105A (specific for AChE_586-599 in β-sheet conformation). Marker proteins are indicated. Arrows indicate low abundance oligomeric species. Due to the strength of the signal for the oligomeric species, Y₉/A was loaded at a third of the amount of the other peptides (Y₉/A*). The signal resulting from loading equal amount to the other peptides can be seen on the individual lane on the right hand side of the top panel (Y₉/A). On the right hand side of the bottom panel, an overexposure of the signal for W₁₃/A shows multiple oligomeric species not seen at normal exposure.</p