Host responses and metabolic profiles of wood components in Dutch elm hybrids with a contrasting tolerance to Dutch elm disease

Background and Aims

Changes occurring in the macromolecular traits of cell wall components in elm wood following attack by Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), are poorly understood. The purpose of this study was to compare host responses and the metabolic profiles of wood components for two Dutch elm (Ulmus) hybrids, ‘Groeneveld’ (a susceptible clone) and ‘Dodoens’ (a tolerant clone), that have contrasting survival strategies upon infection with the current prevalent strain of DED.

Methods

Ten-year-old plants of the hybrid elms were inoculated with O. novo-ulmi ssp. americana × novo-ulmi. Measurements were made of the content of main cell wall components and extractives, lignin monomer composition, macromolecular traits of cellulose and neutral saccharide composition.

Key Results

Upon infection, medium molecular weight macromolecules of cellulose were degraded in both the susceptible and tolerant elm hybrids, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels, but rarely in the fibres. The ¹³C nuclear magnetic resonance spectra revealed that loss of crystalline and non-crystalline cellulose regions occurred in parallel. The rate of cellulose degradation was influenced by the syringyl:guaiacyl ratio in lignin. Both hybrids commonly responded to the medium molecular weight cellulose degradation with the biosynthesis of high molecular weight macromolecules of cellulose, resulting in a significant increase in values for the degree of polymerization and polydispersity. Other responses of the hybrids included an increase in lignin content, a decrease in relative proportions of d-glucose, and an increase in proportions of d-xylose. Differential responses between the hybrids were found in the syringyl:guaiacyl ratio in lignin.

Conclusions

In susceptible ‘Groeneveld’ plants, syringyl-rich lignin provided a far greater degree of protection from cellulose degradation than in ‘Dodoens’, but only guaiacyl-rich lignin in ‘Dodoens’ plants was involved in successful defence against the fungus. This finding was confirmed by the associations of vanillin and vanillic acid with the DED-tolerant ‘Dodoens’ plants in a multivariate analysis of wood traits.     

Leaf trait co-ordination in relation to construction cost, carbon gain and resource-use efficiency in exotic invasive and native woody vine species

Background and Aims

Success of invasive plant species is thought to be linked with their higher leaf carbon fixation strategy, enabling them to capture and utilize resources better than native species, and thus pre-empt and maintain space. However, these traits are not well-defined for invasive woody vines.

Methods

In a glass house setting, experiments were conducted to examine how leaf carbon gain strategies differ between non-indigenous invasive and native woody vines of south-eastern Australia, by investigating their biomass gain, leaf structural, nutrient and physiological traits under changing light and moisture regimes.

Key Results

Leaf construction cost (CC), calorific value and carbon : nitrogen (C : N) ratio were lower in the invasive group, while ash content, N, maximum photosynthesis, light-use efficiency, photosynthetic energy-use efficiency (PEUE) and specific leaf area (SLA) were higher in this group relative to the native group. Trait plasticity, relative growth rate (RGR), photosynthetic nitrogen-use efficiency and water-use efficiency did not differ significantly between the groups. However, across light resource, regression analyses indicated that at a common (same) leaf CC and PEUE, a higher biomass RGR resulted for the invasive group; also at a common SLA, a lower CC but higher N resulted for the invasive group. Overall, trait co-ordination (using pair-wise correlation analyses) was better in the invasive group. Ordination using 16 leaf traits indicated that the major axis of invasive-native dichotomy is primarily driven by SLA and CC (including its components and/or derivative of PEUE) and was significantly linked with RGR.

Conclusions

These results demonstrated that while not all measures of leaf resource traits may differ between the two groups, the higher level of trait correlation and higher revenue returned (RGR) per unit of major resource need (CC) and use (PEUE) in the invasive group is in line with their rapid spread where introduced.     

Thermal tolerance,net CO2 exchange and growth of a tropical tree species, Ficus insipida, cultivated at elevated daytime and nighttime temperatures

Global warming and associated increases in the frequency and amplitude of extreme weather events, such as heat waves, may adversely affect tropical rainforest plants via significantly increased tissue temperatures. In this study, the response to two temperature regimes was assessed in seedlings of the neotropical pioneer tree species, Ficus insipida. Plants were cultivated in growth chambers at strongly elevated daytime temperature (39 °C), combined with either close to natural (22 °C) or elevated (32 °C) nighttime temperatures. Under both growth regimes, the critical temperature for irreversible leaf damage, determined by changes in chlorophyll a fluorescence, was approximately 51 °C. This is comparable to values found in F. insipida growing under natural ambient conditions and indicates a limited potential for heat tolerance acclimation of this tropical forest tree species. Yet, under high nighttime temperature, growth was strongly enhanced, accompanied by increased rates of net photosynthetic CO₂ uptake and diminished temperature dependence of leaf-level dark respiration, consistent with thermal acclimation of these key physiological parameters.     

Early responses to cadmium of two poplar clones that differ in stress tolerance

Soil cadmium (Cd) contamination is becoming a matter of great global concern. The identification of plants differentially sensitive to Cd excess is of interest for the selection of genotype adaptive to grow and develop in polluted areas and capable of ameliorating or reducing the negative environmental effects of this toxic metal. The two poplar clones I-214 (Populus × canadensis) and Eridano (Populus deltoides × maximowiczii) are, respectively, tolerant and sensitive to ozone (O₃) exposure. Because stress tolerance is mediated by an array of overlapping defence mechanisms, we tested the hypothesis that these two clones differently sensitive to O₃ stress factor also exhibit different tolerance to Cd. With this purpose, an outdoor pot experiment was designed to study the responses of I-214 and Eridano to the distribution of different Cd solutions enriched with CdCl₂ (0, 50 and 150 μM) for 35 days. Changes in leaf area, biomass allocation and Cd uptake, photosynthesis, chlorophyll fluorescence, leaf concentration of nutrients and pigments, hydrogen peroxide (H₂O₂) and nitric oxide (NO) production and thiol compounds were investigated. The two poplar clones showed similar sensitivity to excess Cd in terms of biomass production, photosynthesis activity and Cd accumulation, though physiological and biochemical traits revealed different defence strategies. In particular, Eridano maintained in any Cd treatment the number of its constitutively wider blade leaves, while the number of I-214 leaves (with lower size) was reduced. H₂O₂ increased 4.5- and 13-fold in I-214 leaves after the lowest (L) and highest (H) Cd treatments, respectively, revealing the induction of oxidative burst. NO, constitutively higher in I-214 than Eridano, progressively increased in both clones with the enhancement of Cd concentration in the substrate. I-214 showed a more elevated antioxidative capacity (GSH/GSSG) and higher photochemical efficiency of PSII (F_v/F_m) and de-epoxidation degree of xantophylls-cycle (DEPS). The glutathione pool was not affected by Cd treatment in both clones, while non-protein thiols and phytochelatins were reduced at L Cd treatment in I-214. Overall, these two clones presented high adaptability to Cd stress and are both suitable to develop and growth in environments contaminated with this metal, thus being promising for their potential use in phytoremediation programmes.     

A radish seed antifungal peptide with a high amyloid fibril-forming propensity

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.     

Stomatal and Mesophyll Limitations to Photosynthesis in One Evergreen and One Deciduous Mediterranean Oak Species

In the evergreen Quercus rotundifolia and the co-existing deciduous Q. faginea we studied the diurnal variations in photosynthetic capacity (P _max), measured as the rate of O₂ evolution at photon and CO₂ saturation, and in the rate of net CO₂ assimilation (P _N) in the field during the period of maximum photosynthetic activity. Our aim was to check the contribution of stomatal and non-stomatal limitations to the diurnal variation in photosynthesis, and to study the differences between both species. Q. faginea leaves displayed lower mass per unit area and higher nitrogen content than Q. rotundifolia leaves. The maximum stomatal conductance and P _N in the field were higher in Q. faginea than in Q rotundifolia. Also P _max of Q. faginea was higher than that of Q. rotundifolia. Both species attained in the field a high percentage of the P _max (around 82 % for Q. faginea and 73 % for Q. rotundifolia). This indicates reduced stomatal limitation of photosynthesis under favourable conditions, especially in Q. faginea. P _N underwent a sharp decrease towards mid-day in association with increase in the atmospheric vapour pressure deficit and decrease in the leaf water potential. P _max was also reduced during mid-day. This demonstrated the contribution of mesophyll limitations to the P _N in the two species under stress. The mesophyll limitation of photosynthesis seemed to be similar for both species, independently from the differences in leaf traits between them.     

Structures formed by a cell membrane-associated arabinogalactan-protein on graphite or mica alone and with Yariv phenylglycosides

Background

Certain membrane-associated arabinogalactan-proteins (AGPs) with lysine-rich sub-domains participate in plant growth, development and resistance to stress. To complement fluorescence imaging of such molecules when tagged and introduced transgenically to the cell periphery and to extend the groundwork for assessing molecular structure, some behaviours of surface-spread AGPs were visualized at the nanometre scale in a simplified electrostatic environment.

Methods

Enhanced green fluorescent protein (EGFP)-labelled LeAGP1 was isolated from Arabidopsis thaliana leaves using antibody-coated magnetic beads, deposited on graphite or mica, and examined with atomic force microscopy (AFM).

Key Results

When deposited at low concentration on graphite, LeAGP can form independent clusters and rings a few nanometres in diameter, often defining deep pits; the aperture of the rings depends on plating parameters. On mica, intermediate and high concentrations, respectively, yielded lacy meshes and solid sheets that could dynamically evolve arcs, rings, ‘pores’ and ‘co-pores’, and pits. Glucosyl Yariv reagent combined with the AGP to make very large and distinctive rings.

Conclusions

Diverse cell-specific nano-patterns of native lysine-rich AGPs are expected at the wall–membrane interface and, while there will not be an identical patterning in different environmental settings, AFM imaging suggests protein tendencies for surficial organization and thus opens new avenues for experimentation. Nanopore formation with Yariv reagents suggests how the reagent might bind with AGP to admit Ca²⁺ to cells and hints at ways in which AGP might be structured at some cell surfaces.     

Cooperative unfolding of a metastable serpin to a molten globule suggests a link between functional and folding energy landscapes

Alpha-1 antitrypsin (alpha(1)-AT) is a member of the serpin class of protease inhibitors, and folds to a metastable state rather than its thermodynamically most stable native state. Upon cleavage by a target protease, alpha(1)-AT undergoes a dramatic conformational change to a stable form, translocating the bound protease more than 70 A to form an inhibitory protease-serpin complex. Numerous mutagenesis studies on serpins have demonstrated the trade-off between the stability of the metastable state on the one hand and the inhibitory efficiency on the other. Studies of the equilibrium unfolding of serpins provide insight into this connection between structural plasticity and metastability. We studied equilibrium unfolding of wild-type alpha(1)-AT using hydrogen-deuterium/exchange mass spectrometry to characterize the structure and the stability of an equilibrium intermediate that was observed in low concentrations of denaturant in earlier studies. Our results show that the intermediate observed at low concentrations of denaturant has no protection from hydrogen-deuterium exchange, indicating a lack of stable structure. Further, differential scanning calorimetry of alpha(1)-AT at low concentrations of denaturant shows no heat capacity peak during thermal denaturation, indicating that the transition from the intermediate to the unfolded state is not a cooperative first-order-like phase transition.. Our results show that the unfolding of alpha(1)-AT involves a cooperative transition to a molten globule form, followed by a non-cooperative transition to a random-coil form as more guanidine is added. Thus, the entire alpha(1)-AT molecule consists of one cooperative structural unit rather than multiple structural domains with different stabilities. Furthermore, our results together with previous mutagenesis studies suggest a possible link between an equilibrium molten globule and a functional intermediate that may be populated during the protease inhibition.     

Differences in leaf proteome response to cold acclimation between Lolium perenne plants with distinct levels of frost tolerance

Perennial ryegrass (Lolium perenne) is a high quality forage and turf grass mainly due to its excellent nutritive values and rapid establishment rate. However, this species has limited ability to perform in harsh winter climates. Though winter hardiness is a complex trait, it is commonly agreed that frost tolerance (FT) is its main component. Species growing in temperate regions can acquire FT through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation (CA). The research on molecular basis of FT has been performed on the model plants, but they are not well adapted to extreme winter climates. Thus, the mechanisms of cell response to low temperature in winter crops and agronomically important perennial grasses have yet to be revealed. Here, two L. perenne plants with contrasting levels of FT, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on analyses of leaf protein accumulation before and after 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA, using a high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry (MS). Analyses of 580 protein profiles revealed a total of 42 (7.2%) spots that showed at a minimum of 1.5-fold differences in protein abundance, at a minimum of at one time point of CA between HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analyzed plants appeared most often on the 5th (18 proteins) and the 7th (19 proteins) day of CA. The proteins derived from 35 (83.3%) spots were successfully identified by the use of MS and chloroplast proteins were shown to be the major group selected as differentially accumulated during CA. The functions of the identified proteins and their probable influence on the level of FT in L. perenne are discussed.     

Interaction of discoidin domain receptor 1 with collagen type 1

Discoidin domain receptor 1 (DDR1) is a widely expressed tyrosine kinase receptor which binds to and gets activated by collagens including collagen type 1. Little is understood about the interaction of DDR1 with collagen and its possible functional implications. Here, we elucidate the binding pattern of the DDR1 extracellular domain (ECD) to collagen type 1 and its impact on collagen fibrillogenesis. Our in vitro assays utilized DDR1-Fc fusion proteins, which contain only the ECD of DDR1. Using surface plasmon resonance, we confirmed that further oligomerization of DDR1-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Single-molecule imaging by means of atomic force microscopy revealed that DDR1 oligomers bound at overlapping or adjacent collagen molecules and were nearly absent on isolated collagen molecules. Interaction of DDR1 oligomers with collagen was found to modulate collagen fibrillogenesis both in vitro and in cell-based assays. Collagen fibers formed in the presence of DDR1 had a larger average diameter, were more cross-linked and lacked the native banded structure. The presence of DDR1 ECD resulted in "locking" of collagen molecules in an incomplete fibrillar state both in vitro and on surfaces of cells overexpressing DDR1. Our results signify an important functional role of the DDR1 ECD, which occurs naturally in kinase-dead isoforms of DDR1 and as a shedded soluble protein. The modulation of collagen fibrillogenesis by the DDR1 ECD elucidates a novel mechanism of collagen regulation by DDR1.     

Stem extension and mechanical stability of Xanthium canadense grown in an open or in a dense stand

Background and Aims

Plants in open, uncrowded habitats typically have relatively short stems with many branches, whereas plants in crowded habitats grow taller and more slender at the expense of mechanical stability. There seems to be a trade-off between height growth and mechanical stability, and this study addresses how stand density influences stem extension and consequently plant safety margins against mechanical failure.

Methods

Xanthium canadense plants were grown either solitarily (S-plants) or in a dense stand (D-plants) until flowering. Internode dimensions and mechanical properties were measured at the metamer level, and the critical buckling height beyond which the plant elastically buckles under its own weight and the maximum lateral wind force the plant can withstand were calculated.

Key Results

Internodes were longer in D- than S-plants, but basal diameter did not differ significantly. Relative growth rates of internode length and diameter were negatively correlated to the volumetric solid fraction of the internode. Internode dry mass density was higher in S- than D-plants. Young''s modulus of elasticity and the breaking stress were higher in lower metamers, and in D- than in S-plants. Within a stand, however, both moduli were positively related to dry mass density. The buckling safety factor, a ratio of critical buckling height to actual height, was higher in S- than in D-plants. D-plants were found to be approaching the limiting value 1. Lateral wind force resistance was higher in S- than in D-plants, and increased with growth in S-plants.

Conclusions

Critical buckling height increased with height growth due mainly to an increase in stem stiffness and diameter and a reduction in crown/stem mass ratio. Lateral wind force resistance was enhanced due to increased tissue strength and diameter. The increase in tissue stiffness and strength with height growth plays a crucial role in maintaining a safety margin against mechanical failure in herbaceous species that lack the capacity for secondary growth.     

Intramolecular hydrogen bonding in articaine can be related to superior bone tissue penetration: a molecular dynamics study

Local anesthetics (LAs) are drugs that cause reversible loss of nociception during surgical procedures. Articaine is a commonly used LA in dentistry that has proven to be exceptionally effective in penetrating bone tissue and induce anesthesia on posterior teeth in maxilla and mandibula. In the present study, our aim was to gain a deeper understanding of the penetration of articaine through biological membranes by studying the interactions of articaine with a phospholipid membrane. Our approach involves Langmuir monolayer experiments combined with molecular dynamics simulations. Membrane permeability of LAs can be modulated by pH due to a titratable amine group with a pKa value close to physiological pH. A change in protonation state is thus known to act as a lipophilicity switch in LAs. Our study shows that articaine has an additional unique lipophilicity switch in its ability to form an intramolecular hydrogen bond. We suggest this intramolecular hydrogen bond as a novel and additional solvent-dependent mechanism for modulation of lipophilicity of articaine which may enhance its diffusion through membranes and connective tissue.     

Structure and binding mechanism of vascular endothelial cadherin: a divergent classical cadherin

Vascular endothelial cadherin (VE-cadherin), a divergent member of the type II classical cadherin family of cell adhesion proteins, mediates homophilic adhesion in the vascular endothelium. Previous investigations with a bacterially produced protein suggested that VE-cadherin forms cell surface trimers that bind between apposed cells to form hexamers. Here we report studies of mammalian-produced VE-cadherin ectodomains suggesting that, like other classical cadherins, VE-cadherin forms adhesive trans dimers between monomers located on opposing cell surfaces. Trimerization of the bacterially produced protein appears to be an artifact that arises from a lack of glycosylation. We also present the 2.1-Å-resolution crystal structure of the VE-cadherin EC1-2 adhesive region, which reveals homodimerization via the strand-swap mechanism common to classical cadherins. In common with type II cadherins, strand-swap binding involves two tryptophan anchor residues, but the adhesive interface resembles type I cadherins in that VE-cadherin does not form a large nonswapped hydrophobic surface. Thus, VE-cadherin is an outlier among classical cadherins, with characteristics of both type I and type II subfamilies.     

Isolation, Characterisation and Molecular Imaging of a High-Molecular-Weight Insect Biliprotein, a Member of the Hexameric Arylphorin Protein Family

The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI ∼ 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

The surface location of individual residues in a bacterial S-layer protein

Bacterial surface layer (S-layer) proteins self-assemble into large two-dimensional crystalline lattices that form the outermost cell-wall component of all archaea and many eubacteria. Despite being a large class of self-assembling proteins, little is known about their molecular architecture. We investigated the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 to identify residues located at the subunit-subunit interface and to determine the S-layer's topology. Twenty-three single cysteine mutants, which were previously mapped to the surface of the SbsB monomer, were subjected to a cross-linking screen using the photoactivatable, sulfhydryl-reactive reagent N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide. Gel electrophoretic analysis on the formation of cross-linked dimers indicated that 8 out of the 23 residues were located at the interface. In combination with surface accessibility data for the assembled protein, 10 residues were assigned to positions at the inner, cell-wall-facing lattice surface, while 5 residues were mapped to the outer, ambient-exposed lattice surface. In addition, the cross-linking screen identified six positions of intramolecular cross-linking within the assembled protein but not in the monomeric S-layer protein. Most likely, these intramolecular cross-links result from conformational changes upon self-assembly. The results are an important step toward the further structural elucidation of the S-layer protein via, for example, X-ray crystallography and cryo-electron microscopy. Our approach of identifying the surface location of residues is relevant to other planar supramolecular protein assemblies.     

Effects of the known pathogenic mutations on the aggregation pathway of the amyloidogenic peptide of apolipoprotein A-I

The 93-residue N-terminal fragment of apolipoprotein A-I (ApoA-I) is the major constituent of fibrils isolated from patients affected by the amyloidosis caused by ApoA-I mutations. We have prepared eight polypeptides corresponding to all the currently known amyloidogenic variants of the N-terminal region of ApoA-I, other than a truncation mutation, and investigated their aggregation kinetics and the associated structural modifications. All the variants adopted a monomeric highly disordered structure in solution at neutral pH, whereas acidification of the solution induced an unstable α-helical conformation and the subsequent aggregation into the cross-β structure aggregate. Two mutations (Δ70-72 and L90P) almost abrogated the lag phase of the aggregation process, three mutations (Δ60-71, L75P, and W50R) significantly accelerated the aggregation rate by 2- to 3-fold, while the remaining three variants (L64P, L60R, and G26R) were not significantly different from the wild type. Therefore, an increase in aggregation propensity cannot explain per se the mechanism of the disease for all the variants. Prediction of the protection factors for hydrogen exchange in the native state of full-length protein reveals, in almost all the variants, an expansion of the conformational fluctuations that could favour the proteolytic cleavage and the release of the amyloidogenic peptide. Such an event seems to be a necessary prerequisite for ApoA-I fibrillogenesis in vivo, but the observed increased aggregation propensity of certain variants can have a strong influence on the severity of the disease, such as an earlier onset and a faster progression.