Aβ Peptide Fibrillar Architectures Controlled by Conformational Constraints of the Monomer

Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided “dock and lock” mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1–40 Glu22Gly (Aβ40Arc) and wild type Aβ1–40 (Aβ40WT). The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ40WT is significantly impaired to acquire the fibrillar architecture of Aβ40Arc. A detailed characterisation demonstrated that Aβ40WT has a restricted ability to dock and isomerise with high binding affinity onto Aβ40Arc fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence

International audienceThe BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease

Molecular dissection of established and proposed members of the Op18/Stathmin family of tubulin binding proteins

My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis. To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis. To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex

Determination of the critical free concentration of Aβ required for fibril polymerisation.

<p>2 µM Aβ solution was injected for 30 s at a flow rate of 20 µl/min in PBS at 25°C. 100 µl of Aβ (0–400 nM) was then immediately injected. Measurements were carried out to determine the critical free concentrations for (A) Aβ^40WT monomers with Aβ^40WT fibrils, (B) Aβ^40Arc monomers with Aβ^40Arc fibrils, and (C) Aβ^40Arc monomers with Aβ^40WT fibrils.</p

AFM analysis of fibrils immobilised on a C1 chip.

<p>Mature fibrils were briefly sonicated prior to immobilisation on C1 chip followed by continuous polymerisation with free monomers until the total mass doubled. (A) Monomeric Aβ^40WT seeded with Aβ^40WT fibrils, (B) monomeric Aβ^40Arc seeded with Aβ^40Arc fibrils, and (C) monomeric Aβ^40Arc seeded with Aβ^40WT fibrils. Scale bar is 0.5 µm.</p

Solvent protection ratios for backbone amide protons as determined by quenched H/D exchange monitored by NMR spectroscopy.

<p>Protection is defined as the ratio of the observed signal intensity after a 24 h pre-incubation period in D₂O to the signal intensity in a completely protonated reference sample. Protection in the reference sample is defined as 100%. Circles correspond to residues with 0% protection and crosses to residues where exchange was too fast for detection. Pale grey bars indicate overlapping residues with ambiguously assigned protection ratios. Error bars indicate the experimental uncertainty given by the measurements. (A) Aβ^40Arc fibrils, (B) Aβ^40WT fibrils, and (C) Aβ^40Arc seeded with Aβ^40WT fibrils.</p

Competition between Aβ^40WT and Aβ^40Arc for polymerisation with Aβ^40Arc or Aβ^40WT fibrils.

<p>(A) Dose response for Aβ^40WT binding to Aβ^40Arc fibrils (filled squares) or Aβ^40WT fibrils (filled circles). A range of concentrations of Aβ^40WT were injected over Aβ^40Arc or Aβ^40WT fibrils in PBS at 25°C. The maximum response at the end of each injection was plotted against the concentration of Aβ^40WT. (B) Competition between Aβ^40WT and Aβ^40Arc for polymerisation to Aβ^40Arc fibrils. Aβ^40WT was injected for 30 s at a flow rate of 20 µl/min in PBS at 25°C. 5 µM Aβ^40Arc (filled circles), 5 µM Aβ^40Arc+10 µM Aβ^40WT (open triangles), 10 µM Aβ^40WT (open squares).</p

SPR study of fibril elongation.

<p>Pre-formed Aβ fibrils were immobilised on a CM5 chip and probed with 2 µM monomeric Aβ for 1 min at a flow rate of 20 µl/min in PBS at 25°C. (A) Monomeric Aβ^40WT seeded with Aβ^40WT fibrils, (B) monomeric Aβ^40Arc seeded with Aβ^40Arc fibrils, (C) monomeric Aβ^40WT seeded with Aβ^40Arc fibrils, and (D) monomeric Aβ^40Arc seeded with Aβ^40WT fibrils.</p