Amyloid Formation by the Pro-Inflammatory S100A8/A9 Proteins in the Ageing Prostate

BACKGROUND: The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer's disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon--prostate tissue remodelling in middle-aged and elderly men. METHODOLOGY/PRINCIPAL FINDINGS: By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions. CONCLUSIONS/SIGNIFICANCE: These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.Original Publication:Kiran Yanamandra, Oleg Alexeyev, Vladimir Zamotin, Vaibhav Srivastava, Andrei Shchukarev, Ann-Christin Brorsson, Gian Gaetano Tartaglia, Thomas Vogl, Rakez Kayed, Gunnar Wingsle, Jan Olsson, Christopher M Dobson, Anders Bergh, Fredrik Elgh and Ludmilla A Morozova-Roche, Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the ageing prostate., 2009, PloS one, (4), 5, e5562.http://dx.doi.org/10.1371/journal.pone.000556

Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study

Correction: Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study. PLoS ONE 9(1): 10.1371/annotation/dbb84118-9ada-43e4-8734-8f8322be1a59. doi: 10.1371/annotation/dbb84118-9ada-43e4-8734-8f8322be1a59Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.This work was financed by the Andalucía Government (grant FQM-02838), the Spanish Ministry of Science and Innovation (grant BIO2009-07317), and the European Regional Development Fund of the European Union. D. Ruzafa is recipient of a research fellowship from the F.P.U. program of the Spanish Ministry of Education. L. Varela is financed by the G.R.E.I.B. program of the University of Granada

A coarse-grained Monte Carlo approach to diffusion processes in metallic nanoparticles

A kinetic Monte Carlo approach on a coarse-grained lattice is developed for the simulation of surface diffusion processes of Ni, Pd and Au structures with diameters in the range of a few nanometers. Intensity information obtained via standard two-dimensional transmission electron microscopy imaging techniques is used to create three-dimensional structure models as input for a cellular automaton. A series of update rules based on reaction kinetics is defined to allow for a stepwise evolution in time with the aim to simulate surface diffusion phenomena such as Rayleigh breakup and surface wetting. The material flow, in our case represented by the hopping of discrete portions of metal on a given grid, is driven by the attempt to minimize the surface energy, which can be achieved by maximizing the number of filled neighbor cells

Quantifying stickiness: thermodynamic characterization of intramolecular domain interactions to guide the design of forster resonance energy transfer sensors

Item does not contain fulltextThe introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Forster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)-linker-enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (DeltaG0(o-c) = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange.linker.mCherry fusion proteins yielded a toolbox of new mOrange-mCherry combinations that allowed tuning of their intramolecular interaction from very weak (DeltaG0(o-c) = .0.39 kCal/mol) to relatively stable (DeltaG0(o-c) = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean-Citrine pair by mOrange.mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategies

Autoimmune responses to amyloid structures of A beta(25-35) peptide and human lysozyme in the serum of patients with progressive Alzheimer's disease

We have found an increased level of serum antibodies to the prefibrillar structures of both Abeta(25-35) peptide and human lysozyme in Alzheimer's disease (AD) patients compared to age-matched controls, indicating that autoimmunity is implicated in AD. In the serum of AD patients with a long-term duration (>15 years) the titer of serum antibodies to aggregates of Abeta(25-35) peptide increased by approximately 5-fold, whilst the antibody titer to lysozyme protofilaments decreased by approximately 8-fold compared to patients with AD duration of 15 years. An increase in the concentration of immune complexes and higher lysozyme activity was detected in the serum of all patients and this was suggestive of an inflammatory reaction. We propose that the autoimmune response to different amyloid structures in AD can be viewed as a clearance pathway targeting amyloid development. Autoimmune response can be exploited as a marker of ongoing protein aggregation and hence be used as a diagnostic feature of AD

Comprehensive approach for identification of functional FCGR2C alleles resulting in protein expression as a determinant for predicting predisposition to autoimmunity

Abstract The balance of activating and inhibitory signals from the low affinity Fc gamma receptors modulates immune responses triggered by IgG antibody‐immune complexes. In homeostasis, this leads to antigen clearance, while in autoimmune diseases to unwanted immune response. Besides the activating receptors FcɣRIIa, FcɣRIIIa, and the inhibitory FcɣRIIb receptor, a third activating receptor, FcɣRIIc, was shown to be expressed on several immune cell types, however, only in the presence of a functional FCGR2C‐ORF allele. FcɣRIIc expression is associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus or systemic sclerosis. Thus, the determination of the functional FCGR2C gene resulting in protein expression on immune cells becomes highly relevant, particularly in the context of unwanted immune responses through inadvertent FcɣRIIc activation by molecules targeting stimulation of the inhibitory receptor FcɣRIIb, currently pursued by several pharmaceutical companies. The high degree of homology within the FCGR2/3 gene cluster complicates development of an accurate method for identification of FcɣRIIc expression. Here we describe a comprehensive approach to characterize genetic status of the FCGR2C gene locus consisting of cDNA sequencing, SNaPshot genotyping and low‐coverage next‐generation sequencing. This might enable Mendelian randomization hypothesis testing across autoimmune diseases to personalize therapies and enhance treatment outcomes

Templating S100A9 amyloids on Aβ fibrillar surfaces revealed by charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses.

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aβ42 peptide in Alzheimer's disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aβ42 amyloids. Kinetic analysis further corroborates that the surfaces available for the Aβ42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aβ42 fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research