Self-assembly of human latexin into amyloid-like oligomers

Background: In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations. Results: Here, we show that human latexin, a protein that shares the same fold with cystatin C, assembles into stable spherical amyloid-like oligomers that bind thioflavin-T and congo red similarly to common amyloid structures but do not evolve into fibrils. Latexin self-assembly correlates with the formation of a mostly denaturated state rather than with the population of partially structured intermediates during the unfolding process. The results suggest that unfolding of α-helix 3 might be involved in the transition of latexin toward amyloidotic species, supporting the notion of the protective role of the native protein structure against polymerization. Conclusion: Overall the data herein indicate that latexin could be a good model for the study of the structural and sequential determinants of oligomeric assemblies in protein aggregation processes

Prediction of "hot spots" of aggregation in disease-linked polypeptides

BACKGROUND: The polypeptides involved in amyloidogenesis may be globular proteins with a defined 3D-structure or natively unfolded proteins. The first class includes polypeptides such as β2-microglobulin, lysozyme, transthyretin or the prion protein, whereas β-amyloid peptide, amylin or α-synuclein all belong to the second class. Recent studies suggest that specific regions in the proteins act as "hot spots" driving aggregation. This should be especially relevant for natively unfolded proteins or unfolded states of globular proteins as they lack significant secondary and tertiary structure and specific intra-chain interactions that can mask these aggregation-prone regions. Prediction of such sequence stretches is important since they are potential therapeutic targets. RESULTS: In this study we exploited the experimental data obtained in an in vivo system using β-amyloid peptide as a model to derive the individual aggregation propensities of natural amino acids. These data are used to generate aggregation profiles for different disease-related polypeptides. The approach detects the presence of "hot spots" which have been already validated experimentally in the literature and provides insights into the effect of disease-linked mutations in these polypeptides. CONCLUSION: The proposed method might become a useful tool for the future development of sequence-targeted anti-aggregation pharmaceuticals

A pyrene-inhibitor fluorescent probe with large stokes shift for the staining of Aβ1–42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing staphylococcus aureus biofilms

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer, Parkinson or Type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity towards that diversity of amyloid fibrils, or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here, we show the feasibility of the approach by designing, synthesizing and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The more soluble derivative, compound 1D, stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein or amylin, provides a sensitivity only slightly lower than that of Thioflavin T, displays a large Stokes shift, allows an efficient excitation in the UV spectral region,and it is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by Staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining,S. aureus biofilms containing amyloid forming phenol soluble modulins from those lacking them.IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SVis supported by grant BIO2016-783-78310-R and by ICREA (ICREA Academia 2015). MDD is supported by the Government of Aragon (GA E-102). JS is supported by grants BFU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). JS and IL acknowledge financial support from grant CI-2017/001-3 (Campus Iberus, Spain). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government

Prediction of "hot spots" of aggregation in disease-linked polypeptides-1

<p><b>Copyright information:</b></p><p>Taken from "Prediction of "hot spots" of aggregation in disease-linked polypeptides"</p><p>BMC Structural Biology 2005;5():18-18.</p><p>Published online 30 Sep 2005</p><p>PMCID:PMC1262731.</p><p>Copyright © 2005 de Groot et al; licensee BioMed Central Ltd.</p>ncide are colored in green. Those identified experimentally to be relevant for amyloid formation but not predicted by the present approach are colored in blue. The regions predicted to be important for amyloid formation from which experimental data are not available or indicates that they are not involved in aggregation are shown in yellow

Structure of Activated Thrombin-Activatable Fibrinolysis Inhibitor, a Molecular Link between Coagulation and Fibrinolysis

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that TAFIa conforms to the α/β-hydrolase fold of MCPs and displays two unique flexible loops on the molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition, point mutations reported to enhance protein stability in vivo are mainly located in the first loop and in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency. © 2008 Elsevier Inc. All rights reserved.This study was supported by the following grants: BIO2007-68046, BIO2006-02668, BFU2006-09593, PSE-010000-2007-1, and the CONSOLIDER-INGENIO 2010 Project “La Factoría de Cristalización” (CSD2006-00015) from Spanish ministries; EU FP6 Strep Project LSHG-CT-2006-018830 “CAMP”; and 2005SGR00280 and 2005SGR01027 from the Generalitat de Catalunya. Additional funding was obtained by J.J.E. from the Danish National Science Research Council. L.S. and I.P. enjoyed Ph.D. fellowships from the Spanish Ministry of Education and Science. M.S. and J.L.A. are, respectively, beneficiaries of the “Ramón y Cajal” and “Juan de la Cierva” Programs of the Spanish Ministry of Education and SciencePeer Reviewe

Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of α-synuclein aggregation with potentially improved BBB permeability

The treatment of Parkinson’s disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent’s compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.This research was funded by Spanish Ministry of Science and Innovation (grants PID2019-107293GB-I00 to JS and PID2019-105017RB-I00 to SV), by Gobierno de Aragón, Spain (grants LMP30_18 and E45_20R to JS), by ICREA (ICREA-Academia 2015 to SV) and by “la Caixa” Banking Foundation, Spain (grant CaixaImpulse CI18-00019, to SV). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government.Peer reviewe