AMYLOID HOT SPOTS · Cellular and structural determinants of amyloid toxicity

Cellular and structural determinants of amyloid toxicity

Project context and objectives Parkinson's disease (PD) is a progressive, neurodegenerative and age-related movement disorder affecting approximately close to 7 million people worldwide. In Europe there are more than 800 000 people afflicted and about 75 000 new cases diagnosed every year, according to the European Parkinson's Disease Association. The aetiology of PD is not yet fully understood, but genetic analysis, neuropathologic investigations and experimental models of PD have provided fundamental insights into its pathogenesis. It is now widely accepted that a fundamental role in the development of the disease is played by the deposition of Lewy Bodies, which are protein-rich amyloid-like aggregates mainly composed by the pre-synaptic protein alpha-Synuclein (aSyn). Protein aggregation is a common characteristic of many neurodegenerative disorders including PD, Alzheimer's disease, Hungtinton's disease and Prion diseases. Therefore the study of how the interaction between pathological/toxic proteins with other cellular proteins causes neurodegeneration is a hot topic of current neuroscience research. The aim of this project was to bridge the gap between structural and functional studies on the protein aSyn, and to rationalise the role of protein-associated factors in amyloid toxicity. The development of the project was divided in four major milestones: - we determined a protein-protein interaction network of aSyn; - we characterised structurally at high resolution one of these protein complexes to understand the molecular and structural basis of the interaction; - using structure calculations, we later identified the characteristics of regions in aSyn most relevant for protein-protein interaction regions (termed amyloid hot spots); - we implemented and validated a structure-based drug discovery strategy employing these amyloid hot spots in experimental models of disease. Accordingly, this project has an impact on both basic and translational research in PD. New basic knowledge on the intersection of molecular pathways, linking pathogenic mechanisms and protein aggregation, are the major outcome of the first two milestones. Due to the successful achievement of the last two milestones, novel targets for translational medicinal research and the design of novel therapeutic agents may be available in the near future after further development of this project. Work performed Task 1. Functional characterisation of the interactome of the protein aSyn The functional interactome of aSyn comprises a validated set of 85 proteins that can: - interact with aSyn and co-aggregate in pathogenic inclusions (set A); - interact with aSyn and do not aggregate in pathogenic inclusions (set B); - do not interact with aSyn but are found in pathogenic aggregates (set C). The analysis of the interactome of aSyn shows a highly interconnected network of proteins involved in key cellular functions, indicating that protein aggregation in disease carries profound consequences with far-reaching metabolic perturbations. Task 2. Structural characterisation of the complex between aSyn and the Ca2+ regulatory protein Calmodulin At nearly atomic resolution, our results depict the reorganisation of the ensemble of conformations that an intrinsically disordered protein (IDP) such as aSyn experiences upon molecular recognition of a short motif by an interacting partner. This data may help to explain signal transduction pathways based on IDPs, which, in this particular case, constitute an important step forward in the description of Ca2+ signalling pathways in neurodegeneration. Task 3. Structural characterisation of amyloid hot spots in native monomeric aSyn Our study shows that, at least for aSyn, the molecular basis for anti-amyloidogenicity mediated by protein-protein interactions involves targeting regions that have a high amyloidogenic character and that are engaged in pro-amyloidogenic long-range tertiary interactions. Targeting these amyloid hot spots is a promising anti-amyloid strategy, as was validated by studies on poly-aromatic small molecules that target protein-protein interaction regions in aSyn. Task 4. Targeting amyloid hot spots in native monomeric aSyn by means of small molecules as a therapeutic strategy for Parkinson's disease The development and application of high-throughput docking calculations, coupled with an experimental binding assay, has resulted in the discovery of a novel, biologically active, drug-like small molecule binder to the native monomeric state of the IDP aSyn. We show that this discovery results from the existence of small molecule binding pockets within selected members of the heterogeneous conformational ensemble of this amyloidogenic IDP despite an overall lack of persistent structure. Such binding pockets coincide with protein-protein interaction hot spots and residues engaged in long-range interactions. No significant deviations from the work plan proposed at the start of the fellowship were necessary and objectives were achieved up to a relatively high degree. Work is continuing in the direction of the project as the fellow has received a Ramón y Cajal research fellowship to continue his research at the host institution. Main results In particular, the outcome of this project highlights the role that disordered regions have as hot spots for binding in the network of protein-protein interactions that regulate the concentration of misfolding-prone protein species. We expect the basic knowledge produced throughout this work to be capable of leading to the discovery of a range of alternative lead molecules for Parkinson's disease therapeutics. In addition, the methodology developed here is also applicable to other IDP systems involved in the onset or promulgation of the molecular events associated with major diseases, such as neurodegeneration or cancer. Our research suggests, therefore, that targeting protein-protein interaction hot spots in IDPs by small molecules represents a promising drug discovery strategy for combating neurodegenerative and other misfolding disorders, a finding that could have pronounced consequences in the context of the rapid proliferation of these highly debilitating and currently incurable diseases.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз