Proteins have a generic tendency to convert from their soluble states into insoluble highly structured aggregates known as amyloid fibrils. Since these aggregates are toxic to the cells, especially in the form of early forming oligomers, the cellular machinery has evolved, among other requirements, to inhibit uncontrolled protein aggregation. Failure to inhibit protein aggregation often results in pathology. To date, a large number of very diverse human diseases, including Alzheimer's, Parkinson's disease and type II diabetes, have been recognised to be associated with the formation of amyloid fibrils or protein inclusions with amyloid-like properties. Amyloid fibril formation is also exploited by living organisms for the formation of fibrillar structures with specific biological functions, provided that this occurs under controlled conditions. Finally, the recent findings that the inclusion bodies formed after overexpression of heterologous proteins in bacteria are amyloid-like in structure and the increasing awareness that an enormous number of novel materials based on the amyloid motif can be constructed has expanded the interest of amyloid formation to biotechnology.
The research of this lab uses a multi angle approach to investigate the mechanisms by which soluble proteins assemble into ordered aggregates and by which the resulting aggregates cause cell dysfunction. Here is a list of past and ongoing projects:
- Study, at a molecular level, of the process by which native proteins convert into partially folded states necessary to initiate aggregation
- Study, in molecular depth, of the conversion of partially folded states of proteins into amyloid fibrils and their precursor oligomers
- Study of the relationship between structure and toxicity of protein oligomers
- Investigation of molecular chaperones as inhibitors of the toxicity of protein oligomers
- Investigation of small molecules as inhibitors on the formation and toxicity of protein oligomers
- Study, at a molecular level, of the process of native-like aggregation, involving directly the folded state of a protein
- Study of the effect of agents of the extracellular matrix, such as glycosamynoglycans, on the formation of amyloid fibrils, precursor oligomers and their toxicity
- Editing of mathematical algorithms able to predict fundamental aspects of protein aggregation in vitro and in vivo
- Elucidation of protein oligomers on neuronal function of primary neurons and rat brains
In addition to classical molecular biology and biochemical techniques for manipulation/mutation of genes and purification of their resulting proteins, the experimental work involves the utilisation of biophysical techniques for the characterisation of the folding and aggregation processes. These include turbidometry, fluorescence, circular dichroism and Fourier-transform infra-red spectroscopies, static and dynamic light scattering, stopped-flow devices. Studies of the interaction of protein aggregates with cells are based on the utilisation of cell cultures and specific protocols to assess their viability, oxidative stress and calcium homeostasis. Confocal microscopy and FACS are widely used. Projects involving nuclear magnetic resonance (NMR), atomic force microscopy (AFM), transmission electron microscopy (TEM), mass spectrometry (MS) and limited proteolysis are feasible through established collaborations.