Unraveling functional and pathological protein aggregation in cells
Images representing typical data produced in the Picotti laboratory.
Top left panel: visualization of amyloid aggregates.
Top right panel: light microscopy image of yeast cells expressing a GFP-tagged protein involved in protein aggregation disease.
Bottom panel: sample of data generated using targeted proteomics analysis.
In protein aggregation diseases, insoluble aggregates of abnormally folded proteins accumulate intra-or extra-cellularly and are critically involved in cellular damage. A range of disorders (for example many neurodegenerative diseases) of previously unknown cause now falls into this category. In these conditions, a susceptible protein, usually prone to misfolding and self-assembly, adopts an abnormal three-dimensional conformation, rich in β-sheet structure (also called amyloid fold), even when the monomeric protein is mostly disordered or α-helical. These abnormally folded proteins can associate to molecules of the same type, forming oligomers and large insoluble aggregates of regular structural organization, also called amyloid fibrils. Such aggregated structures are highly stable and detergent insoluble, and their core is resistant to protease cleavage. While in some pathologies cellular damage results from the subtraction of the misfolded cytoprotective factor, other amyloidogenic proteins clearly acquire a cytotoxic function. Despite extensive studies, little is known about the in vivo kinetics of such detrimental conformational change, its modulating factors and its connection to the triggered toxicity cascade.
Beside their pathological implications described above, assemblies of proteins are increasingly gaining attention in light of their possible functional roles in cells. In the last decade, different types of functional protein aggregates or assemblies, formed upon conformational switch of their constitutive protein have been described. Notable examples of this are yeast prion proteins, which exist in very different stable conformational states.
In our laboratory we apply proteomic techniques to quantitatively study the formation of protein aggregates in cells and their functional and pathological consequences.
Methodologies used in the lab: Unbiased and targeted (selected reaction monitoring) proteomics, mass spectrometry, cell biology (e.g. yeast growth, mammalian cell culture, live cell imaging), protein biochemistry (e.g. preparation of protein aggregates and their structural characterization by spectroscopic techniques and biochemical assays).