Biophysical investigation on therapeutic proteins (Chaperonins, Hsp60 and CCT/TRiC) involved in human diseases

Molecular chaperones are indispensable cellular components that assist folding and assembly of newly synthesized proteins, translocation of proteins across membranes, as well as refolding and degrading of misfolded and aggregated proteins. In the last few years, innovative therapeutic strategies targeting stability and functionality of chaperones have received great attention, particularly in the field of neurodegenerative diseases. Moreover, the growing number of diseases found linked to chaperone mutations, testifies to the importance of their role in the cellular protein-quality control mechanism. The investigation of the biophysical interactions between chaperones and specific proteins involved in diseases, including their structural and functional properties, are therefore a crucial step for both validating the chaperones’ role in physiological and pathological state, and developing effective chaperones-based treatment approaches. In the present PhD thesis work, we studied two representative examples of human molecular chaperones, Hsp60 and CCT/TRiC, appertaining to the class of the so-called “chaperonins” (Cpns). They are large, hollow, ATP-dependent nanomachines that promote correct folding of a wide range of proteins. Heat shock protein (Hsp60) is a molecular chaperone that assists protein folding in mitochondria. Hsp60 can accumulate in the cytosol, in various pathological conditions (i.e., cancer and chronic inflammatory diseases). Here we studied its functional oligomeric equilibrium as compared to that of its well-known bacterial homolog GroEL. We also show that Hsp60 is capable of inhibiting the fibrillogenesis of Aβ peptide (the protein involved in Alzheimer’s disease). The probable inhibition mechanism operating at molecular level is discussed. CCT/TriC is a chaperon universally found in Archaea and eukaryotes. Several chaperonopathies linked to CCT loci are clinically well characterized and, for those due to non-lethal genetic mutations, there is considerable information on their mode of inheritance. A protein model has been recently developed to investigate the mechanism of a crippling hereditary sensory neuropathy, due to a point mutation (His147Arg) in CCT5, one of the eight subunits of the human CCT. Here we report quantitative information on the loss of structural stability impaired by the pathogenic mutation. Finally, we tested if an efficient inhibition of amyloid formation can be achieved by chaperone-like systems, like as α-Caseins, which is known to exert a stabilizing function through direct interaction (similar to small Hsps). In fact, the evaluation of the mechanism of chaperone-like activity proved helpful for better understanding the structural basis of the substrate binding in sHsps.