title: Mutations of Profilin-1 Associated with Amyotrophic Lateral Sclerosis Promote Aggregation Due to Structural Changes of Its Native State.
abstract: The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as three mutations, namely C71G, M114T, and G118V, have been found in patients with familial forms of the disease and another, E117G, has been proposed to be a moderate risk factor for disease onset. In this work, we have purified the four profilin-1 variants along with the wild-type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular β-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all four variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation, followed by the G118V and M114T substitutions and then the E117G mutation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all four mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Section of Biochemistry, University of Florence , Viale Morgagni 50, I-50134, Florence, Italy.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Del Poggetto
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Section of Biochemistry, University of Florence , Viale Morgagni 50, I-50134, Florence, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bemporad
authors.affiliation: Institute of Applied Physics Nello Carrara, National Research Council , Via Madonna del Piano 10, I-50019, Sesto Fiorentino (FI), Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Tatini
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Section of Biochemistry, University of Florence , Viale Morgagni 50, I-50134, Florence, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
mesh_terms: D000682:Amyloid / Q000737:chemistry; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics*; D002942:Circular Dichroism; D006801:Humans; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D051304:Profilins / Q000737:chemistry* / Q000235:genetics*; D066263:Protein Aggregation, Pathological / Q000235:genetics*; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D000682:Amyloid; D051304:Profilins