Abstract
Recently, it has been found that the CacyBP/SIP protein acts as HSP90 co-chaperone and exhibits chaperone properties itself. Namely, CacyBP/SIP has been shown to protect citrate synthase from aggregation and to recover the activity of thermally denatured luciferase in vitro. In the present work, we have analyzed the influence of CacyBP/SIP on aggregation of α-synuclein, a protein present in Lewy bodies of Parkinson's disease brain. By applying a thioflavin T (ThT) fluorescence assay, we have found that CacyBP/SIP protects α-synuclein from aggregation and that the fragment overlapping the N-terminal part and the CS domain of CacyBP/SIP is crucial for this activity. This protective effect of CacyBP/SIP has been confirmed by results obtained using high-speed ultracentrifugation followed by dot-blot and by transmission electron microscopy (TEM). Interestingly, CacyBP/SIP exhibits the protective effect only at the initial phase of α-synuclein aggregation. In addition, we have found that, in HEK293 cells overexpressing CacyBP/SIP, there are less α-synuclein inclusions than in control ones. Moreover, these cells are more viable when treated with rotenone, an agent that mimics PD pathology. By applying proximity ligation assay (PLA) on HEK293 cells and in vitro assays with the use of purified recombinant proteins, we have found that CacyBP/SIP directly interacts with α-synuclein. Altogether, in this work, we show for the first time that CacyBP/SIP is able to protect α-synuclein from aggregation in in vitro assays. Thus, our results point to an important role of CacyBP/SIP in the pathology of Parkinson's disease and other synucleinopathies.