Molecular chaperones are vital proteins that maintain protein homeostasis by assisting in protein folding, activation, degradation, and stress protection. Among them, heat-shock protein 90 (Hsp90) stands out as an essential proteostasis hub in eukaryotes, chaperoning hundreds of ‘clients’ (substrates). After decades of research, several ‘known unknowns’ about the molecular function of Hsp90 remain unanswered, hampering rational drug design for the treatment of cancers, neurodegenerative, and other diseases. We highlight three fundamental open questions, reviewing the current state of the field for each, and discuss new opportunities, including single-molecule technologies, to answer the known unknowns of the Hsp90 chaperone.

Previously, we reported that α-synuclein (α-syn) clusters synaptic vesicles (SV) Diao et al., 2013, and neutral phospholipid lysophosphatidylcholine (LPC) can mediate this clustering Lai et al., 2023. Meanwhile, post-translational modifications (PTMs) of α-syn such as acetylation and phosphorylation play important yet distinct roles in regulating α-syn conformation, membrane binding, and amyloid aggregation. However, how PTMs regulate α-syn function in presynaptic terminals remains unclear. Here, based on our previous findings, we further demonstrate that N-terminal acetylation, which occurs under physiological conditions and is irreversible in mammalian cells, significantly enhances the functional activity of α-syn in clustering SVs. Mechanistic studies reveal that this enhancement is caused by the N-acetylation-promoted insertion of α-syn’s N-terminus and increased intermolecular interactions on the LPC-containing membrane. N-acetylation in our work is shown to fine-tune the interaction between α-syn and LPC, mediating α-syn’s role in synaptic vesicle clustering.