Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Joint Public Review:

The manuscript by Lolicato and colleagues characterizes the role of FGF2 dimerization in the unconventional secretion of this signaling molecule using a combination of cell-based and in vitro assays. FGF2 is secreted from the cell via an unconventional mechanism because it lacks a signal sequence. Previous studies by the same group have established a compelling model in which FGF2 forms an oligomer in a PIP2-dependent manner at the plasma membrane, which drives its translocation to the cell exterior. The same group also identified two cysteine residues (C77 and C95) critical for FGF2 oligomerization and secretion.

In this study, the authors analyzed the impact of single cysteine to alanine substitution on the oligomerization and secretion of FGF2. They found that C95 but not C77 is required for PIP2-dependent membrane binding, FGF2 oligomerization, and secretion. On the other hand, C77 regulates the interaction of FGF2 with the plasma membrane Na, K-ATPase, which is thought to enhance the FGF2-PIP2 interaction. Using a set of bi-functional crosslinkers, the authors were able to capture an FGF2 homo-dimer whose formation is dependent on C95.

They propose that FGF2 forms a disulfide-bridged dimer via C95, the building block for FGF2 oligomerization in the plasma membrane.

While most experiments were carefully designed and the data are of high quality, a few issues need further clarification.

A significant concern is a need for more direct evidence for the proposed disulfide-bridged FGF2 dimer in the cytoplasm despite multiple assays highlighting the critical role of C95 in FGF2 oligomerization and secretion. The crosslinking experiments only suggest that C95 is close to another C95 in crosslinked FGF2 dimers. Given that the reducing cytosolic environment does not usually support disulfide bond formation and that no electron acceptor has been identified to support this unusual model, the reviewers feel that the authors should consider an alternative and more plausible explanation for their observations, which is that the C95A mutation disrupts the dimerization interface. This is actually the author's explanation for why the C77A FGF2 mutant fails to bind Na, K-ATPase. For these reasons, the reviewers feel it is an overstatement to claim that FGF2 forms a disulfide dimer in the cytoplasm.

Furthermore, the authors propose that FGF2 dimers can assemble into a transient higher-order FGF2 oligomer to form a toroidal pore for protein secretion. This is supported by a computational simulation study, which suggests that FGF2 dimers exhibit a higher affinity for PI(4,5)P2 than monomers. However, the model would be much stronger if the authors could provide additional experimental validation.

Additionally, the authors propose that C95-dependent FGF2 dimerization may generate a signaling module. They cited a few structure papers on page 9 (Plotnikov et al., 1999; Plotnikov et al., 2000; Schlessinger et al., 2000), suggesting that the FGF2 dimer reported here may be the primary signaling unit. However, this statement may mislead the reader, as it has been clearly stated in these papers that FGF2 does not form a dimer directly. Instead, heparin facilitates the dimerization of the FGF receptor, which results in the recruitment of two FGF2 molecules.