Nucleation-dependent amyloid fibrillation of human GRASP55 in aqueous solution

GRASP55, one of the two human GRASP proteins, has been implicated in the organization of Golgi stacks and in unconventional protein secretion. However, the detailed molecular mechanisms supporting GRASP55 participation in those processes remain mostly unclear. We have shown that GRASP55 exists as monomers in solution, which transitions to amorphous aggregates with increasing temperatures. Here, we further investigated the formation of higher order structures of GRASP55 by exploring its amyloid fibrillation at 37 °C. Sequence-based AGGRESCAN analysis revealed that GRASP55 has ten aggregation "hot spots", preferentially concentrated in its N-terminal half. Congo Red, ThT, and circular dichroism assays suggested GRASP55 formed amyloid-like fibrils in a time-dependent manner at 37 °C. Dynamic light scattering showed the mean hydrodynamic radius of GRASP55 amyloid-like fibrils increased with increasing incubation times at 37 °C. Transmission electron microscopy and intrinsic fluorescence lifetime imaging showed that, upon increasing incubation time at 37 °C, GRASP55 yielded amyloid-like fibrils in a nucleation-dependent process via a sequence of events: lag-phase (monomers to oligomers), growth phase (oligomers to organized protofibrils), and plateau phase (protofibrils to amyloid-like fibrils). The insights gained herein may help in better understanding the mechanisms of GRASP55 amyloid fibrillation in vivo and its potential association with neurological disorders.