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Nucleation-dependent amyloid fibrillation of human GRASP55 in aqueous solution

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Abstract

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.

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Abbreviations

CD:

Circular dichroism

CR:

Congo Red

FLIM:

Fluorescence lifetime imaging microscopy

GRASPs:

Golgi-reassembly and stacking proteins

ID:

Intrinsic disorder

TEM:

Transmission electron microscopy

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Acknowledgements

The authors thank the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support through Grant nos. 2015/50366-7, 2012/20367-3, and 308380/2013-4. STR thanks FAPESP for the post-doctoral fellowships (Grant no. 2017/12146-0).

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Correspondence to Antonio Jose Costa-Filho.

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Reddy, S.T., Uversky, V.N. & Costa-Filho, A.J. Nucleation-dependent amyloid fibrillation of human GRASP55 in aqueous solution. Eur Biophys J (2020). https://doi.org/10.1007/s00249-019-01419-7

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Keywords

  • GRASP55
  • Aggregation “hot spots”
  • Spectroscopy
  • Microscopy
  • Amyloid fibrils