Protein Misfolding and Aggregation

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

We have carried out detailed investigations of membrane interactions and amyloid formation of alpha-synuclein that have provided residue-specific information and molecular insights into the mechanism of aggregation. Due to the complexity of the amyloid problem, the tools with which we attack have included molecular biology, steady-state and time-resolved fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, electron microscopy, neutron reflectometry, and mass spectrometry. Through our work, we are developing a chemical understanding in how specific biomolecular interactions and cellular environments modulate protein structure and aggregation propensity. In the last review period, we continued our efforts in understanding the effect of post-translational modifications and processing on alpha-synuclein fibril formation and structure. Specifically, we investigated N-terminally truncated variants in order to examine the role of the N-terminus in the mechanism of amyloid formation and its effect on alpha-synuclein fibril structure. Using three different N-terminal truncations, we showed that the deletions of 13-, 35- and 40-residues in the N-terminus modulated both aggregation kinetics and fibril morphologies of alpha-synuclein as determined by thioflavin-T assays and transmission electron microscopic imaging. Importantly, the removal of N-terminal residues outside the canonical amyloid core weakens the fibril seeding efficiency and hinders propagation. Remarkably, a unique, asymmetric amyloid core is revealed by cryoelectron microscopy in the N-terminally truncated 41140 variant, highlighting the strong influence of the N-terminus in dictating fibril structure. Collectively, these results establish the important role of N-terminal residues in fibril formation and structure. With the removal of N-terminal residues modulating aggregation kinetics as well as fibril structure and propagation, a central question is whether these species are beneficial or detrimental in a cellular environment. It is known that both N- and C-terminal truncations are prevalent in both PD and healthy individuals and some of their generation can be attributed to lysosomal protease activity. While 41140 has not been identified in patients, it is related to another known truncation, 39140, which can be generated by CtsD activity. These proteases have long been implicated in -syn turnover and given the opposing fibril propagation behaviors of N-terminally and C-terminally truncated alpha-syn, it is imperative that future studies consider contributions from both fibril variants.