Protein Misfolding and Aggregation

National Heart, Lung, And Blood Institute

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Abstract

We have carried out detailed investigations of membrane interactions and amyloid formation of alpha-syn 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 investigated the effect of post-translational modifications and processing on alpha-synuclein fibril formation and structure. Specifically, we chose to study N-terminal acetylation and C-terminal truncations because acetylation of the alpha-amino N-terminus is constitutive and a significant amount of alpha-syn in Lewy bodies is C-terminally truncated, respectively. We find N-terminally acetylated alpha-synuclein aggregates more slowly than non-acetylated alpha-synuclein with significantly reduced sensitivity to thioflavin T (ThT), a widely used fluorescent amyloid probe. Fibril differences were characterized by transmission electron microscopy, circular dichroism spectroscopy and limited-proteolysis. Interestingly, the low-ThT N-terminally acetylated alpha-synuclein fibrils seed both acetylated and non-acetylated alpha-synuclein and faithfully propagate the low-ThT character through several generations, indicating a stable fibril polymorph. In contrast, the high-ThT non-acetylated alpha-synuclein seeds lose fidelity over subsequent generations. Despite being outside of the amyloid core, the chemical nature of the N-terminus modulates alpha-synuclein aggregation and fibril polymorphism. The observation that a small acetyl group at a single residue outside the amyloid core can impact alpha-synuclein fibril structure is surprising, and highlights the potential importance of post-translational modifications in modulating alpha-synuclein fibril polymorphism. In a collaborative effort with Ni and Jiang, we carried out a study to elucidate the effect of C-terminal truncations on alpha-synuclein fibril structures. Structural differences between N-terminally acetylated full length and two Lewy bodies-derived deltaC-alpha-synuclein species, Ac1-122 and Ac1-103, were investigated by cryoelectron microscopy, coupled to measurements of aggregation kinetics, Raman spectroscopic characterization, and limited-proteolysis experiments. We find that the loss of C-terminal residues strongly correlates to accelerated aggregation and increased helical twist of alpha-synuclein fibrils. Interestingly, fibril helical twists can be propagated through cross-seeding. Within the commonly observed Greek-key like topology, molecular differences and interaction changes generate a more compact core for the twisted Ac1-103 fibril structure. This work highlights a more complex picture of alpha-synuclein fibril polymorphism from the residue to the ultrastructural level, where the helical twist is not dictated by the core structure. Importantly, since Ac1-103 efficiently seeds Ac1-140, this twisted fibril polymorph poses a greater threat in promoting alpha-synuclein amyloid formation and explains their presence in Lewy bodies in Parkinsons disease.