Protein Misfolding and Aggregation

我们对α-SYN的膜相互作用和淀粉样蛋白的形成进行了详细的研究，为聚集机制提供了残基特异性信息和分子洞察力。由于淀粉样蛋白问题的复杂性，我们攻击的工具包括分子生物学、稳态和时间分辨荧光光谱、核磁共振光谱、电子显微镜、中子反射仪和质谱仪。通过我们的工作，我们正在发展对特定生物分子相互作用和细胞环境如何调节蛋白质结构和聚集倾向的化学理解。 在上一期综述中，我们研究了翻译后修饰和加工对α-突触核蛋白原纤维形成和结构的影响。具体地说，我们选择研究N-末端的乙酰化和C-末端的截断，因为α-氨基N-末端的乙酰化是结构性的，而路易体中相当数量的α-syn是C-末端截断的。我们发现，N末端乙酰化的α-突触核蛋白聚集体比非乙酰化的α-突触核蛋白聚集得更慢，对广泛使用的荧光淀粉样蛋白硫黄素T(THT)的敏感性显著降低。用透射电子显微镜、圆二色谱和有限蛋白水解法对纤维差异进行了表征。有趣的是，低THT N末端乙酰化的α-突触核蛋白纤维既有乙酰化的，也有非乙酰化的，并在几代中忠实地传播低THT的特征，表明有稳定的纤维多晶型。相比之下，高THT的非乙酰化α-突触核蛋白种子在随后的世代中会失去保真度。尽管在淀粉样蛋白核心之外，N-末端的化学性质调节α-突触核蛋白聚集和纤维多态。观察到淀粉样蛋白核心外单个残基上的一个小乙酰基可以影响α-突触核蛋白原纤维结构，这一观察结果令人惊讶，并突显了翻译后修饰在调节α-突触核蛋白原纤维多态方面的潜在重要性。 在与倪和酱的合作中，我们开展了一项研究，以阐明C末端截断对α-突触核蛋白原纤维结构的影响。用低温电子显微镜结合聚集动力学测量、拉曼光谱表征和有限蛋白降解实验，研究了N-末端乙酰化全长和两个路易体衍生的deltaC-α-突触核蛋白物种AC1-122和AC1-103的结构差异。我们发现，C-末端残基的丢失与α-突触核蛋白纤维的加速聚集和螺旋扭曲增加密切相关。有趣的是，原纤状螺旋扭曲可以通过交叉播种来传播。在通常观察到的希腊键相似的拓扑结构中，分子差异和相互作用的变化为扭曲的AC1-103纤维结构产生了更紧密的核心。这项工作突出了从残基到超微结构水平的α-突触核蛋白原纤维多态的更复杂的画面，其中螺旋扭曲不是由核心结构决定的。重要的是，由于AC1-103有效地为AC1-140提供种子，这种扭曲的纤维多态在促进α-突触核蛋白淀粉样蛋白的形成方面构成了更大的威胁，并解释了它们在帕金森病患者路易体中的存在。

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.