From the Departments of Physiology and Neuroscience and of Biochemistry and Molecular Medicine. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90033.
From the Departments of Physiology and Neuroscience and of Biochemistry and Molecular Medicine. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90033
J Biol Chem. 2018 Feb 16;293(7):2597-2605. doi: 10.1074/jbc.M117.813667. Epub 2017 Dec 27.
Aggregation of huntingtin protein arising from expanded polyglutamine (polyQ) sequences in the exon-1 region of mutant huntingtin plays a central role in the pathogenesis of Huntington's disease. The huntingtin aggregation pathways are of therapeutic and diagnostic interest, but obtaining critical information from the physiologically relevant htt exon-1 (Httex1) protein has been challenging. Using biophysical techniques and an expression and purification protocol that generates clean, monomeric Httex1, we identified and mapped three distinct aggregation pathways: 1) unseeded in solution; 2) seeded in solution; and 3) membrane-mediated. In solution, aggregation proceeded in a highly stepwise manner, in which the individual domains (N terminus containing 17 amino acids (N17), polyQ, and proline-rich domain (PRD)) become ordered at very different rates. The aggregation was initiated by an early oligomer requiring a pathogenic, expanded Gln length and N17 α-helix formation. In the second phase, β-sheet forms in the polyQ. The slowest step is the final structural maturation of the PRD. This stepwise mechanism could be bypassed by seeding, which potently accelerated aggregation and was a prerequisite for prion-like spreading Remarkably, membranes could catalyze aggregation even more potently than seeds, in a process that caused significant membrane damage. The N17 governed membrane-mediated aggregation by anchoring Httex1 to the membrane, enhancing local concentration and promoting collision via two-dimensional diffusion. Considering its central roles in solution and in membrane-mediated aggregation, the N17 represents an attractive target for inhibiting multiple pathways. Our approach should help evaluate such inhibitors and identify diagnostic markers for the misfolded forms identified here.
由突变亨廷顿蛋白外显子 1 区域中扩展的多聚谷氨酰胺（polyQ）序列引起的亨廷顿蛋白聚集在亨廷顿病的发病机制中起核心作用。亨廷顿蛋白聚集途径具有治疗和诊断意义，但从生理相关的 htt 外显子 1（Httex1）蛋白中获取关键信息一直具有挑战性。使用生物物理技术和一种表达和纯化方案，该方案产生清洁的单体 Httex1，我们确定并绘制了三种不同的聚集途径：1）无种子在溶液中；2）在溶液中接种；3）膜介导。在溶液中，聚集以高度逐步的方式进行，其中各个结构域（包含 17 个氨基酸的 N 端（N17）、polyQ 和脯氨酸丰富结构域（PRD））以非常不同的速率变得有序。聚合由需要致病性、扩展的 Gln 长度和 N17α-螺旋形成的早期寡聚体引发。在第二阶段，polyQ 中形成β-折叠。最慢的步骤是 PRD 的最终结构成熟。通过接种可以绕过这种逐步机制，接种可有效加速聚集，是形成类朊病毒样传播的先决条件。值得注意的是，与种子相比，膜甚至可以更有效地催化聚集，从而导致膜严重损伤。N17 通过将 Httex1 锚定在膜上、增强局部浓度并通过二维扩散促进碰撞，来控制膜介导的聚集。考虑到它在溶液中和膜介导的聚集中的核心作用，N17 代表了抑制多种途径的有吸引力的靶标。我们的方法应该有助于评估此类抑制剂，并识别此处鉴定的错误折叠形式的诊断标志物。
