Mechanisms of Functional Amyloid Formation

功能性淀粉样蛋白形成机制

• 批准号: 8939823
• 负责人: Jennifer Lee
• 金额: $ 12.19万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939823
• 关键词: Address; Agreement; Alzheimer' s Disease; Amides; Amyloid; Amyloid Fibrils; Behavior; Benign; Biological; C-terminal; Carboxylic Acids; Charge; Chemicals; Data; Dependence; Deposition; Development; Disease; Electrostatics; Employee Strikes; Event; Exhibits; Filament; Glutamic Acid; Goals; Growth; Human; Hydrogen Bonding; Kinetics; Melanins; Melanosomes; Modification; Molecular; Mutation; Organelles; Parkinson Disease; Pigments; Play; Positioning Attribute; Process; Protein Precursors; Proteins; Resistance; Role; Series; Site; Solutions; Staging; Structure; Time; Work; alanylglutamine; amyloid formation; amyloid structure; cytotoxic; human disease; in vivo; insight; methyl group; mutant; polymerization; polypeptide; prevent; protonation; research study; self assembly

The emerging concept of functional amyloids is challenging the way we view amyloids, which have been previously thought as either a cause or consequence of human diseases as in Alzheimers and Parkinsons. In our work, we have studied a crucial fibril forming domain termed the repeat domain (RPT, residues 315444) derived from the human functional amyloid, Pmel17, to gain insights into what may differentiate functional from pathological amyloid. Pmel17 is a transmembrane precursor protein that is proteolytically processed to form intralumenal fibrils in melanosomes upon which melanin is deposited. Pmel17 is highly regulated in vivo, undergoing a series of post-translational and proteolytic modifications whereby the timing and sequence of these events permit amyloid formation. RPT is essential for the amyloid structures observed in melanosomes. Fibrils are formed during the early stages of melanosome development and once formed are responsible for the deposition of the pigment melanin. Since melanin precursors are cytotoxic, sequestering their synthesis on fibrils prevents potential detriment to the organelle. A distinguishing feature that we have discovered is that not only does RPT form amyloid at a mildly acidic, melanosomal pH regime (4.5-5.5) but these fibrils completely dissolve at pH ≥ 6. This reversible polymerization behavior highly contrasts those exhibited by disease-related amyloids, which only upon the harshest treatments will disassemble, e.g. chemical denaturants and non-physiological pH. A potential biological implication for this observed disaggregation process is that if RPT filaments were to escape from the melanosome, they would dissolve under neutral cytosolic pH, and thus remain benign. While this is a compelling hypothesis, there is no current data supporting fibril dissolution in vivo and other domains may be involved. Nevertheless, our results support the requirement of the acidic melanosome pH for amyloid assembly where protonation of specific carboxylic acids promotes key interactions for RPT fibril formation by reducing either intra- or inter-molecular electrostatic repulsion. We have identified specific carboxylic acids (protonation sites) that are necessary for aggregation and assessed the role of hydrogen bonding in fibril formation by utilizing Ala- and Gln-mutants, respectively. Specifically, effects of mutations at residues, E404, E422, E425 and E430 on RPT aggregation kinetics and pH dependence of amyloid formation were studied. Protonation of the C-terminal glutamic acids is shown to be vital, likely through the inhibition of intra/intermolecular electrostatic repulsion. Particularly, both charge neutralization and hydrogen bonding play key roles at position E422, where the introduction of an amide (-NH2 vs. -OH) sidechain accelerates aggregation via the increase of hydrogen bonding capability. This is in strong agreement with the inhibitory effect of the Ala mutation where hydrogen bonding donor (-OH) and acceptor (C=O) are removed. However, the difference in residue size, i.e. sidechain packing, cannot be ruled out as a contributing factor in kinetics modulation. By comparison, hydrogen bonding and/or size are not as critical at E404 where Ala/Gln both stimulate aggregation. Mutations at both E425 and E430 have a similar negative effect on aggregation, prolonging fibril growth. Upon protonation, these residues influence the self-assembly process perhaps through the formation of local noncovalent interactions and thus, retarding aggregation. Only E422 mutants had a substantial impact where fibrils now form at pH 6.5. Consistently, dissolution experiments conducted on E422Q fibrils verified that it is more stable than WT fibrils and are resistant to disassembly up to pH 7. We note that E404A/Q occasionally aggregated at pH 6 suggesting that it may play an ancillary role. Taken together, our data suggest that residue 422 is the critical sidechain in controlling the pH sensitivity of RPT amyloid formation. From a structural perspective, we propose that E404 and E422 reside within the amyloid-forming region of RPT. Here, Glu sidechains are oriented with E404 positioned outside and E422 inside the filament. Having E422 sidechains within the filament core, suggests that upon protonation, both intra- and inter-sheet contacts are facilitated and essential in stabilizing filament structure. In the absence of a net charge, filaments can form at higher pH. The reduced aggregation rates associated with E422A indicate that either hydrogen bonding or size is involved in inter-sheet packing and stability. Protonation of the outwardly facing E404 would prevent intra-sheet electrostatic repulsion. The increased aggregation propensity associated with E404A also may suggest a role for sidechain interdigitation as the small methyl groups would allow tighter packing between filaments.

功能性淀粉样蛋白的新概念正在挑战我们看待淀粉样蛋白的方式，淀粉样蛋白以前被认为是阿尔茨海默病和帕金森病等人类疾病的原因或后果。在我们的工作中，我们已经研究了一个重要的原纤维形成域称为重复结构域（RPT，残基315444）来自人类功能性淀粉样蛋白，Pmel 17，以获得洞察什么可能区分功能性病理性淀粉样蛋白。Pmel 17是一种跨膜前体蛋白，其被蛋白水解加工以在黑素体中形成管腔内原纤维，黑素沉积在所述管腔内原纤维上。Pmel 17在体内受到高度调节，经历一系列翻译后和蛋白水解修饰，由此这些事件的时间和顺序允许淀粉样蛋白形成。RPT对于在黑素体中观察到的淀粉样结构是必需的。原纤维在黑素体发育的早期阶段形成，并且一旦形成就负责色素黑色素的沉积。由于黑色素前体是细胞毒性的，因此将它们的合成隔离在原纤维上可以防止对细胞器的潜在损害。 我们发现的一个显著特征是RPT不仅在弱酸性的黑素体pH范围（4.5-5.5）下形成淀粉样蛋白，而且这些原纤维在pH 6下完全溶解。这种可逆的聚合行为与疾病相关的淀粉样蛋白所表现出的聚合行为形成鲜明对比，这些淀粉样蛋白仅在最苛刻的处理下才会分解，例如化学变性剂和非生理pH。这种观察到的解聚过程的潜在生物学意义是，如果RPT细丝从黑素体中逃逸，它们将在中性胞质pH下溶解，从而保持良性。虽然这是一个令人信服的假设，但目前没有数据支持体内原纤维溶解，可能涉及其他领域。 然而，我们的研究结果支持淀粉样蛋白组装的酸性黑素体pH的要求，其中特定羧酸的质子化通过减少分子内或分子间的静电排斥促进RPT原纤维形成的关键相互作用。我们已经确定了特定的羧酸（质子化位点），是必要的聚集和评估的作用，通过利用丙氨酸和谷氨酰胺突变体，分别在原纤维形成的氢键。具体而言，研究了残基E404、E422、E425和E430处的突变对RPT聚集动力学和淀粉样蛋白形成的pH依赖性的影响。 质子化的C-末端谷氨酸被证明是至关重要的，可能通过抑制内/分子间的静电排斥。特别地，电荷中和和氢键合在位置E422处起关键作用，其中酰胺（-NH 2对-OH）侧链的引入通过增加氢键合能力加速聚集。这与其中氢键供体（-OH）和受体（C=O）被去除的Ala突变的抑制作用非常一致。然而，不能排除残留物大小的差异，即侧链堆积，作为动力学调节的一个影响因素。相比之下，氢键和/或大小在E404处不那么关键，其中Ala/Gln都刺激聚集。E425和E430处的突变对聚集具有类似的负面影响，延长原纤维生长。质子化后，这些残基可能通过形成局部非共价相互作用影响自组装过程，从而延缓聚集。只有E422突变体具有实质性影响，其中原纤维现在在pH 6.5下形成。因此，对E422 Q原纤维进行的溶解实验证实，它比WT原纤维更稳定，并且在高达pH 7的条件下耐分解。我们注意到E404 A/Q偶尔在pH 6下聚集，这表明它可能起辅助作用。总之，我们的数据表明，残基422是控制RPT淀粉样蛋白形成的pH敏感性的关键侧链。 从结构的角度来看，我们建议E404和E422驻留在RPT的淀粉样蛋白形成区域内。在此，Glu侧链被取向为E404位于细丝外部，E422位于细丝内部。在细丝芯内具有E422侧链表明，在质子化后，片内和片间接触都是促进的，并且在稳定细丝结构中是必不可少的。在没有净电荷的情况下，长丝可以在较高的pH值下形成。与E422 A相关的降低的聚集速率表明，氢键或尺寸参与片间包装和稳定性。面向外的E404的质子化将防止片内静电排斥。与E404 A相关的增加的聚集倾向也可能表明侧链交错的作用，因为小的甲基基团将允许细丝之间更紧密的堆积。