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兴概念正在挑战我们观察淀粉样蛋白的方式，淀粉样蛋白以前被认为是人类疾病的原因或后果，就像阿尔茨海默氏症和帕金森一家一样。在我们的工作中，我们研究了一个至关重要的原纤维形成结构域，该结构域称为重复结构域（RPT，残基315444），该结构域源自人类功能性淀粉样蛋白PMEL17，以了解可能将功能与病理淀粉样蛋白区分开的见解。 PMEL17是一种跨膜前体蛋白，在黑色素沉积的黑色素体中被蛋白水解处理以形成肉内纤维。 PMEL17在体内受到了高度调节，经历了一系列翻译后和蛋白水解修饰，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至关重要。纤维是在黑色素体发育的早期阶段形成的，一旦形成，是颜料黑色素的沉积。由于黑色素前体是细胞毒性的，因此将其在原纤维上的合成隔离会防止潜在的细胞器损害。 我们发现的一个显着特征是，RPT不仅以酸性，黑色素体pH状态形成淀粉样蛋白（4.5-5.5），而且这些原纤维完全溶解在pH≥6时。这种可逆的聚合行为高度对比，高度对比是通过疾病相关的淀粉样蛋白，仅在疾病相关的淀粉样蛋白上，这只会贬低Harshestement，E.G E.G E.G。化学变性剂和非生理pH。对这一观察到的分类过程的潜在生物学意义是，如果rpt丝从黑色素体中逃脱，它们会在中性的胞质pH下溶解，从而保持良性。尽管这是一个引人注目的假设，但目前可能涉及体内纤维溶解和其他域的当前数据。 然而，我们的结果支持酸性黑色素体pH的要求对淀粉样蛋白组装的需求，其中特定羧酸的质子化通过减少分子内或分子间静电抑制来促进RPT Fibril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），这些羧酸（质子化位点）分别通过利用ALA-和GLN突变剂来评估氢键在原纤维形成中的作用。具体而言，研究了残基的突变，E404，E422，E425和E430对RPT聚集动力学和淀粉样蛋白形成的pH依赖性的影响。 C末端谷氨酸的质子化可能是通过抑制/分子间静电排斥而言至关重要的。特别是，电荷中和和氢键在E422位置起关键作用，其中引入酰胺（-nh2 vs. -OH）Sidechain通过氢键能力的增加加速了聚集。这与ALA突变的抑制作用有很强的一致，其中氢键供体（-OH）和受体（C = O）被去除。但是，残留大小的差异（即侧chain填料）不能排除为动力学调制的促成因素。相比之下，在ALA/GLN都刺激聚集的E404时，氢键和/或大小并不那么重要。 E425和E430的突变对聚集的负面影响相似，延长了原纤维生长。质子化后，这些残基可能会通过形成局部非共价相互作用，从而影响延迟聚集来影响自组装过程。只有E422突变体在pH 6.5处形成原纤维时会产生重大影响。一致地，在E422Q纤维上进行的溶出实验证实了它比WT纤维更稳定，并且可以抵抗拆卸至pH 7。我们注意到，E404A/Q有时在pH 6中聚集了，这表明它可能起辅助作用。综上所述，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，我们提出E404和E422位于RPT的淀粉样蛋白形成区域内。在这里，Glu Sidechains的定位是在灯丝内部放置的E404和E422的E404。在细丝芯内具有e422侧级技术，表明质子化后，内部和间室间接触均可促进，对于稳定细丝结构至关重要。在没有净电荷的情况下，可以在较高的pH下形成丝。与E422A相关的降低的聚合速率表明，氢键或大小参与了板间包装和稳定性。向外面向E404的质子化将防止骨内静电排斥。与E404A相关的聚集倾向的增加也可能表明Sidechain互插的作用，因为小甲基将允许细丝之间的更紧密的堆积。