Mechanisms of Functional Amyloid Formation

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，此前人们认为淀粉样蛋白要么是人类疾病的原因，要么是人类疾病的后果，就像阿尔茨海默病和帕金森病一样。在我们的工作中，我们研究了来自人类功能性淀粉样蛋白PMel17的一个关键的纤维形成结构域，称为重复结构域(Rpt，残基315444)，以深入了解什么可能区分功能性和病理性淀粉样蛋白。PMel17是一种跨膜前体蛋白，在黑素小体中被蛋白水解性加工形成腔内纤维，黑色素在其上沉积。Pmel17在体内受到高度调控，经历了一系列翻译后和蛋白质降解修饰，从而这些事件的时间和序列允许淀粉样蛋白的形成。RPT对于观察到的黑素小体中的淀粉样结构是必不可少的。纤维是在黑素小体发育的早期阶段形成的，一旦形成，就负责黑色素的沉积。由于黑色素前体具有细胞毒性，将它们的合成隔离在纤维上可以防止对细胞器的潜在损害。 我们已经发现的一个显著特征是，RPT不仅在中等酸性的黑素体pH区(4.5-5.5)形成淀粉样蛋白，而且这些纤维在pH=6完全溶解。这种可逆的聚合行为与疾病相关的淀粉样蛋白表现出的高度对比，只有经过最严厉的处理，例如化学变性剂和非生理性的pH，淀粉样蛋白才会分解。这种观察到的解聚过程的一个潜在的生物学意义是，如果RPT细丝从黑小体中逃逸，它们将在中性胞浆pH下溶解，从而保持良性。虽然这是一个令人信服的假设，但目前还没有数据支持体内纤维溶解，可能涉及其他领域。 然而，我们的结果支持酸性黑素小体对淀粉样蛋白组装的要求，其中特定羧酸的质子化通过减少分子内或分子间的静电斥力来促进RPT纤维形成的关键相互作用。我们已经确定了聚集所必需的特定羧酸(质子化位置)，并分别利用Ala-和Gln-突变体评估了氢键在纤维形成中的作用。具体地说，研究了E404、E422、E425和E430残基突变对RPT聚集动力学和淀粉样蛋白形成的pH依赖性的影响。 C末端谷氨酸的质子化被证明是至关重要的，可能是通过抑制分子内/分子间的静电斥力。特别是，电荷中和和氢键都在E422位置起着关键作用，在E422位置，酰胺(-NH2与-OH)侧链的引入通过增加氢键能力来加速聚集。这与ALA突变的抑制作用非常一致，在ALA突变中，氢键供体(-OH)和受体(C=O)被移除。然而，残基大小的差异，即侧链堆积，不能排除是动力学调节的一个贡献因素。相比之下，在Ala/Gln都刺激聚集的E404中，氢键和/或大小并不那么关键。E425和E430的突变对聚集有相似的负面影响，延长了纤维的生长。质子化后，这些残基可能通过形成局部非共价相互作用而影响自组装过程，从而延缓聚集。只有E422突变对现在pH 6.5时形成纤维的地方产生了实质性影响。在E422Q纤维上进行的溶解实验一致地证明，它比WT纤维更稳定，在pH 7以下耐拆解。我们注意到E404A/Q在pH 6时偶尔聚集，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，我们认为E404和E422位于RPT的淀粉样蛋白形成区域。这里，Glu侧链定位为E404定位在灯丝外部，E422定位在灯丝内部。在灯丝核心内有E422侧链，这表明质子化后，片内和片间接触都得到了促进，这对稳定灯丝结构是必不可少的。在没有净电荷的情况下，细丝可以在较高的pH下形成。与E422A相关的降低的聚集率表明氢键或尺寸与片间堆积和稳定性有关。向外的E404的质子化将防止片内静电斥力。与E404A相关的聚集倾向的增加也可能表明侧链交错的作用，因为较小的甲基将允许细丝之间更紧密的堆积。

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.