Mechanisms of Functional Amyloid Formation

功能性淀粉样蛋白形成机制

• 批准号: 9157372
• 负责人: Jennifer Lee
• 金额: $ 8.45万
• 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/9157372
• 关键词: Address; Adverse effects; Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Behavior; Benign; Biological; C-terminal; Cell membrane; Charge; Chemicals; Circular Dichroism; Cone; Cytosol; Cytotoxic agent; Data; Deposition; Development; Disease; Electrons; Employee Strikes; Event; Exhibits; Fluorescence Spectroscopy; Glutamic Acid; Goals; Human; Kinetics; Location; Lysophosphatidylcholines; Lysophospholipids; Melanins; Melanosomes; Membrane; Membrane Fusion; Membrane Lipids; Micelles; Modification; Organelles; Parkinson Disease; Phospholipids; Physiological; Pigments; Play; Population; Process; Property; Protein Precursors; Proteins; Proteolytic Processing; Recycling; Regulation; Relative (related person); Role; Route; Series; Shapes; Solutions; Speed; Staging; Structure; Surface; Time; Transmission Electron Microscopy; Tryptophan; Vesicle; Work; amyloid formation; amyloid structure; biophysical techniques; coping; cytotoxic; human disease; in vivo; insight; interest; light scattering; monomer; polypeptide; preference; prevent; surfactant; trafficking

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 315-444) 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 &#8805; 6. Most recently, our mutational study showed that a single C-terminal glutamic acid, E422, is predominantly responsible for this pH behavior; neutralization of the negative charge at E422 accelerates fibril formation and increases fibril stability by a full pH unit. This highly reversible aggregation/disaggregation process under physiological pH is a unique property of RPT and contrasts those exhibited by disease-related amyloids, which only upon the harshest treatments will disassemble, e.g. chemical denaturants and non-physiological pH. We speculate that this is a potential way for keeping these amyloids benign if they were to escape melanosomes into the cytosol. Moreover, it may be plausible to recycle amyloid fibrils via this mechanism. While this is a compelling hypothesis, there is no current data supporting fibril dissolution in vivo and other domains may be involved. Studies have shown that Pmel17 trafficking and its subsequent proteolytic processing in melanosomes is important for melanin formation; however, the precise series of events and players involved in initiating and propagating fibril formation remain to be defined. Electron tomographic analyses of early stage melanosomes suggest that fibril formation is initiated on intralumenal membrane vesicles (ILVs). In another study, we sought to determine the effects of membrane lipids on RPT fibril formation, including vesicles and micelles formed from phospholipids and lysophospholipids (lysolipids), respectively. Lysolipids are particularly interesting due to their high content in melanosomal membranes (> 10%) as compared to plasma membranes (< 2%). With a combination of biophysical techniques, including circular dichroism and tryptophan fluorescence spectroscopy, dynamic light scattering as well as transmission electron microscopy, mechanistic insights were gained for the modulation of RPT amyloid formation by two specific lysolipids, negatively-charged lysophosphatidylglycerol (LPG) and zwitterionic lysophosphatidylcholine (LPC). Negatively charged LPG has dual and opposing effects on RPT aggregation: monomers accelerate whereas micelles retard kinetics. For the zwitterionic LPC, both monomers and micelles stimulate fibril formation, with micelles exerting a stronger effect. Lysolipids are structurally closer to surfactants than to phospholipids. Lysolipids are inverted cone-shaped due to their large headgroup and relatively small acyl chain, a structure that contributes to positive spontaneous curvature. As a result, it is not surprising to see a higher lysolipid content in melanosomes because these structural features of lysolipids would favor the formation of highly curved small ILVs and stabilize the ellipsoid-shaped melanosomes. Therefore, it supports the proposal that the fibril initiation occurs on the highly curved ILV membrane surface. Moreover, the preference of lysolipids for positive spontaneous curvature has been shown to promote membrane fusion by minimizing the bending energy. We conjecture that a high concentration of lysolipids is needed in ILVs to aid their merging with melanosomal membranes. Because the intermediate species (oligomers) en-route to fibrils are proposed to be the most potent cytotoxic agents, one way for melanosomes to cope with the adverse effect of amyloid formation is through the spatial and temporal regulation of aggregation. By using different lysolipids and their relative distribution and concentrations on ILVs, both the location and the speed of fibrillation can be controlled. With faster aggregation kinetics, over-population of oligomers may also be circumvented as well as the prompt sequestration of melanin and its associated toxic intermediates on the fibrils may be achieved. Taken together, our data suggest that lysolipids may play a key role in modulating Pmel17 fibril formation and possibly be involved in melanosome maturation.

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315-444）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和病理性淀粉样蛋白。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。最近，我们的突变研究表明，一个单一的C-末端谷氨酸，E422，是主要负责这种pH值的行为;在E422的负电荷的中和加速原纤维的形成，并增加原纤维的稳定性由一个完整的pH值单位。这种高度可逆的聚集/解聚过程下的生理pH值是一个独特的属性RPT和对比那些表现出的疾病相关的淀粉样蛋白，只有在最苛刻的治疗将解体，例如化学变性剂和非生理pH值。我们推测，这是一个潜在的方式保持这些淀粉样蛋白良性的，如果他们逃避黑素体进入细胞质。此外，通过这种机制回收淀粉样纤维可能是合理的。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。 研究表明，Pmel 17运输及其随后在黑素体中的蛋白水解加工对于黑素形成是重要的;然而，参与启动和传播原纤维形成的一系列事件和参与者仍有待确定。早期黑素体的电子断层扫描分析表明，原纤维的形成开始于腔内膜囊泡（ILV）。在另一项研究中，我们试图确定膜脂质对RPT原纤维形成的影响，包括分别由磷脂和溶血磷脂（lysolipids）形成的囊泡和胶束。由于与质膜（< 2%）相比，溶血脂质在黑素体膜（> 10%）中的含量高，因此它们特别令人感兴趣。结合生物物理技术，包括圆二色谱和色氨酸荧光光谱，动态光散射以及透射电子显微镜，获得了两个特定的溶血磷脂，带负电荷的溶血磷脂酰甘油（LPG）和两性离子溶血磷脂酰胆碱（LPC）的RPT淀粉样蛋白形成的调制机制的见解。 带负电荷的LPG对RPT聚集具有双重和相反的影响：单体加速而胶束延迟动力学。对于两性离子LPC，单体和胶束都刺激原纤维形成，胶束发挥更强的作用。溶血脂在结构上更接近于表面活性剂而不是磷脂。 由于其较大的头基和相对较小的酰基链，溶血脂呈倒锥形，这是一种有助于正自发曲率的结构。因此，在黑素体中看到较高的溶脂质含量并不令人惊讶，因为溶脂质的这些结构特征将有利于形成高度弯曲的小ILV并稳定椭圆形黑素体。因此，它支持原纤维引发发生在高度弯曲的ILV膜表面上的提议。此外，溶磷脂对正自发曲率的偏好已被证明通过使弯曲能最小化来促进膜融合。我们推测ILV需要高浓度的溶血脂来帮助它们与黑素体膜合并。 由于纤维形成过程中的中间物质（低聚物）被认为是最有效的细胞毒性剂，黑素体科普淀粉样蛋白形成的不利影响的一种方式是通过聚集的空间和时间调节。通过使用不同的溶血脂及其在ILV上的相对分布和浓度，可以控制原纤化的位置和速度。通过更快的聚集动力学，也可以避免寡聚体的过度聚集，以及可以实现黑色素及其相关的有毒中间体在原纤维上的迅速隔离。两者合计，我们的数据表明，溶血脂可能在调节Pmel 17原纤维形成中发挥关键作用，并可能参与黑素体成熟。