Protein Structure, Stability, and Amyloid Formation

蛋白质结构、稳定性和淀粉样蛋白形成

• 批准号: 8552693
• 负责人: Ruth Nussinov
• 金额: $ 53.14万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8552693
• 关键词: Alzheimer' s Disease; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Amyloid; Amyloid Fibrils; Amyloid beta-Protein; Amyloidosis; Apoptosis; Atomic Force Microscopy; Binding; Binding Proteins; Binding Sites; Biological; Birefringence; Calcium; Cell Surface Receptors; Cells; Cellular Membrane; Characteristics; Chronic Disease; Clinical; Congo Red; Cullin Proteins; Data; Deposition; Disease; Distant; Drainage procedure; Drug Design; Exhibits; Extracellular Domain; Face; Filament; Frontotemporal Dementia; Growth; Homeostasis; Human; Image; Individual; Ion Channel; Ions; K-18 conjugate; KRT19 gene; Lead; Length; Ligand Binding; Ligands; Link; Lipid Bilayers; Location; MAPT gene; Mediating; Membrane; Memory Loss; Microbe; Modeling; Molecular; Molecular Conformation; Mutate; Mutation; N-terminal; Nature; Neurodegenerative Disorders; Parkinson Disease; Pathogenesis; Pathology; Pathway interactions; Peptides; Pharmaceutical Preparations; Polyubiquitin; Polyubiquitination; Population; Positioning Attribute; Prevention; Process; Production; Proline; Property; Proteins; Reaction; Role; Scaffolding Protein; Seeds; Senile Plaques; Shapes; Signal Transduction; Simulate; Site; Solutions; Staining method; Stains; Structure; Structure-Activity Relationship; Syndrome; System; Tissues; Toxic effect; Ubiquitin; Ubiquitination; Zinc; abeta accumulation; amyloid beta-protein (1-42); amyloid formation; amyloid peptide; antimicrobial; antimicrobial peptide; base; beta pleated sheet; conformer; design; drug development; extracellular; flexibility; glycine receptor alpha1; insight; interest; killings; molecular dynamics; mutant; paired helical filament; prevent; progressive neurodegeneration; protegrin PG-1; protein aggregation; protein function; protein misfolding; protein structure; receptor; research study; response; scaffold; simulation; tau Proteins; therapeutic target; ubiquitin-protein ligase

Alzheimer's disease (AD) is a misfolded protein disease characterized by the accumulation of beta-amyloid (Abeta) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic pores made of Abeta peptides. Understanding the exact nature by which these pores conduct electrical and molecular signals could aid in identifying potential therapeutic targets for the prevention and treatment of AD. Here using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we compared the imaged pore structures with models to predict channel conformations as a function of amino acid sequence. Site-specific amino acid (AA) substitutions in the wild-type Abeta(1-42) peptide yield information regarding the location and significance of individual AA residues to its characteristic structure-activity relationship. We selected two AAs that our MD simulation predicted to inhibit or permit pore conductance. The substitution of Phe19 with Pro has previously been shown to eliminate conductance in the planar lipid bilayer system. Our MD simulations predict a channel-like shape with a collapsed pore, which is supported by the AFM channel images. We suggest that proline, a known beta-sheet breaker, creates a kink in the center of the pore and prevents conductance via blockage. This residue may be a viable target for drug development studies aiming to inhibit Abeta from inducing ionic destabilization toxicity. The substitution of Phe20 with Cys exhibits pore structures indistinguishable from the wild type in AFM images. MD simulations predict site 20 to face the solvated pore. Overall, the mutations support the previously predicted beta-sheet-based channel structure.In Alzheimer's disease and frontotemporal dementias, the microtubule-associated protein Tau forms intracellular paired helical filaments. The filaments can form not only by the full-length human Tau protein, but also by the three repeated (K19) or four repeated (K18) Tau segments. However, of interest, experimentally, K19 can seed K18, but not vice versa. To obtain insight into the cross-seeding between K18 and K19 aggregates, here, K18 and K19 octamers with repeat 3 (R3) in U-shaped, L-shaped, and long straight line-shaped (SL-shape) conformations are assembled into different structures. The simulation results show that K18-8/K19-8 (K18 and K19 assemblies number 8) with R3 in an L shape and K18-9/K19-9 with R3 in an SL shape are highly populated and present the highest structural similarity among all simulated K18 and K19 octamers, suggesting that similar folding of K18/K19 may serve as structural core for the K18-K19 co-assembled heterogeneous filament. We demonstrate that formation of stable R2 and R3 conformations is the critical step for K18 aggregation, and R3 is critical for K19 fibrillization. The different core units in K18 and K19 may create a cross-seeding barrier for the K18 seed to trigger K19 fibril growth because R2 is not available for K19. Our study provides insights into cross-seeding involving heterogeneous structures. The polymorphic nature of protein aggregation could be magnified in the cross-seeding process. If the seeding conformations lead to too much divergence in the energy landscape, it could impede fibril formation. Such an effect could also contribute to the asymmetric barrier between K18 and K19.More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-beta structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states. A growing body of data has suggested that these toxic species form ion channels in cellular membranes causing disruption of calcium homeostasis, membrane depolarization, energy drainage, and in some cases apoptosis. Amyloid peptide channels exhibit a number of common biological properties including the universal U-shape beta-strand-turn-beta-strand structure, irreversible and spontaneous insertion into membranes, production of large heterogeneous single-channel conductances, relatively poor ion selectivity, inhibition by Congo red, and channel blockade by zinc. Recent evidence has suggested that increased amounts of amyloids not only are toxic to its host target cells but also possess antimicrobial activity. Furthermore, at least one human antimicrobial peptide, protegrin-1, which kills microbes by a channel-forming mechanism, has been shown to possess the ability to form extended amyloid fibrils very similar to those of classic disease-forming amyloids.Mutations, even if not directly in the ligand binding sites of proteins, can lead to disease. In cell surface receptors, this can happen if they uncouple conformational changes that take place upon agonist (or antagonist) binding to the extracellular domain and the intracellular response. Uncoupling can take place by disrupting a major allosteric propagation pathway between the extra- and intracellular domains. Here I provide a mechanistic explanation: I first describe how propagation takes place; second, what can happen in the presence of a disease-related mutation which is distant from the binding site; and finally, how drugs may overcome this disruption and rescue function. The mutations in the glycine receptor alpha1 subunit (alpha1R271Q/L) which cause the neuromotor disorder hyperekplexia are on example of such allosteric mutations. In this issue of the BJP, Shan et al. show that normal function was restored to these mutant receptors by substitution of the segment which contained the mutated position, by a homologous one. An allosteric drug could mimic the effects of such substitution. Within this framework, I highlight the advantages of allosteric drugs and the challenges in their design.How do the cullins, with conserved structures, accommodate substrate-binding proteins with distinct shapes and sizes? Cullin-RING E3 ubiquitin ligases facilitate ubiquitin transfer from E2 to the substrate, tagging the substrate for degradation. They contain substrate-binding, adaptor, cullin, and Rbx proteins. Previously, we showed that substrate-binding and Rbx proteins are flexible. This allows shortening of the E2-substrate distance for initiation of ubiquitination or increasing the distance to accommodate the polyubiquitin chain. However, the role of the cullin remained unclear. Is cullin a rigid scaffold, or is it flexible and actively assists in the ubiquitin transfer reaction? Why are there different cullins, and how do these cullins specifically facilitate ubiquitination for different substrates? To answer these questions, we performed structural analysis and molecular dynamics simulations based on Cul1, Cul4A, and Cul5 crystal structures. Our results show that these three cullins are not rigid scaffolds but are flexible with conserved hinges in the N-terminal domain. However, the degrees of flexibilities are distinct among the different cullins. Of interest, Cul1 flexibility can also be changed by deletion of the long loop (which is absent in Cul4A) in the N-terminal domain, suggesting that the loop may have an allosteric functional role. In all three cases, these conformational changes increase the E2-substrate distance to a specific range to facilitate polyubiquitination, suggesting that rather than being inert scaffold proteins, cullins allosterically regulate ubiquitination.

阿尔茨海默病 (AD) 是一种错误折叠的蛋白质疾病，其特征是 β-淀粉样蛋白 (Abeta) 肽以老年斑形式积累、进行性神经退行性变和记忆丧失。最近的证据表明，AD 病理与由 Abeta 肽制成的有毒孔介导的细胞离子稳态的不稳定有关。了解这些毛孔传导电信号和分子信号的确切性质有助于确定预防和治疗 AD 的潜在治疗靶点。在这里，我们使用原子力显微镜（AFM）和分子动力学（MD）模拟，将成像的孔结构与模型进行比较，以预测通道构象作为氨基酸序列的函数。野生型 Abeta(1-42) 肽中的位点特异性氨基酸 (AA) 取代可提供有关单个 AA 残基的位置和对其特征性结构-活性关系的重要性的信息。我们选择了两种 AA，MD 模拟预测它们会抑制或允许孔隙电导。先前已证明用 Pro 替代 Phe19 可以消除平面脂质双层系统中的电导。我们的 MD 模拟预测了具有塌陷孔的类似通道的形状，这得到了 AFM 通道图像的支持。我们建议，脯氨酸（一种已知的 β-片层破坏剂）会在毛孔中心产生扭结，并通过堵塞来防止电导。该残留物可能是药物开发研究的可行目标，旨在抑制 Abeta 诱导离子不稳定毒性。用 Cys 替代 Phe20 后，在 AFM 图像中表现出与野生型无法区分的孔结构。 MD 模拟预测位点 20 面向溶剂化孔。总体而言，这些突变支持之前预测的基于 β 片层的通道结构。 在阿尔茨海默病和额颞叶痴呆中，微管相关蛋白 Tau 形成细胞内成对的螺旋丝。细丝不仅可以由全长人类 Tau 蛋白形成，还可以由三个重复 (K19) 或四个重复 (K18) Tau 片段形成。然而，有趣的是，通过实验，K19 可以为 K18 播种，但反之则不然。为了深入了解 K18 和 K19 聚集体之间的交叉播种，本文将具有 U 形、L 形和长直线形（SL 形）构象的重复 3 (R3) 的 K18 和 K19 八聚体组装成不同的结构。模拟结果表明，R3 呈 L 形的 K18-8/K19-8（K18 和 K19 组装体编号 8）和 R3 呈 SL 形的 K18-9/K19-9 数量较多，并且在所有模拟的 K18 和 K19 八聚体中表现出最高的结构相似性，表明 K18/K19 的类似折叠可能作为 K18-K19 的结构核心 共组装异质长丝。我们证明稳定的 R2 和 R3 构象的形成是 K18 聚集的关键步骤，而 R3 对于 K19 纤维化至关重要。 K18 和 K19 中的不同核心单元可能会为 K18 种子产生交叉播种障碍，以触发 K19 原纤维生长，因为 R2 不适用于 K19。我们的研究提供了对涉及异质结构的交叉播种的见解。蛋白质聚集的多态性可以在交叉播种过程中放大。如果播种构象导致能量景观出现太大分歧，则可能会阻碍原纤维的形成。这种效应也可能导致 K18 和 K19 之间的不对称屏障。超过两打被称为淀粉样蛋白疾病的临床综合征的特征是组织中扩展的不溶性纤维沉积物的积累。这些被称为淀粉样蛋白的无定形刚果红染色沉积物表现出特征性的绿色双折射和交叉β结构。大量证据表明淀粉样蛋白的寡聚中间体在这些慢性疾病的发病机制中是有毒物质。越来越多的数据表明，这些有毒物质在细胞膜中形成离子通道，导致钙稳态破坏、膜去极化、能量流失，在某些情况下甚至导致细胞凋亡。淀粉样肽通道表现出许多常见的生物学特性，包括通用的 U 形 β 链转角 β 链结构、不可逆和自发插入膜、产生大的异质单通道电导、相对较差的离子选择性、刚果红抑制和锌阻断通道。最近的证据表明，淀粉样蛋白数量的增加不仅对其宿主靶细胞有毒，而且还具有抗菌活性。此外，至少一种人类抗菌肽 protegrin-1 通过通道形成机制杀死微生物，已被证明具有形成延伸淀粉样原纤维的能力，与经典的形成疾病的淀粉样蛋白非常相似。突变，即使不是直接发生在蛋白质的配体结合位点，也可能导致疾病。在细胞表面受体中，如果它们解开激动剂（或拮抗剂）结合胞外结构域和胞内反应时发生的构象变化，就会发生这种情况。解偶联可以通过破坏细胞外和细胞内结构域之间的主要变构传播途径来发生。在这里我提供一个机制解释：我首先描述传播是如何发生的；其次，当存在远离结合位点的疾病相关突变时会发生什么；最后，药物如何克服这种破坏和救援功能。甘氨酸受体 α1 亚基 (α1R271Q/L) 中的突变会导致神经运动障碍亢进症，这是此类变构突变的一个例子。在本期印度人民党中，Shan 等人。表明通过用同源片段替换包含突变位置的片段，这些突变受体恢复了正常功能。变构药物可以模拟这种替代的效果。在此框架内，我强调了变构药物的优势及其设计中的挑战。具有保守结构的cullin如何容纳具有不同形状和大小的底物结合蛋白？ Cullin-RING E3 泛素连接酶促进泛素从 E2 转移到底物，标记底物进行降解。它们含有底物结合蛋白、接头蛋白、cullin 蛋白和 Rbx 蛋白。之前，我们证明底物结合蛋白和 Rbx 蛋白是灵活的。这允许缩短用于起始泛素化的E2-底物距离或增加距离以适应多泛素链。然而，库林的作用仍不清楚。 cullin 是刚性支架，还是灵活且积极协助泛素转移反应？为什么有不同的 cullin，以及这些 cullin 如何专门促进不同底物的泛素化？为了回答这些问题，我们基于Cul1、Cul4A和Cul5晶体结构进行了结构分析和分子动力学模拟。我们的结果表明，这三个 cullin 不是刚性支架，而是具有柔性，在 N 端结构域具有保守的铰链。然而，不同的库林之间的灵活性程度是不同的。有趣的是，Cul1 的灵活性也可以通过删除 N 端结构域中的长环（Cul4A 中不存在）来改变，这表明该环可能具有变构功能作用。在所有三种情况下，这些构象变化将 E2 底物距离增加到特定范围以促进多聚泛素化，这表明 cullin 不是惰性支架蛋白，而是变构调节泛素化。