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）肽积累为老年斑，进行性神经变性和记忆丧失。最近的证据表明，阿尔茨海默病的病理与细胞离子稳态的不稳定有关，这种不稳定是由β肽构成的毒性孔介导的。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，将成像的孔隙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）肽的位点特异性氨基酸（AA）替换产生了关于单个AA残基的位置和对其特征构效关系的重要性的信息。我们选择了两种原子吸收剂，我们的MD模拟预测它们会抑制或允许孔隙传导。先前已经证明，用Pro取代Phe19可以消除平面脂质双分子层系统中的电导。我们的MD模拟预测了一个具有塌陷孔的通道状形状，这得到了AFM通道图像的支持。我们认为脯氨酸是一种已知的破片剂，它会在孔的中心形成一个扭结，并通过堵塞来阻止传导。该残基可能是药物开发研究的可行靶点，旨在抑制β诱导离子不稳定毒性。在AFM图像中，用Cys取代Phe20显示出与野生型难以区分的孔隙结构。MD模拟预测site 20面对溶剂化孔隙。总的来说，这些突变支持先前预测的基于β -薄片的通道结构。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细丝。这些细丝不仅可以由全长的人Tau蛋白形成，也可以由3个重复的（K19）或4个重复的（K18） Tau片段形成。然而，有趣的是，在实验中，K19可以播种K18，反之则不能。为了深入了解K18和K19聚集体之间的交叉播种，本文将u形、l形和长直线形三种构型的重复3 （R3）的K18和K19八聚体组装成不同的结构。模拟结果表明，K18-8/K19-8 （K18和K19组合体编号8）和K18-9/K19-9 （K18和K19组合体编号8）的R3为L型，K18-9/K19-9的R3为SL型的八聚体密度高，结构相似性最高，表明K18/K19的相似折叠可能是K18-K19共组装非均质细丝的结构核心。我们证明了稳定的R2和R3构象的形成是K18聚集的关键步骤，R3是K19成纤维的关键步骤。由于R2不适合K19， K18和K19中不同的核心单位可能为K18种子触发K19原纤维生长创造了交叉播种屏障。我们的研究为涉及异质结构的交叉播种提供了见解。在交叉播种过程中，蛋白质聚集的多态性被放大。如果种子构象导致能量景观中太多的分歧，它可能会阻碍纤维的形成。这种效应也可能导致K18和K19之间的不对称屏障。有二十多种被称为淀粉样蛋白疾病的临床综合征，其特征是组织中延伸的不溶性纤维沉积物的积累。这些无定形刚果红染色沉积物被称为淀粉样物，具有典型的绿色双折射和交叉结构。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的。越来越多的数据表明，这些有毒物质在细胞膜上形成离子通道，导致钙稳态破坏，膜去极化，能量流失，在某些情况下导致细胞凋亡。淀粉样肽通道表现出许多共同的生物学特性，包括普遍的u型β -链-转-链结构、不可逆和自发插入膜、产生大的非均质单通道电导、相对较差的离子选择性、刚果红的抑制作用和锌的通道阻断作用。最近的证据表明，淀粉样蛋白的增加不仅对宿主靶细胞有毒，而且还具有抗菌活性。此外，至少有一种人类抗菌肽，蛋白蛋白-1，通过通道形成机制杀死微生物，已被证明具有形成延伸的淀粉样蛋白原纤维的能力，与经典的形成疾病的淀粉样蛋白原纤维非常相似。突变，即使不是直接发生在蛋白质的配体结合位点，也会导致疾病。在细胞表面受体中，当激动剂（或拮抗剂）结合胞外结构域和细胞内反应时发生的构象变化偶联时，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。这里我提供一个机械的解释：我首先描述传播是如何发生的；第二，在远离结合位点的疾病相关突变存在的情况下会发生什么；最后，药物如何克服这种破坏和拯救功能。导致神经运动障碍过度增生的甘氨酸受体α 1亚基（α 1r271q /L）突变就是这种变构突变的一个例子。在这一期的人民党中，Shan等人。结果表明，通过替换含有突变位置的片段，这些突变受体恢复了正常功能。一种变构药物可以模拟这种取代的效果。在这个框架内，我强调了变构药物的优势和它们设计中的挑战。具有保守结构的cullins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，我们发现底物结合蛋白和Rbx蛋白是灵活的。这允许缩短e2 -底物距离以启动泛素化或增加距离以容纳多泛素链。然而，扑杀的作用仍不清楚。cullin是一种刚性支架，还是一种灵活的、积极协助泛素转移反应的支架？为什么会有不同的cullins，这些cullins如何特别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的晶体结构进行了结构分析和分子动力学模拟。我们的研究结果表明，这三种cullins不是刚性支架，而是在n端结构域中具有保守铰链的柔性支架。然而，在不同的cullins中，灵活性的程度是不同的。有趣的是，Cul1的柔韧性也可以通过删除n端结构域的长环（Cul4A中不存在）而改变，这表明该环可能具有变构功能作用。在这三种情况下，这些构象变化将e2 -底物距离增加到特定范围，以促进泛素化，这表明cullins不是惰性支架蛋白，而是变构调节泛素化。