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: 8349004
• 负责人: Ruth Nussinov
• 金额: $ 64.26万
• 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/8349004
• 关键词: Address; Agreement; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Antibiotics; Atomic Force Microscopy; Attention; Binding; Binding Sites; Biochemical; Biological Models; Cell membrane; Complex; Consensus; Cysteine; DNA; Data; Disease; Drug Design; Enhancers; Growth; Image; Interferon-beta; Ions; Kinetics; Length; Ligands; Lipid Bilayers; Measures; Mediating; Membrane; Mica; Molecular Conformation; Neurodegenerative Disorders; Parkinson Disease; Peptides; Population; Property; Proteins; Reporting; Research; Response Elements; Seeds; Serine; Solutions; Solvents; Specificity; Staging; Staining method; Stains; Structure; Surface; Thioflavin T; Time; Toxic effect; amyloid formation; antimicrobial; antimicrobial peptide; base; beta pleated sheet; combinatorial; conformer; cytotoxic; cytotoxicity; design; interest; molecular dynamics; oxidation; phosphoethanolamine; protegrin PG-1; protein function; protein protein interaction; protein structure; research study; therapeutic target; transcription factor

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich beta-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-beta (Abeta(1-42)) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Abeta(1-42) fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Abeta(1-42) fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing beta-rich AMPs and amyloids such as Abeta, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and beta-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function. Elucidating the structure of Abeta(1-40) fibrils is of interest in Alzheimer's disease research because it is required for designing therapeutics that target Abeta(1-40) fibril formation at an early stage of the disease. M35 is a crucial residue because of its potential oxidation and its strong interactions across beta-strands and across beta-sheets in Abeta fibrils. Experimentally, data for the three-fold symmetry structure of the Abeta(9-40) fibril suggest formation of tight hydrophobic core through M35 interactions across the fibril axis and strong I31-V39 interactions between different cross-beta units. Herein, on the basis of experimental data, we probe conformers with three-fold symmetry of the full-length Abeta(1-40). Our all-atom molecular dynamics simulations in explicit solvent of conformers based on the ssNMR data reproduced experimental observations of M35-M35 and I31-V39 distances. Our interpretation of the experimental data suggests that the observed 5-7 Angstrom M35-M35 distance in the fibril three-fold symmetry structure is likely to relate to M35 interactions along the fibril axis, rather than across the fibril axis, since our measured M35-M35 distances across the fibril axis are consistently above 15 Angstrom. Consequently, we revealed that the unique Abeta(1-40) triangular structure has a large cavity along the fibril axis and that the N-termini can assist in the stabilization of the fibril by interacting with the U-turn domains or with the C-termini domains. Our findings, together with the recent cyroEM characterization of the hollow core in Abeta(1-42) fibrils, point to the relevance of a cavity in Abeta(1-40/1-42) oligomers which should be considered when targeting oligomer toxicity. We addressed the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA. The IFN-beta enhanceosome provides a good model system: it is small; its components crystal structures are available; and there are biochemical and cellular data. In the IFN-beta enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs; the order of the REs can determine which complexes will form; and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization.

Protegrin-1（PG-1）是一种18个残基长的富含半胱氨酸的β折叠抗菌肽（AMP）。PG-1在细胞膜上诱导强烈的细胞毒活性，并作为一种有效的抗生素。早先我们报道了它的细胞毒性是由它的通道形成能力介导的。在这项研究中，我们研究了PG-1与一种定义明确的淀粉样蛋白-β（Abeta（1-42））肽相比的淀粉样蛋白原纤维形成特性。我们已经使用原子力显微镜（AFM）和硫磺素-T染色研究PG-1纤维生长的动力学和分子动力学模拟，以阐明潜在的机制。PG-1在高亲水性表面（云母）上的AFM图像显示出与Abeta（1-42）原纤维具有形态相似性的原纤维。原纤维生长的实时AFM成像表明，与Abeta（1-42）原纤维相比，PG-1原纤维生长遵循相对快的动力学。原子力显微镜的结果是在从硫磺素-T染色数据的结果密切一致。此外，结果表明PG-1在溶液中形成原纤维。值得注意的是，相比之下，我们在阴离子脂质双层2-二油酰基-sn-甘油基-3-磷酸-L-丝氨酸/1-棕榈酰基-2-油酰基-sn-甘油基-3-磷酸乙醇胺上没有检测到PG-1的纤维状结构;只能观察到小的PG-1寡聚体。分子动力学模拟能够识别膜双层上这些小的低聚物的存在。因此，我们目前的研究结果表明，细胞毒性AMP PG-1是淀粉样蛋白和能够形成原纤维。总的来说，比较富含β的AMP和淀粉样蛋白如Abeta，除了细胞毒性和淀粉样蛋白原性之外，它们具有共同的结构基序，并且是通道形成的。这些综合性质支持淀粉样蛋白生成肽和富含β折叠的细胞溶解性AMP之间的功能关系，表明淀粉样蛋白通道可能具有抗菌功能。阐明Abeta（1-40）原纤维的结构在阿尔茨海默病研究中是令人感兴趣的，因为它是设计在疾病早期靶向Abeta（1-40）原纤维形成的治疗剂所必需的。M35是一个关键的残基，因为它的潜在氧化和它的强大的相互作用，跨β链和跨β折叠在Abeta纤维。实验上，Abeta（9-40）原纤维的三重对称结构的数据表明，通过跨原纤维轴的M35相互作用和不同交叉β单元之间的强I31-V39相互作用形成紧密的疏水核心。在此，根据实验数据，我们探测全长Abeta（1-40）的三重对称构象。我们的全原子分子动力学模拟在显式溶剂的构象的基础上ssNMR数据再现实验观察到的M35-M35和I31-V39距离。我们对实验数据的解释表明，在原纤维三重对称结构中观察到的5-7埃M35-M35距离可能与沿着原纤维轴而不是穿过原纤维轴的M35相互作用有关，因为我们测量的穿过原纤维轴的M35-M35距离始终高于15埃。因此，我们揭示了独特的Abeta（1-40）三角形结构沿着原纤维轴具有大的空腔沿着，并且N-末端可以通过与U-转弯结构域或与C-末端结构域相互作用来帮助原纤维的稳定。我们的研究结果，连同最近对Abeta（1-42）原纤维中空核心的cyroEM表征，指出了Abeta（1-40/1-42）寡聚体中空腔的相关性，当靶向寡聚体毒性时应考虑到这一点。我们解决了转录因子（TF）组装的机制，通过特异性沿着增强子DNA。IFN-β增强体提供了一个很好的模型系统：它很小;它的组分晶体结构是可用的;并且有生化和细胞数据。在IFN-β增强体中，即使连续的DNA反应元件（RE）重叠，也很少有蛋白质-蛋白质相互作用。我们对增强体不同基序组合的分子动力学（MD）模拟表明，协同性是通过RE的独特组织实现的：一个TF的特异性结合可以通过RE的重叠来增强另一个TF与相邻RE的结合并限制其他TF; RE的顺序可以决定哪些复合物将形成;共有和非共有RE的交替可以通过优化配偶体之间的相互作用来调节结合特异性。我们的观察结果提供了一个解释，尽管增强子DNA上相邻TF之间的相互作用有限，但特异性和协同性是如何实现的。迄今为止，在解决选择性TF结合时，注意力主要集中在RE序列上。然而，RE在DNA上的顺序和它们之间的间隔区的长度可能是TF在增强子上的特异性组合组装的关键因素，因此在功能上。我们的研究结果强调通过RE结合位点组织的协同性。