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）是一种长18个残基、富含半胱氨酸的β -sheet抗菌肽（AMP）。PG-1对细胞膜具有很强的细胞毒活性，是一种有效的抗生素。早前我们报道了其细胞毒性是由其通道形成能力介导的。在这项研究中，我们检测了PG-1的淀粉样原纤维形成特性，并将其与定义明确的淀粉样蛋白进行了比较，即淀粉样蛋白- β (β(1-42))肽。我们利用原子力显微镜（AFM）和硫黄素- t染色研究PG-1原纤维的生长动力学和分子动力学模拟来阐明其潜在的机制。PG-1在高亲水性表面（云母）上的AFM图像显示出与Abeta（1-42）原纤维形态相似的原纤维。纤维生长的实时AFM成像表明，PG-1纤维的生长与Abeta（1-42）原纤维相比具有相对较快的动力学。AFM结果与硫黄素- t染色数据的结果非常一致。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，我们没有检测到PG-1在阴离子脂质双分子层2-二油基-sn-甘油-3-磷酸- l-丝氨酸/1-棕榈酰-2-油基-sn-甘油-3-磷酸乙醇胺上的纤维状结构；只能观察到小的PG-1低聚物。分子动力学模拟能够识别膜双分子层上这些小的低聚物的存在。因此，我们目前的结果表明，细胞毒性AMP PG-1是淀粉样变性的，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β），除了细胞毒性和淀粉样蛋白原性外，它们还具有共同的结构基序，并且是通道形成。这些综合性质支持淀粉样蛋白生成肽和富含-薄片的细胞溶解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）原纤维的结构对阿尔茨海默病的研究很有意义，因为这是在疾病早期设计针对Abeta（1-40）原纤维形成的治疗方法所必需的。M35是一个至关重要的残基，因为它具有潜在的氧化作用，并且在β -原纤维中β -链和β -片之间具有很强的相互作用。实验数据表明，Abeta（9-40）原纤维的三重对称结构通过原纤维轴上的M35相互作用和不同交叉β单元之间的强I31-V39相互作用形成了紧密的疏水核。在实验数据的基础上，我们探测了具有全长Abeta（1-40）三重对称的构象。基于ssNMR数据的显式溶剂中全原子分子动力学模拟再现了M35-M35和I31-V39距离的实验观测结果。我们对实验数据的解释表明，在纤维三重对称结构中观测到的5-7埃的M35-M35距离可能与沿纤维轴的M35相互作用有关，而不是与沿纤维轴的M35相互作用有关，因为我们测量的M35-M35在纤维轴上的距离始终在15埃以上。因此，我们发现独特的Abeta（1-40）三角形结构沿着纤维轴有一个大的空腔，并且n -末端可以通过与U-turn结构域或c -末端结构域相互作用来帮助纤维的稳定。我们的研究结果，加上最近对Abeta（1-42）原纤维中空核的cyroEM表征，指出了Abeta（1-40/1-42）低聚物中空腔的相关性，这在靶向低聚物毒性时应该考虑到。我们研究了转录因子（tf）沿着增强子DNA特异性组装的机制。ifn - β增强体提供了一个很好的模型系统：它很小；其组成的晶体结构是可用的；还有生化和细胞数据。在ifn - β增强体中，即使连续的DNA反应元件（REs）重叠，也很少有蛋白质-蛋白质相互作用。我们对来自增强体的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的特异性结合可以增强另一个TF与相邻RE的结合，并通过RE的重叠限制其他TF；re的顺序可以决定形成何种配合物；共识REs和非共识REs的交替可以通过优化伙伴之间的相互作用来调节结合特异性。我们的观察结果解释了尽管增强子DNA上邻近tf之间的相互作用有限，但如何获得特异性和协同性。迄今为止，当研究选择性TF结合时，注意力主要集中在RE序列上。然而，DNA上转录因子的顺序和它们之间间隔物的长度可能是转录因子在增强子上特定组合组装的关键因素，从而影响其功能。我们的结果强调通过RE结合位点组织的协同性。