Structural and Biological Characterization of Diverse Oligomers Derived from Abeta

Abeta 衍生的多种低聚物的结构和生物学表征

基本信息

批准号：
10214205
负责人：
JAMES S NOWICK
金额：
$ 137.31万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10214205
关键词：
Address Affect Alzheimer&apos s Disease Amyloid beta-Protein Antibodies Apoptosis Astrocytes Biochemical Biological Biology Biophysics Brain C-terminal Calcium Cell Separation Diagnostic Engineering Event Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Heterogeneity Homeostasis Immunologics Knowledge Laboratories Length Light Link Microglia Modeling Molecular Molecular Sieve Chromatography Molecular Target Mus N-terminal NMR Spectroscopy Necrosis Nerve Degeneration Neurites Neurons Oxidative Stress Peptide Fragments Peptides Positioning Attribute Property Proteins Research Roentgen Rays Site Slice Spectrum Analysis Structural Models Structure Tail Vertebral column X-Ray Crystallography abeta accumulation abeta oligomer amyloid peptide analytical ultracentrifugation base brain cell cell type crosslink design dimer disulfide bond endoplasmic reticulum stress experimental study immunoreactivity insight methyl group polyclonal antibody protein aminoacid sequence response structural biology synthetic peptide tau aggregation tau phosphorylation uptake

项目摘要

Project Summary/Abstract: Understanding the aggregation of the β-amyloid peptide (Aβ) to form toxic oligomers is fundamental to understanding the molecular basis of Alzheimer’s disease (AD). Despite decades of research, the structures of Aβ oligomers remain a mystery, constituting a significant gap in understanding AD. This proposal seeks to address this knowledge gap through the structural, biophysical, and biological profiling of a diverse group of Aβ oligomer models and correlation of these models with biogenic Aβ oligomers. My laboratory has developed an approach for create structurally defined Aβ oligomer models composed of peptide fragments from Aβ constrained into a β-hairpin. The X-ray crystallographic structures of these Aβ β- hairpin peptides reveal the structures of oligomers that the peptides can form and key intermolecular contacts that the peptides make in the oligomeric state. These contacts reveal sites that can be crosslinked to create covalently stabilized Aβ oligomer models that mimic the crystallographic oligomers. Studying the crosslinked oligomers then allows detailed correlation between oligomer structure and biophysical and biological properties. We will characterize how our Aβ oligomer models interact with and affect neurons, microglia, and astrocytes, to provide detailed insights into how our Aβ oligomer models impact different brain cell types and thus help shed light on the relationship between Aβ oligomer structure and cellular events that occur in AD. We will use fluorescence microscopy and fluorescence-assisted cell sorting (FACS) to visualize and quantify the interactions and uptake of our Aβ oligomer models with neurons, microglia, and astrocytes. We will evaluate downstream biochemical and cellular effects elicited by Aβ oligomer models in neurons, microglia, and astrocytes and evaluate how treatment affects apoptosis, necrosis, calcium homeostasis, endoplasmic reticulum stress, oxidative stress, neurite length, tau phosphorylation and aggregation, and proinflammatory responses in microglia and astrocytes. To elucidate the relationship between the structures of our Aβ oligomer models and biogenic Aβ oligomers, we will generate polyclonal antibodies against each Aβ oligomer model and then examine the immunoreactivity of these antibodies with brain protein extract and brain slices from 5XFAD mice. We will discover new Aβ oligomer models, by creating new Aβ β-hairpin peptides that contain more of the Aβ peptide sequence and alternate β-strand alignments. We will create new crosslinked Aβ oligomer models by identifying key contacts in existing and newly discovered Aβ oligomer models and then engineering in disulfide bonds to stabilize the oligomers. To characterize the structures and oligomerization properties of the new Aβ oligomer models that we generate, we will use X-ray crystallography and a variety of other biophysical experiments, including CD spectroscopy, SDS-PAGE, size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), NMR spectroscopy, and Förster resonance energy transfer (FRET) studies. The biological and immunological properties of these new Aβ oligomer models will be studied as described above.

项目摘要/摘要：了解 β-淀粉样肽 (Aβ) 的聚集形成有毒物质尽管几十年来，低聚物对于理解阿尔茨海默病 (AD) 的分子基础至关重要。在研究中，Aβ 寡聚物的结构仍然是个谜，这在理解 AD 方面构成了重大差距。该提案旨在通过结构、生物物理和生物学分析来解决这一知识差距一组不同的 Aβ 寡聚物模型以及这些模型与生物 Aβ 寡聚物的相关性。我的实验室开发了一种创建结构明确的 Aβ 寡聚体模型的方法，该模型由以下组成：来自 Aβ 的肽片段被限制在 β-发夹中这些 Aβ β- 的 X 射线晶体结构。发夹肽揭示了肽可以形成的寡聚物的结构以及关键的分子间接触这些接触揭示了可以交联以产生的位点。研究交联低聚物的共价稳定 Aβ 低聚物模型。然后，低聚物允许低聚物结构与生物物理和生物学特性之间的详细关联。我们将描述我们的 Aβ 寡聚体模型如何与神经元、小胶质细胞和星形胶质细胞相互作用并影响它们，提供有关我们的 Aβ 寡聚物模型如何影响不同脑细胞类型的详细见解，从而帮助摆脱我们将使用 Aβ 寡聚体结构与 AD 中发生的细胞事件之间的关系。荧光显微镜和荧光辅助细胞分选 (FACS) 来可视化和量化相互作用我们将评估下游的 Aβ 寡聚体模型与神经元、小胶质细胞和星形胶质细胞的摄取。 Aβ 寡聚体模型在神经元、小胶质细胞和星形胶质细胞中引起的生化和细胞效应评估治疗如何影响细胞凋亡、坏死、钙稳态、内质网应激、氧化应激、神经突长度、tau 磷酸化和聚集以及促炎反应阐明我们的 Aβ 寡聚体模型的结构与小胶质细胞和星形胶质细胞之间的关系。生物 Aβ 寡聚体，我们将针对每个 Aβ 寡聚体模型生成多克隆抗体，然后检查这些抗体与 5XFAD 小鼠脑蛋白提取物和脑切片的免疫反应性。我们将通过创建新的 Aβ β-发夹肽来发现新的 Aβ 寡聚物模型，其中包含更多 Aβ 肽序列和替代 β 链排列我们将通过以下方式创建新的交联 Aβ 寡聚物模型。识别现有和新发现的 Aβ 寡聚体模型中的关键接触点，然后进行二硫键改造稳定寡聚物的键来表征新 Aβ 的结构和寡聚特性。我们生成的低聚物模型，我们将使用 X 射线晶体学和各种其他生物物理实验，包括 CD 光谱、SDS-PAGE、尺寸排阻色谱 (SEC)、分析超速离心 (AUC)、NMR 光谱和福斯特共振能量转移 (FRET) 研究。这些新的 Aβ 寡聚体模型的生物学和免疫学特性将如上所述进行研究。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Macrocyclic Peptides Derived from Familial Alzheimer's Disease Mutants Show Charge-Dependent Oligomeric Assembly and Toxicity.

源自家族性阿尔茨海默病突变体的大环肽表现出电荷依赖性寡聚组装和毒性。

DOI：
发表时间：
2022-03-16
期刊：
影响因子：
5
作者：
Howitz, William J;Guaglianone, Gretchen;McKnelly, Kate J;Haduong, Katelyn;Ashby, Shareen N;Laayouni, Mohamed;Nowick, James S
通讯作者：
Nowick, James S

Effects of Familial Alzheimer's Disease Mutations on the Assembly of a β-Hairpin Peptide Derived from Aβ16-36.

家族性阿尔茨海默氏病突变对 Aβ16-36 衍生的 β-发夹肽组装的影响。

DOI：
发表时间：
2022-03-15
期刊：
影响因子：
2.9
作者：
McKnelly, Kate J;Kreutzer, Adam G;Howitz, William J;Haduong, Katelyn;Yoo, Stan;Hart, Candace;Nowick, James S
通讯作者：
Nowick, James S

Correction to "β-Hairpin Alignment Alters Oligomer Formation in Aβ-Derived Peptides".

更正“β 发夹排列改变 Aβ 衍生肽中的寡聚体形成”。

DOI：
发表时间：
2024-03-05
期刊：
影响因子：
2.9
作者：
Ruttenberg, Sarah M;Kreutzer, Adam G;Truex, Nicholas L;Nowick, James S
通讯作者：
Nowick, James S

A β-barrel-like tetramer formed by a β-hairpin derived from Aβ.

由源自 Aβ 的 β 发夹形成的 β 桶状四聚体。

DOI：
发表时间：
2023-12-20
期刊：
影响因子：
8.4
作者：
Samdin, Tuan D;Jones, Chelsea R;Guaglianone, Gretchen;Kreutzer, Adam G;Freites, J Alfredo;Wierzbicki, Michał;Nowick, James S
通讯作者：
Nowick, James S

An Improved Turn Structure for Inducing β-Hairpin Formation in Peptides.

一种改进的转弯结构，用于诱导肽中β-发夹的形成。