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-淀粉样胡椒（Aβ）的聚集以形成有毒低聚物是理解阿尔茨海默氏病（AD）的分子基础的基础。尽管数十年研究，Aβ低聚物的结构仍然是一个神秘的，构成了理解AD的显着差距。该建议旨在通过结构，生物物理和生物学分析来解决这一知识差距 Aβ低聚物模型的潜水组以及这些模型与生物Aβ低聚物的相关性。我的实验室已经开发了一种创建结构定义的Aβ低聚物模型的方法来自Aβ的肽片段被约束成β-发蛋白。这些Aβ-β-的X射线晶体结构发夹petides揭示了宠物可以形成和关键分子间接触的低聚物的结构肽在低聚状态下产生的。这些触点揭示了可以交联以创建的站点共价稳定模仿晶体学寡聚物的Aβ低聚物模型。研究交联然后，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。我们将表征我们的Aβ低聚物模型如何与神经元，小胶质细胞和星形胶质细胞相互作用并影响为了提供有关我们的Aβ低聚物模型如何影响不同脑细胞类型的详细见解，从而有助于脱落关于Aβ低聚物结构与AD中发生的细胞事件之间的关系。我们将使用荧光显微镜和荧光辅助细胞分选（FACS），以可视化和量化相互作用并用神经元，小胶质细胞和星形胶质细胞吸收我们的Aβ低聚物模型。我们将评估下游 Aβ低聚物模型在神经元，小胶质细胞和星形胶质细胞中引起的生化和细胞作用，以及评估治疗如何影响凋亡，坏死，钙稳态，内质网应激，在小胶质细胞和星形胶质细胞。阐明我们Aβ低聚物模型的结构与生物Aβ低聚物，我们将对每个Aβ低聚物模型产生多克隆抗体，然后检查这些抗体用脑蛋白提取物和5xFAD小鼠的脑切片的免疫反应性。我们将通过创建新的Aββ-发peperides来发现新的Aβ低聚物模型，该模型包含更多 Aβ肽序列和替代β链比对。我们将通过在现有和新发现的Aβ低聚物模型中识别关键接触，然后在二硫化物中进行工程粘结以稳定低聚物。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，尺寸排除色谱法（SEC），分析超速离心（AUC），NMR光谱和Förster共振能量转移（FRET）研究。如上所述，这些新Aβ低聚物模型的生物学和免疫学特性将进行研究。

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）

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：
10.1021/acs.biochem.4c00037
发表时间：
2024
期刊：
Biochemistry
影响因子：
2.9
作者：
Ruttenberg,SarahM;Kreutzer,AdamG;Truex,NicholasL;Nowick,JamesS
通讯作者：
Nowick,JamesS

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

DOI：
10.1021/acs.biochem.3c00526
发表时间：
2024-01-16
期刊：
BIOCHEMISTRY
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.

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

DOI：
10.1021/acschemneuro.1c00833
发表时间：
2022-03-16
期刊：
ACS chemical neuroscience
影响因子：
5
作者：
Howitz WJ;Guaglianone G;McKnelly KJ;Haduong K;Ashby SN;Laayouni M;Nowick JS
通讯作者：
Nowick JS

Enantiomeric β-sheet peptides from Aβ form homochiral pleated β-sheets rather than heterochiral rippled β-sheets.

DOI：
10.1039/d2sc02080g
发表时间：
2022-07-06
期刊：
Chemical science
影响因子：
8.4
作者：
通讯作者：

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

DOI：
10.1039/d3sc05185d
发表时间：
2023-12-20
期刊：
CHEMICAL SCIENCE
影响因子：
8.4
作者：
Samdin, Tuan D.;Jones, Chelsea R.;Guaglianone, Gretchen;Kreutzer, Adam G.;Freites, J. Alfredo;Wierzbicki, Michal;Nowick, James S.
通讯作者：
Nowick, James S.