权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Cellular, molecular and functional characterization of proteasomes in EAE

EAE 中蛋白酶体的细胞、分子和功能表征

基本信息

批准号：
8723318
负责人：
OSCAR A BIZZOZERO
金额：
$ 18.69万
依托单位：
UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8723318
关键词：
Active Immunization Acute Affect Animal Model Antibodies Astrocytes Binding Biological Assay C57BL/6 Mouse Cell Survival Cells Cerebellum Chronic Complex Data Demyelinating Diseases Disease Disease Progression Excision Experimental Autoimmune Encephalomyelitis Female Gel Gel Chromatography Goals Hemoglobin Immunization Immunohistochemistry Impairment Inflammation Inflammatory Laboratories Measures Microglia Molecular Multiple Sclerosis Muramidase Mus Nerve Degeneration Nerve Tissue Neurologic Neurons Nucleosome Core Particle Oligodendroglia Oxidative Stress Pattern Peptide Hydrolases Peptides Phase Polyacrylamide Gel Electrophoresis Preparation Proteins Series Spinal Cord Staging Stress Structure Testing Therapeutic Tissues Ubiquitin Western Blotting base cell type design fast protein liquid chromatography inhibitor/antagonist multicatalytic endopeptidase complex nervous system disorder novel protein misfolding public health relevance research study young adult

项目摘要

DESCRIPTION (provided by applicant): Our laboratory has previously shown that multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are characterized by severe oxidative stress and the accumulation of oxidized proteins in the CNS, which in turn reduces cell viability and leads to tissue damage. Build-up of oxidized/misfolded proteins often occurs as a consequence of deficiencies in the proteasome, the enzymatic machinery responsible for their proteolytic removal. We have also discovered that in both MS and chronic EAE there is a significant reduction in the peptidolytic activities of the proteasome, which may explain the accumulation of oxidized proteins observed in the neurodegenerative stages of these diseases. While very important, all of these studies were carried out in tissue homogenates using fluorescent peptide substrates, and a critical issue that remains to be established is whether these deficits are localized to specific cell types or affect all CNS cells o the same extent. Furthermore, the proteolytic activity of the standard (s) and immuno (i) proteasome core particles (i.e. s-20S and i-20S) are greatly affected by the binding of regulatory caps (19S and 11S) and inhibitors, which may be expressed differentially in the various CNS cells during the course of the disease. In this proposal, we hypothesize that as a result of disease activity neurons, oligodendrocytes, astrocytes and microglia express unique patterns of proteasome complexes, making some CNS cells less equipped than others to remove abnormal proteins and thereby more susceptible to stress. To test this idea, we will first determine by double-immunohistochemistry the temporal/spatial pattern of proteasome core (s-20S and i-20S) and proteasome cap (19S and 11S) expression and their relationship to the extent of inflammation and oxidative stress during the course of EAE. We will then isolate neurons, oligodendrocytes, astrocytes and microglial cells from the CNS of control and EAE mice and measure the levels of each complex (20S, 11S- 20S-11S, 19S-20S-19S and 19S-20S-11S) by blue native-polyacrylamide gel electrophoresis and quantitative western blot analysis. Finally, we will compare the ability of the various proteasome complexes isolated from control and EAE tissues to digest oxidized and ubiquitinated proteins. These exploratory studies are essential for understanding how the various CNS cells regulate the expression and assembly of functional proteasome complexes as EAE progresses from the inflammatory to the neurodegenerative stages, and for determining the molecular basis for proteasome impairment in chronic EAE. Identification of the cell(s) with altered proteasome function will ultimately serve to design a therapeutic approach geared to decrease the toxic consequence of oxidative burden in EAE and MS.

描述（由申请人提供）：我们的实验室先前已经表明多发性硬化症（MS）及其动物模型实验性自身免疫性脑脊髓炎（EAE）的特征在于严重的氧化应激和CNS中氧化蛋白的积累，这反过来降低了细胞活力并导致组织损伤。氧化/错误折叠蛋白质的积累通常是由于蛋白酶体（负责蛋白水解去除的酶机制）缺陷而发生的。我们还发现，在MS和慢性EAE中，蛋白酶体的肽水解活性显著降低，这可以解释在这些疾病的神经退行性阶段观察到的氧化蛋白的积累。虽然非常重要，但所有这些研究都是使用荧光肽底物在组织匀浆中进行的，并且仍有待确定的关键问题是这些缺陷是否局限于特定细胞类型或以相同程度影响所有CNS细胞。此外，标准和免疫蛋白酶体核心颗粒（即s-20 S和i-20 S）的蛋白水解活性受到调节帽（19 S和11 S）和抑制剂的结合的极大影响，调节帽和抑制剂在疾病过程中可能在各种CNS细胞中差异表达。在这个提议中，我们假设，由于疾病活动的神经元，少突胶质细胞，星形胶质细胞和小胶质细胞表达蛋白酶体复合物的独特模式，使一些CNS细胞比其他细胞更不具备去除异常蛋白质，从而更容易受到压力。为了验证这一想法，我们将首先通过双重免疫组织化学确定蛋白酶体核心（s-20 S和i-20 S）和蛋白酶体帽（19 S和11 S）表达的时间/空间模式及其与EAE过程中炎症和氧化应激程度的关系。然后，我们将从对照和EAE小鼠的CNS中分离神经元、少突胶质细胞、星形胶质细胞和小胶质细胞，并通过蓝色天然聚丙烯酰胺凝胶电泳和定量蛋白质印迹分析测量每种复合物（20 S、11 S-20 S-11 S、19 S-20 S-19 S和19 S-20 S-11 S）的水平。最后，我们将比较从对照和EAE组织中分离的各种蛋白酶体复合物消化氧化和泛素化蛋白的能力。这些探索性的研究是必不可少的了解如何不同的中枢神经系统细胞调节功能性蛋白酶体复合物的表达和组装EAE的进展，从炎症到神经退行性阶段，并确定蛋白酶体损伤的分子基础在慢性EAE。鉴定具有改变的蛋白酶体功能的细胞将最终用于设计适合于降低EAE和MS中氧化负荷的毒性后果的治疗方法。