Immunoproteasome: A key component of the cellular stress response

免疫蛋白酶体：细胞应激反应的关键组成部分

• 批准号: 7510560
• 负责人: Deborah Ann Ferrington
• 金额: $ 14.72万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7510560
• 关键词: Age of Onset; Antigen Presentation; Apoptotic; Atrophic; Attenuated; Biochemistry; Biological Models; Biology; Brain; Catalytic Domain; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cells; Cellular Stress; Cellular Stress Response; Cellular biology; Chemicals; Chronic stress; Collaborations; Condition; Cultured Cells; Disease; Disease Progression; Endoplasmic Reticulum; Epithelial Cells; Exhibits; Exposure to; Half-Life; Hindlimb; Immune; Injury; Knockout Mice; Legitimacy; Modification; Molecular Chaperones; Molecular Genetics; Mus; Muscle; Muscle function; Muscular Atrophy; Myosin ATPase; Nature; Oxidative Stress; Peptides; Physiology; Play; Predisposition; Protein Isoforms; Proteins; Research; Research Proposals; Retina; Role; Signal Transduction; Skeletal Muscle; Stress; System; Techniques; Testing; Tissues; Toxic effect; Transgenic Mice; Tunicamycin; Up-Regulation; Work; ataxin-1; cytotoxic; immune function; immunogenic; in vitro Model; injured; lens; multicatalytic endopeptidase complex; mutant; novel; oxidation; polyglutamine; protein aggregate; protein degradation; protein misfolding; research study; response; stressor; transcription factor

DESCRIPTION (provided by applicant): The proteasome plays a central role in maintaining cell viability in response to stressors, such as protein overload or endoplasmic reticulum (ER) stress, by regulating signal transduction that promotes cell survival (i.e., NF-?B) and degrading potentially cytotoxic proteins (i.e., defective ribosomal products or DRiPs). The proposed work investigates the interplay between specific stressors (Protein Overload and ER Stress) and the immunoproteasome by defining conditions that induce immunoproteasome expression and testing specific hypotheses regarding immunoproteasomes role in responding to cellular stress. While its role in generating immunogenic peptides for antigen presentation has been clearly established, the expression of immunoproteasome subunits in non-inflammed immune-privileged tissue such as retina and brain implies other non-immune functions are possible. Notably, the upregulation of immunoproteasome in diseased retina and brain suggests it responds to challenges that induce stress and injury. The central hypothesis is that the immunoproteasome is a key component of the cellular response to stress. We have made several predictions that can be experimentally tested using techniques and strategies already familiar to our lab and the labs of our collaborators using knock-out mice lacking two (lmp7-/-/mecl-1-/-, L7M1) catalytic subunits of the immunoproteasome and conditional SCA1 transgenic mice expressing either the WT [30Q] or the mutant [82Q] ataxin-1 protein. Cell lines generated from WT and L7M1 mice and transfected with either the WT [30Q] or mutant [82Q] ataxin protein provide excellent in vitro model systems for testing our hypotheses. (Aim 1) Hypothesis: Immunoproteasome is required for effective muscle remodeling. Muscle remodeling will be induced by hindlimb unweighting WT and L7M1 mice to test if immunoproteasome (a) helps maintain muscle function during muscle remodeling, (b) provides protection from cellular damage, and (c) is directly involved in muscle remodeling. (Aim 2) Hypothesis: Immunoproteasome helps protect cells from the toxic effects of misfolded proteins. Using the conditional SCA1 transgenic mouse and cultured cells (WT and immunoproteasome-deficient) transfected with the WT [30Q] or mutant [82Q] ataxin protein, we will test if (a) the presence of a mutant, misfolded protein induces expression of the immunoproteasome, and (b) immunoproteasome provides protection from misfolded proteins. (Aim 3) Hypothesis: Reduced immunoproteasome expression increases the susceptibility to ER stress-induced damage. Using WT and immunoproteasome-deficient cell lines, we will test if immunoproteasome (a) is upregulated under conditions of ER stress, (b) protects from ER stress, and (c) immunoproteasome is involved in activation of the stress- sensitive transcription factor NF-?B. The proposed research brings together a powerful team with expertise in proteasome biology (Ferrington), muscle physiology (Thompson), and age-onset polyglutamine diseases (Orr). This collaboration incorporates techniques in molecular genetics, cell biology, biochemistry, and muscle physiology in pursuing evidence consistent with novel roles for the immunoproteasome in protecting the cell from a variety of stressors. PROJECT NARRATIVE: The proposed work investigates the interplay between specific stressors (Protein Overload and ER Stress) and the immunoproteasome, a specialized proteasome subtype, by defining conditions that induce immunoproteasome expression and testing specific hypotheses regarding immunoproteasomes role in responding to cellular stress. The approach is to use knock-out mice lacking two (lmp7-/-/mecl-1-/-, L7M1) catalytic subunits of the immunoproteasome and conditional SCA1 transgenic mice expressing either the wildtype [30Q] or the mutant [82Q] ataxin-1 protein. Cell lines generated from WT and L7M1 mice and transfected with either the WT [30Q] or mutant [82Q] ataxin protein provide excellent in vitro model systems for testing our hypotheses.

描述（由申请人提供）：蛋白酶体通过调节促进细胞存活的信号转导（即，NF-？B）和降解潜在的细胞毒性蛋白（即，缺陷核糖体产物或DRiP）。拟议的工作调查特定的压力（蛋白质过载和ER压力）和免疫蛋白酶体之间的相互作用，通过定义诱导免疫蛋白酶体表达的条件和测试有关免疫蛋白酶体在响应细胞应激中的作用的特定假设。虽然它在产生用于抗原递呈的免疫原性肽中的作用已经明确，但免疫蛋白酶体亚基在非炎症免疫特权组织（例如视网膜和大脑）中的表达意味着其他非免疫功能是可能的。值得注意的是，患病视网膜和大脑中免疫蛋白酶体的上调表明它对诱导应激和损伤的挑战做出反应。核心假设是免疫蛋白酶体是细胞对应激反应的关键组成部分。我们已经做出了几个预测，可以使用我们实验室和我们合作者的实验室已经熟悉的技术和策略进行实验测试，使用缺乏免疫蛋白酶体的两个（lmp 7-/-/mecl-1-/-，L7 M1）催化亚基的敲除小鼠和表达WT [30 Q]或突变[82 Q]共济失调蛋白-1蛋白的条件性SCA 1转基因小鼠。从WT和L7 M1小鼠产生并用WT [30 Q]或突变型[82 Q]共济失调蛋白转染的细胞系为测试我们的假设提供了极好的体外模型系统。(Aim 1）假设：免疫蛋白酶体是有效的肌肉重塑所必需的。将通过后肢减重WT和L7 M1小鼠诱导肌肉重塑，以测试免疫蛋白酶体是否（a）在肌肉重塑期间帮助维持肌肉功能，（B）提供免受细胞损伤的保护，以及（c）直接参与肌肉重塑。(Aim 2）假设：免疫蛋白酶体有助于保护细胞免受错误折叠蛋白质的毒性作用。使用条件性SCA 1转基因小鼠和用WT [30 Q]或突变型[82 Q]共济失调蛋白转染的培养细胞（WT和免疫蛋白酶体缺陷型），我们将测试（a）突变型错误折叠蛋白的存在是否诱导免疫蛋白酶体的表达，以及（B）免疫蛋白酶体是否提供对错误折叠蛋白的保护。(Aim 3）假说：免疫蛋白酶体表达减少增加了对ER应激诱导的损伤的易感性。使用WT和免疫蛋白酶体缺陷细胞系，我们将测试免疫蛋白酶体（a）是否在ER应激条件下上调，（B）是否保护免受ER应激，以及（c）免疫蛋白酶体是否参与应激敏感性转录因子NF-？B。拟议的研究汇集了一个强大的团队，他们在蛋白酶体生物学（Ferrington），肌肉生理学（Thompson）和年龄发作性多聚谷氨酰胺疾病（Orr）方面具有专业知识。这项合作结合了分子遗传学，细胞生物学，生物化学和肌肉生理学的技术，以寻求与免疫蛋白酶体在保护细胞免受各种应激源中的新作用一致的证据。项目叙述：拟议的工作调查之间的相互作用特定的压力（蛋白质过载和ER应力）和免疫蛋白酶体，一个专门的蛋白酶体亚型，通过定义诱导免疫蛋白酶体表达的条件和测试特定的假设免疫蛋白酶体的作用，在响应细胞应激。该方法是使用缺乏免疫蛋白酶体的两个（lmp 7-/-/mecl-1-/-，L7 M1）催化亚基的敲除小鼠和表达野生型[30 Q]或突变型[82 Q]共济失调蛋白-1蛋白的条件性SCA 1转基因小鼠。从WT和L7 M1小鼠产生并用WT [30 Q]或突变型[82 Q]共济失调蛋白转染的细胞系为测试我们的假设提供了极好的体外模型系统。