Immunoproteasome: A key component of the cellular stress response

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-κB）和降解潜在的细胞毒性蛋白（即有缺陷的核糖体产物或 DRiP），在维持细胞活力以应对应激源（例如蛋白质超载或内质网 (ER) 应激）方面发挥着核心作用。拟议的工作通过定义诱导免疫蛋白酶体表达的条件并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，研究特定应激源（蛋白质过载和内质网应激）与免疫蛋白酶体之间的相互作用。虽然其在产生用于抗原呈递的免疫原性肽中的作用已明确确定，但免疫蛋白酶体亚基在非炎症免疫豁免组织（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会对引起压力和损伤的挑战做出反应。中心假设是免疫蛋白酶体是细胞应激反应的关键组成部分。我们已经做出了一些预测，可以使用我们的实验室和我们的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（lmp7-/-/mecl-1-/-，L7M1）催化亚基的敲除小鼠和表达 WT [30Q] 或突变体 [82Q] ataxin-1 蛋白的条件 SCA1 转基因小鼠。从 WT 和 L7M1 小鼠中产生并用 WT [30Q] 或突变体 [82Q] 共济失调蛋白转染的细胞系为测试我们的假设提供了出色的体外模型系统。 （目标 1）假设：有效的肌肉重塑需要免疫蛋白酶体。将通过后肢减重 WT 和 L7M1 小鼠诱导肌肉重塑，以测试免疫蛋白酶体 (a) 是否有助于在肌肉重塑过程中维持肌肉功能，(b) 提供细胞损伤保护，以及 (c) 直接参与肌肉重塑。 （目标 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。使用转染有 WT [30Q] 或突变体 [82Q] 共济失调蛋白的条件 SCA1 转基因小鼠和培养细胞（WT 和免疫蛋白酶体缺陷型），我们将测试 (a) 突变体、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针对错误折叠蛋白的保护。 （目标 3）假设：免疫蛋白酶体表达减少会增加对 ER 应激诱导损伤的易感性。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）在内质网应激条件下是否上调，（b）保护免受内质网应激，以及（c）免疫蛋白酶体参与应激敏感转录因子NF-κB的激活。拟议的研究汇集了一支强大的团队，他们在蛋白酶体生物学（Ferrington）、肌肉生理学（Thompson）和年龄发病的多聚谷氨酰胺疾病（Orr）方面拥有专业知识。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方面的技术，以寻求与免疫蛋白酶体在保护细胞免受各种应激源侵害方面的新作用相一致的证据。项目叙述：拟议的工作通过定义诱导免疫蛋白酶体表达的条件并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，研究特定应激源（蛋白质过载和内质网应激）与免疫蛋白酶体（一种特殊的蛋白酶体亚型）之间的相互作用。该方法是使用缺乏免疫蛋白酶体的两个 (lmp7-/-/mecl-1-/-, L7M1) 催化亚基的敲除小鼠和表达野生型 [30Q] 或突变型 [82Q] ataxin-1 蛋白的条件 SCA1 转基因小鼠。从 WT 和 L7M1 小鼠中产生并用 WT [30Q] 或突变体 [82Q] 共济失调蛋白转染的细胞系为测试我们的假设提供了出色的体外模型系统。

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.