Cellular Response to Misfolded Rhodopsins.

描述（由申请人提供）：该项目的长期目标是了解细胞如何应对常染色体显性视网膜色素变性（ADRP）果蝇模型中与年龄相关的视网膜变性相关的视紫红质-1 （Rh-1）蛋白突变。该模型中的Rh-1等位基因破坏编码蛋白的折叠特性，并对内质网（ER）施加压力。健康的年轻细胞表现出强大的内质网应激反应机制，使受影响的光感受器存活到老年。另一方面，衰老细胞通过激活细胞死亡途径屈服于压力，导致与年龄相关的视网膜变性。更好地了解这些途径可能有助于开发针对这种疾病的有效治疗策略。通过我们独特的基于果蝇的方法，我们确定了许多意想不到的突变Rh-1的细胞反应机制，这些机制可能在ADRP中发挥重要作用。在这里，我们提出了三个具体目标来研究这些机制。在Aim 1中，我们计划研究细胞如何降解错误折叠的视紫红质以减少内质网中的应激。我们之前已经证实，参与这一过程的中心泛素连接酶HRD1可以显著延缓果蝇ADRP模型的视网膜变性过程。HRD1需要其他蛋白质的合作来帮助识别和降解错误折叠的Rh-1。通过遗传筛选，我们确定了一种特征不佳的羧肽酶，作为帮助减少错误折叠的Rh-1水平的最有效因素之一。在这里，我们提出验证hrd1介导的错误折叠视紫红质的降解需要这种功能未知的羧肽酶这一有趣的想法。在目标2中，我们建议确定翻译抑制剂在内质网应激反应中的作用。翻译抑制是细胞应激的常见结果，特别是在内质网中错误折叠的蛋白质过载时，细胞激活翻译抑制剂PERK以减轻内质网蛋白质折叠系统的负担。出乎意料的是，我们发现另一种翻译抑制剂4E-BP在PERK的下游被诱导。由于4E-BP具有增强抗逆性和延长寿命的作用，我们计划在ADRP模型中测试4E-BP诱导的功能意义。此外，我们建议确定4E-BP如何保护细胞免受内质网应激。在Aim 3中，我们提出研究内质网应激如何诱导细胞死亡。许多细胞死亡调节因子的功能定位于线粒体外膜，我们假设一个特定的细胞死亡途径将应激信号从内质网传递到线粒体。通过基因筛选，我们发现了两种细胞周期蛋白依赖性激酶（CDKs），它们出乎意料地参与了这种细胞死亡信号传导。在这里，我们提出测试这些CDKs形成线性途径的想法，促进线粒体外膜的促凋亡事件。我们将进一步研究这些基因在果蝇ADRP模型中与年龄相关的视网膜变性中的作用。这一建议的成功结果有望为内质网应激反应的三个鲜为人知的领域带来概念上的进步，这些领域与ADRP的病理直接相关。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how cells cope with mutant Rhodopsin-1 (Rh-1) proteins that underlie age-related retinal degeneration in the Drosophila model for Autosomal Dominant Retinitis Pigmentosa (ADRP). The Rh-1 alleles in this model impair the encoded protein's folding property and impose stress to the endoplasmic reticulum (ER). Healthy young cells exhibit robust ER stress-response mechanisms that allow the afflicted photoreceptors to survive until old age. On the other hand, old cells succumb to stress by activating cell death pathways, leading to age-related retinal degeneration. A better understanding of these pathways may allow the development of effective therapeutic strategies against this disease. Through our unique Drosophila-based approach, we identified a number of cellular response mechanisms to mutant Rh-1 that were unexpected, yet likely to play important roles in ADRP. Here, we propose three Specific Aims to investigate those mechanisms. In Aim 1, we plan to examine how cells degrade misfolded rhodopsins to reduce stress in the ER. We had previously established that a central ubiquitin ligase involved in this process, HRD1, can significantly delay the course of retinal degeneration in the Drosophila ADRP model. HRD1 requires the cooperation of other proteins that help recognize and degrade misfolded Rh-1. Through a genetic screen, we identified a poorly characterized carboxypeptidase as one of the most potent factors that help reduce misfolded Rh-1 levels. Here, we propose to validate the intriguing idea that HRD1-mediated degradation of misfolded rhodopsin requires this carboxypeptidase of unknown function. In Aim 2, we propose to determine the role of translational inhibitors in the ER stress response. Translational inhibition s a common outcome of cellular stress, and specifically in response to misfolded protein overload in the ER, cells activate the translational inhibitor PERK to reduce the burden on the ER protein folding system. Unexpectedly, we discovered that another translational inhibitor, 4E-BP, is induced downstream of PERK. As 4E-BP is known for its effects in enhancing stress resistance and prolonging lifespan, we plan to test the functional significance of 4E-BP induction in the ADRP model. Moreover, we propose to determine how 4E-BP protects cells against ER stress. In Aim 3, we propose to study how ER stress induces cell death. Many cell death regulators localize to the mitochondrial outer membrane for their function, and we hypothesize that a specific cell death pathway conveys stress signals from the ER to the mitochondria. Through a genetic screen, we identified two Cyclin Dependent Kinases (CDKs) that are unexpectedly involved in this cell death signaling. Here, we propose to test the idea these CDKs form a linear pathway that promotes pro-apoptotic events at the mitochondrial outer membrane. The genes of interest here will be further examined for their roles in age-related retinal degeneration in a Drosophila model of ADRP. A successful outcome of this proposal is expected to bring conceptual advances to the three poorly understood areas of ER stress response that are directly related to the pathology of ADRP.