ER stress-induced translational regulation in retinal degeneration

项目总结 常染色体显性遗传性视网膜色素变性(ADRP)是一种遗传性视网膜变性疾病，可导致 进行性视力丧失和随后的失明。近三分之一的ADRP患者在 感光G蛋白偶联受体视紫红质。这些突变中的许多会导致视紫红质错误折叠 在内质网(ER)导致内质网功能障碍，从而导致视网膜变性。与年龄相关的 这种疾病的本质可能是因为内质网应激反应通路保护视紫红质不被错误折叠 随着年龄的增长而下降。因此，对这些通路的更好理解可能有助于治疗策略 针对ADRP和其他内质网应激介导的疾病的研究进展。在这里，我将重点放在一个特殊的ER压力上 由跨膜蛋白激酶PERK启动的反应通路。在检测到错误折叠的蛋白质后，PERK 磷酸化失活翻译起始因子eIF2α，抑制蛋白质合成并降低内质网 负担。有趣的是，这些抑制条件刺激转录因子ATF4的翻译，这是由于 它不同寻常的5‘UTR。我们对ATF4诱导的理解在很大程度上是基于对酵母和残骸的研究 不完整，因此仍有可能存在尚未确定的翻译监管机构，具体 影响ATF4翻译，但不影响规范的mRNA翻译。除了由磷酸-2调节的翻译外 EIF2α，PERK途径通过4E-BP参与第二种翻译抑制机制，这是一种直接的 ATF4的转录靶点。通过PERK信号激活两种翻译抑制机制， 如何翻译压力反应转录本(缓解内质网压力所需的转录本)？ 这项提案旨在通过以下方式解决关于PERK/ATF4途径的这一重大悬而未决的问题 使用果蝇ADRP模型来确定ATF4信号的病理后果，其中 突变的视紫红质-1(Rh1G69D)施加内质网应激，导致视网膜变性。初步研究是 通过筛选针对ATF4报告基因缺失的各种翻译起始因子的RNAi系进行的 活动。这导致了一个特征不佳的翻译启动因素被确定为意想不到的 ATF4的调节器。在培养的小鼠胚胎成纤维细胞(MEF)中的实验显示了一个系统发育 这一新因子在调节ATF4翻译中的保守作用。更多的初步证据表明 新发现的因子调控5‘UTRATF4的翻译。其中概述的实验的一部分 一项提案旨在确定该因子如何利用尖端核糖体调节ATF4的翻译 侧写技术。提案的其余部分详细说明了理解第二个角色的战略 PERK下游的翻译抑制物4E-BP在视网膜变性中的作用。如果实现，这个项目将 大大加深了我们对视网膜变性过程中翻译控制的理解，除了提供 治疗内质网应激介导疾病的有价值的药物靶点。

PROJECT SUMMARY Autosomal Dominant Retinitis Pigmentosa (adRP) is a heritable retinal degeneration disorder that results in progressive vision loss and subsequent blindness. Nearly a third of adRP patients possess a mutation in the light-sensing G-protein coupled receptor, Rhodopsin. Many of these mutations cause the rhodopsin to misfold in the Endoplasmic Reticulum (ER) resulting ER dysfunction that leads to retinal degeneration. The age-related nature of the disease is likely because ER stress response pathways that protect against misfolded rhodopsins decline with age. A better understanding of these pathways may therefore contribute to therapeutic strategy development against adRP and other ER stress-mediated maladies. Here, I focus on one particular ER stress response pathway that is initiated by a transmembrane kinase, PERK. Upon sensing misfolded proteins, PERK phospho-inactivates a translation initiation factor, eIF2α, which inhibits protein synthesis and reduces ER burden. Interestingly, these inhibitory conditions stimulate the translation of a transcription factor, ATF4, due to its unusual 5'UTR. Our understanding of ATF4 induction is largely based on studies in yeast and remains incomplete, thus it remains possible that there are as yet unidentified translation regulators that specifically affect ATF4 translation but not canonical mRNA translation. In addition to translation regulation by phospho- eIF2α, the PERK pathway engages a second translational inhibition mechanism via 4E-BP, which is a direct transcriptional target of ATF4. With two translational inhibition mechanisms being activated by PERK signaling, how are stress responsive transcripts (that are required to ameliorate ER stress) translated? This proposal aims to address this major unanswered question regarding the PERK/ATF4 pathway by employing a Drosophila model of adRP to determine the pathological consequence of ATF4 signaling wherein mutant Rhodopsin-1 (Rh1G69D) imposes ER stress and leads to retinal degeneration. Preliminary studies were conducted by screening RNAi lines targeting various translation initiation factors for loss of an ATF4 reporter activity. This lead to the identification of a poorly characterized translational initiation factor as an unexpected regulator of ATF4. Experiments in cultured mouse embryonic fibroblasts (MEFs) shows a phylogenetically conserved role for this new factor in regulating ATF4 translation. Additional preliminary evidence indicates that the newly identified factor regulates translation at the 5' UTR ATF4. Part of the experiments outlined in this proposal is designed to determine how this factor regulates translation of ATF4 utilizing cutting-edge ribosome profiling techniques. The remainder of the proposal details a strategy to understand the role of the second translational inhibitor downstream of PERK, 4E-BP, in retinal degeneration. If realized, this project will significantly further our understanding of translation control during retinal degeneration in addition to providing valuable pharmaceutical targets for ER stress-mediated maladies.