中文摘要
內質網壓力(ER stress)普遍存在於神經退化性疾病、癌症及糖尿病中，當蛋白質摺疊與代謝過程受阻時，就會在細胞中產生ER stress，持續的ER stress會引發下游訊息並最終導致細胞凋亡。Derlin-1是一個位在ER上的膜蛋白，在ER associated degradation擔任極為重要的角色，ER stress會加強Derlin-1表現，此外，Derlin-1過量表現亦會引發嚴重的ER stress及細胞凋亡。我們利用反向基因體學方法和Derlin-1過量表現之果蠅模式研究ER stress導致細胞凋亡的路徑，在研究中發現glutamyl-prolyl-tRNA synthetase (EPRS)參與Derlin-1誘導之細胞凋亡。減低EPRS的表現不但會加強Derlin-1誘導之果蠅眼睛退化，同時也會加劇ER stress誘劑tunicamycin引發之細胞毒性，另一方面，增加EPRS的表現能減緩Derlin-1誘導之眼睛退化和tunicamycin引發之細胞毒性，此現象指出EPRS在ER stress的細胞調適扮演極重要的角色。經由GST pull-down和co-immunoprecipitation分析，證實內生性Derlin-1和EPRS在ER stress時可形成複合體，我們認為此作用與細胞適應ER stress具有密切關聯，進而可左右細胞生死。此外，為闡明此ER stress適應之分子機轉，我們探討了EPRS參與的multiple tRNA synthetase complex以及一群蛋白質體學資料庫中的EPRS交互作用蛋白。有趣的是，減少參與核運輸的EPRS交互作用蛋白也會加強Derlin-1誘導之眼睛退化。這些結果指出細胞的核運輸作用在ER stress中扮演顯著的角色。

Abstract
Endoplasmic reticulum (ER) stress has been observed in many types of diseases, including diabetes, cancer, and neurodegeneration. ER stress can be elicited when this organelle fails to fold and degrade proteins properly. If not mended, prolonged ER stress could trigger signals that ultimately lead to cell death. Derlin-1 is an ER membrane protein plays a crucial role in ER associated degradation. ER stress enhances Derlin-1 expression, and its ectopic overexpression induces serious ER stress and cell death. To understand how ER stress results in cell death, we use reverse genetic approach in Derlin-1 overexpression model and identify glutamyl-prolyl-tRNA synthetase (EPRS) which is involved in Derlin-1 induced cell death. EPRS downregulation enhances Derlin-1-induced rough eye phenotype, and this effect appears to be generic because its knockdown also escalates ER stressor tunicamycin induced toxicity. Conversely, overexpression of EPRS can rescue Derlin-1-induced rough eye phenotype and suppress tunicamycin-induced toxicity. These results indicate EPRS may play an important role in cell survival when cell adopts to ER stress. Through GST pull-down assay and co-immunoprecipitation, we further confirm that endogenous Derlin-1 can interact with EPRS specifically under ER stress condition, suggesting their interaction may closely link to life-or-death adaptation upon ER stress. To understand the underlying molecular mechanism, we have tested EPRS-involved pathways such as the multiple tRNA synthetase complex, and also investigated a group of physical interactors depicted in the proteomic database. Interestingly, knockdown EPRS interactors involved in nuclear transport strongly enhance Derlin-1-induced rough eye phenotype. These results suggest that nuclear transport has a pronounced role in ER stress.

Table of contents

Introduction 8
Material & Methods 13
Drosophila stocks and genetics 13
Heat shock condition 13
Tunicamycin treatment 13
Scanning electron micrograph 14
Immunohistochemistry 14
RNA extraction and RT-PCR analysis 15
Cloning and construct the recombinant plasmid 15
Drosophila protein extraction 15
IPTG induction 16
GST pull down assay 16
Immunoprecipitation 17
Immunoblotting analysis 18
Results 19
Identify Derlin-1 associates in ER stress 19
EPRS affects Derlin-1-induced eye degeneration 20
EPRS modifies Derlin-1-induced phenotype is a ER stress-specific condition 22
EPRS and Derlin-1 are co-localized in Derlin-1-expressed cells 23
EPRS interact with Derlin-1 under ER stress 24
EPRS is an ER stress response protein 26
EPRS affect the adaptation to ER stress 27
Regulation of translation enhance Derlin-1 induced degeneration 28
EPRS associated nuclear transport can strongly enhance Derlin-1-induced cytotoxicity 29
Discussion 31
A potential signaling regulation of EPRS in ER stress response 32
Derlin-1 play reverse roles 33
Translational regulation in ER stress 34
Nuclear transport in ER stress 35
Figure 37
Reference 52
Appendix 58

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