內質網中產生錯誤摺疊的蛋白質時，會產生內質網壓力(ER stress)進而引發複雜的細胞內信號路徑來重建ER內部平衡。先前研究發現ER膜上的蛋白Derlin-1，過度表現會誘發細胞凋亡。我們發現glutamyl-prolyl-tRNA synthetase (EPRS) 不僅可以緩解Derlin-1所誘導的毒性，且和Derlin-1有直接的相互作用，但詳盡機制還不明瞭。因此我們利用遺傳篩選和藥物刺激來探討EPRS和ER stress之間的關聯。利用果蠅當樣本，將Derlin-1過度表現或是給予ER stress 誘導劑tunicamycin (TM)，發現兩者都可讓EPRS表達上升，和上述結果相符。再者抑制內生性EPRS表達會加劇Derlin-1誘導的毒性，另外在抑制EPRS後給予TM後會縮短果蠅的壽命。相反的過度表現EPRS可以舒緩Derlin-1過度表現和TM的效果，結果顯示當細胞產生ER stress時EPRS可能具有保護的能力。EPRS主要為tRNA合成酶，而研究發現EPRS參與MSC (multi-aminoacyl-tRNA synthetase) and GAIT (IFN-γ-activated inhibitor of translation) complex的形成。為了進一步瞭解，這些和EPRS相關的複合物是否因參與ER stress調節，我們在過度表現Derlin-1的背景下抑制MSC和GAIT相關的基因。結果顯示抑制三個和MSC相關的骨架蛋白Aminoacyl-tRNA synthetase-interacting multifunctional proteins 1-3 (AIMPs)可減緩Derlin-1所誘導的毒性。MSC主要是用來和tRNAs結合幫助蛋白質合成，但抑制AIMPs會阻礙蛋白的合成外同時有效緩解ER stress。此外抑制AIMPs使EPRS被釋放對Derlin-1所造成的細胞凋亡和調節細胞內部的平衡有所幫助。總結來說我們提供一個新穎機制對於調節ER stress中Derlin-1和蛋白合成之間的連接。

The production of misfolded proteins in the Endoplasmic reticulum (ER) triggers ER stress response, complex intracellular signaling pathways for re-establishing ER homeostasis. We have previously identified an ER membrane protein Derlin-1 whose expression is induced by ER stress, and its overexpression would elicit apoptosis. In this process, we found glutamyl-prolyl-tRNA synthetase (EPRS) appeared to modify Derlin-1-induced cytotoxicity and directly interact with Derlin-1, but the detailed mechanism was unclear. Here, I harness genetic screens and pharmacological assays to dissect the underlying mechanism of EPRS in ER stress response. Flies with ectopic expression of Derlin-1 or ER stressor Tunicamycin (TM) treatment both induce EPRS expression, supporting a role of EPRS in ER stress response. Genetic knockdown of endogenous EPRS could exacerbate Derlin-1-induced cytotoxicity and shorten TM-induced life span. Conversely, enhancing EPRS level by expressing wildtype transgene could suppress Derlin-1- and TM-induced phenotype, indicating EPRS may have a protective role in ER stress signaling. EPRS is a canonical tRNA synthetase, but additional roles have been reported, which include the involvement of MSC (multi-aminoacyl-tRNA synthetase) and GAIT (IFN-γ-activated inhibitor of translation) complex formation. To further delineate whether these EPRS-involved complexes might participate in ER stress regulation, I use RNA-inhibition approach to selectively knockdown MSC and GAIT-associated genes in Derlin-1 overexpression model. The results show the reduction of three MSC scaffold components, Aminoacyl-tRNA synthetase-interacting multifunctional proteins 1-3 (AIMPs), could ameliorate Derlin-1-induced cytotoxicity, supporting a role of MSC in regulating ER stress response. Because MSC is reserved for recharging tRNAs and thus required for protein synthesis, the knockdown of AIMPs could hinder protein production which is beneficial when cells are under ER stress. Moreover, the release of EPRS upon AIMPs knockdown may help to divert Derlin-1-mediated apoptosis and leave the cells to re-establish homeostasis. Altogether, our data provide a novel mechanism of ER stress regulation in connecting to Derlin-1 signaling and protein synthesis.

Abstract I
中文摘要 III
Introduction 1
Material and methods 8
Drosophila genetics and molecular biology 8
Scanning electron micrographs 8
Immunohistochemistry 8
Tunicamycin (TM) treatment 9
Heat shock condition 9
Drosophila protein extraction and Western blot 10
Result 11
Screening Derlin-1 association protein 11
ER stress induces Derlin-1 and EPRS expression 12
Modulation of EPRS does not affect Derlin-1 expression 13
GAIT complex lacks a role in modifying ER stress 14
MSC may have a close link to the ER stress regulation 15
AIMPs distribution can be regulated by ER stress 17
Discussion 19
Other MSC associate protein in ER stress response 19
AIMPs and EPRS relationship in ER stress condition 20
Figure 21
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