內質網(ER)是一個負責調控細胞內鈣離子平衡及儲存分泌性蛋白和膜蛋白的重要胞器， 因此當鈣離子失去平衡或是錯誤折疊蛋白堆積在內質網內，就會引發ER stress並藉由活化unfolded protein response (UPR)下游反應來調節內質網內的平衡。目前已知UPR透過起始下游的三條訊息傳遞鏈IRE1、PERK 和ATF6，去調控細胞適應ER stress或是死亡。在哺乳類動物中，Derlin-1這個位於ER 膜上的通道蛋白可以將摺疊的蛋白送出ER，利用ERAD的機制將這些不正常蛋白降解。在實驗室先前的研究中，我們發現過度表現Derlin-1，可以造成果蠅眼睛的退化並且活化UPR。當餵食tunicamycin或是給予cold shock刺激果蠅，使其產生ER stress時，我們發現Derlin-1的表現量也明顯上升，這證明了Derlin-1可能參與在UPR的下游反應，進而促使細胞凋亡。為了更了解Derlin-1與UPR之間的關連，我們利用western blotting分析並發現了PERK和ATF6可能造成ER stress時Derlin-1的上升。我們進一步利用遺傳方式分析果蠅的UPR訊息傳遞鏈去找尋Derlin-1參與在UPR下的機制。根據結果推斷，我們認為PERK-eIF2α下游的GADD34可能影響Derlin-1所引起的細胞凋亡機制，我們的實驗結果也指出PERK的訊息傳遞鏈可以決定ER stress情況下的細胞命運。這個研究可以讓我們進一步了解ER stress的調控，且有助於發展ER stress相關疾病的治療方法。

Endoplasmic reticulum (ER) is an important organelle that stores the intracellular calcium and manufactures secretory and membrane proteins. When the ER calcium homeostasis or the protein biosynthesis is disturbed, accumulation of misfolded proteins obstructs the ER homeostasis, which could activate the intracellular signaling pathways termed the unfolded protein response (UPR). Current knowledge distinguishes UPR as three signaling pathways, IRE1α (inositol-requiring protein-1α), PERK (protein kinase RNA (PKR)-like ER kinase), and ATF6 (activating transcription factor 6). These three pathways regulate the fate of those cells under the ER stress to either strive for adaptation or commit for apoptosis. In mammal, Derlin-1 (Der1-like domain family, member 1) is an integral ER membrane protein that functions as a retro-translocation channel to help misfolded ER proteins crossing the membrane for proteasome degradation, a process known as endoplasmic reticulum-associated protein degradation (ERAD). In a Drosophila model, we found that overexpression of fly Derlin-1 causes eye degeneration, a phenotype also coincide with the UPR activation. As Derlin-1 expression level is increased upon ER stressor tunicamycin treatment or cold shock condition, this data suggest that Derlin-1 may involve in UPR pathway to promote cell apoptosis. To further investigate the relationship of Derlin-1 and UPR, I performed Western blotting and the data suggests that PERK and ATF6 might contribute to Derlin-1 increasing upon ER stress. I used genetic approach aiming to delineate Derlin-1 regulation in the UPR pathway and apoptosis, and identified Derlin-1-mediated cell death is closely associated with GADD34, a gene downstream of the PERK-eIF2α pathway. Our data indicate that ER stress-induced signaling may exploit Derlin-1 and PERK pathway to control the cell fate determination. This study could advance our understanding on ER stress regulation, and potentially benefit the development of new clinical strategies for ER-stress related diseases.

Introduction 1
Materials and methods 6
Drosophila genetics and molecular biology 6
Cloning and construct of the transgenic plasmid 6
Scanning electron micrographs 6
Immunohistochemistry 7
Tunicamycin (TM) treatment 7
Heat shock condition 8
Cold shock condition 8
Drosophila protein extraction 8
Immunoblotting analysis 9
mRNA extraction and RT-PCR analysis 9
Results 11
Aberrant Derlin-1 expression causes ERAD defect and cytotoxicity 11
Derlin-1 is an essential gene 12
Overexpressing Derlin-1 caused rough eye phenotype is associated with the activation of apoptotic cell death 13
Derlin-1 responds to ER stress and involves in UPR pathway to promote cell death 15
PERK and ATF6 pathway may regulate Derlin-1-mediated apoptosis 17
PERK pathway protein GADD34 modifies Derlin-1-mediated neurodegeneration 18
GADD34 modulates Derlin-1-induced apoptosis 20
Akt is a potent pro-survival regulator in Derlin-1-mediated apoptosis 22
Discussion 24
PERK is required during development 24
Overexpressing of elF2α could suppress Derlin-1-mediated cytotoxicity 25
The relationship between GADD34 and PP1 26
Figure 28
Appendix 48
References 55

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