Endoplasmic reticulum-associated protein degradation (ERAD)是重要的細胞機制。它主要用以降解細胞中發生錯誤摺疊的蛋白。錯誤摺疊的蛋白在內質網中被辨識後會經由膜上的通道蛋白送至細胞質中，再經過泛化作用(Ubiquitination)後經由蛋白酶體(proteasome)降解。ERAD執行的過程有許多蛋白成員參與，其中我們相當關注名為Derlin的蛋白家族。Derlin蛋白具有數個穿膜蛋白序列，因此被認為可能構成ERAD的通道，使得錯誤摺疊蛋白能夠經由通道送出內質網。此外，這個作用還需要一種被稱為VCP的蛋白參與。VCP是一種AAA ATPase他會與幾種不同的協同因子(cofactors)形成複合體，將錯誤摺疊的蛋白帶往蛋白酶體(proteasome)降解。VCP是如何前往內質網並帶離錯誤摺疊蛋白的機制仍不清楚，不過藉由免疫螢沉澱(immunoprecipitation)以及pull down實驗，我驗證了Derlin-1 C端的SHP box在形成Derlin-1/TER94(VCP在果蠅上的同源蛋白)複合物的作用上相當重要。有趣的是Derlin家族的的另一個蛋白Derlin-2也有類似的SHP box序列，但它沒有辦法與TER94形成複合物。而這個樣的差異可能是Derlin-2沒有辦法像Derlin-1一樣去抑制TER94A229E病變的原因。在我的研究中發現利用點突變修改Derlin-2的SHP box可以使與TER94形成complex的能力恢復，這個令人振奮的發現可以重新定義對於SHP box序列的認知。此外，在我的研究中也探討了Derlin-1與Derlin-2 C端結構上α螺旋結構的差異以及這個α螺旋結構在調控內質網平衡的重要意義。在觀察ER stress與Derlin之間的關聯性之後，我發現Derlin-1在調控上下游的其他細胞路徑都有重要的功用，而Derlin-2與Derlin-1相比，在ERAD上所扮演的可能是更偏向通道結構的角色

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a cellular process for protein quality control which targets misfolded proteins of the ER for ubiqutination and subsequent proteasome degradation in the cytosol. Derlin family proteins are ER transmembrane protein that could form channel to function as the aforementioned dislocation passage. This process also requires p97/VCP (also known as TER94 in fly), a multi-functional AAA ATPase, which forms complexes with different cofactors to remove the targeted protein from ER. Currently, it is unclear how p97/VCP being recruited to the ER for removing the misfolded proteins. Using immunoprecipitation and pull-down studies, I demonstrated the SHP box (also known as binding site 1 (BS1)) at the C-terminus of fly Derlin-1 is critical for TER94 interaction in which the N-domain of TER94/VCP plays as a binding counterpart of this interaction. Interestingly, another Derlin family protein Derlin-2, which has extensive similarity with Derlin-1, includes the conserved SHP box sequence, and is also involving in ERAD pathway, failed to bind TER94. This TER94-binding discrepancy is functionally important because in a Drosophila model, overexpressing fly Derlin-1, but not Derlin-2, impairs ERAD which suggests that Derlin-1 might negatively regulate ERAD. Furthermore, Derlin-1-mediated dominant phenotype could be suppressed by overexpressing TER94 or human VCP. Surprisingly, I modulated the Derlin-2 SHP box residue and the interaction of Derlin-2 and TER94 dramatically increased, redefining the originally proposed SHP box sequence. This study suggests that Derlin-1 and Derlin-2, despite of their high homology and could cooperatively form a channel for dislocating misfolded protein, may function differently during ERAD processing in terms of recruiting p97/VCP ATPase and feedback regulation.

Table of Contents
Abstract i
中文摘要 iii
致謝 iv
Table of Contents vii
Introduction 1
Materials and Methods 5
Cloning and construct of the recombinant plasmid 5
Drosophila genetics and protein extraction 5
Dissection and Immunohistochemistry 6
IPTG induction 6
GST pull down assay 6
Immunoblotting analysis 7
The mRNA extraction and RT-PCR analysis 8
Results 9
Production of GST-Derlin-1 fusion proteins in bacteria 9
Derlin-1 carboxyl terminus and TER94 N-L1 domain are critical for Derlin-1/TER94 binding 10
ER association is crucial for Derlin-1 to suppress TER94A229E 11
Modified Derlin-2 does bind to TER94 directly 12
Derlin-2 expression fails to induce ER stress and cytotoxicity 12
ER stress induces Derlin-1 but not Derlin-2 expression 13
A novel ERAD reporter GFP-PRC1-1 for ERAD-L 14
Discussion 16
The counterbalance between Derlin-1 and TER94/VCP 16
Analysis of Derlin-2 and its function 17
Figure legend 19
Figure 1. Description of the ERAD mechanism 19
Figure 2. Derlin-1 C-terminus interacts with TER94 through SHP box in Derlin-1 and N-L1 domain in TER94. 22
Figure 3. The genetic test for Derlin-1 and TER94A229E expression 24
Figure 4. Modified Derlin-2 bind to TER94 directly. 26
Figure 5. Derlin-2 expression modification fails to induce ER stress. 28
Figure 6. Tunicamycin treatment induce Derlin-1 and Bip, but fail to rise the Derlin-2 expression. 32
Figure 7. A novel ERAD reporter GFP-PRC1-1 for ERAD-L. 34
References 36

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