臺灣博碩士論文加值系統

Endoplasmic reticulum (ER) is the major site of protein synthesis and maturation in eukaryotes. Inside ER, the inability of newly synthesized secretory and membrane proteins to adopt the native conformations lead to their retrotranslocation into the cytosol and subsequent clearance by 26S proteasome. This pathway is termed the ER-associated degradation (ERAD) pathway. A fundamentally important question to understand the mechanism of ERAD is the identity of retrotranslocon. In this thesis, an ER membrane protein Derlin-2 (Der2) is discovered required for luminal Human Sonic hedgehog (HShh) degradation. SiRNA experiment to knockdown the expression of Derlin-2 blocks the ERAD of HShh precursor and its C-fragment. Co-immunoprecipitation shows that HShh interacts with Derlin-2. Moreover, from the results of co-immunoprecipitation and chemical crosslinking experiments, Derlin-2 is discovered forming homo-oligomers, which raised the possibility that oligomeric Derlin-2 forms the retrotranslocation channel for ERAD of HShh. The oligomerization does not depend on intermolecular disulfide bond. Because of Derlin-2 oligomerization, the domain important for Derlin-2 oligomerization will be examined using various Derlin-2 constructs.

在真核生物體內，蛋白質會在內質網胞器中進行組裝、修飾以及摺疊形成成熟的蛋白質。在內質網中，這些新合成的分泌性蛋白或膜蛋白失去了它們正常的構型，將會被反向運輸從內質網基質送到細胞質內，由細胞質中的26S蛋白酶體降解。這一精密品質管控的過程就稱為內質網相關的蛋白質降解 (ERAD)。 因此，內質網膜上的反向運送通道，在內質網相關的蛋白質降解過程中伴演了不可或缺的角色。在此篇論文中，內質網的膜蛋白Derlin-2 (Der2) 被發現參與人類內質網基質Sonic hedgehog (HShh) 的降解過程。由SiRNA 的實驗，將Derlin-2 的蛋白表現量降低會抑致HShh的precursor以及HShh-C羧基片段進行內質網相關的蛋白質降解過程。由免疫沉澱的實驗，可以同時抓到Derlin-2 以及HShh，證明它們在同一個complex內。其次，在進行免疫沉澱法與化學性交聯作用的實驗中，我們發現Derlin-2可以自已形成多聚體型式，對於此Derlin-2多聚化現象， 我們提出了Derlin-2可能是膜上一個反向運輸的通道蛋白負責轉送HShh到細胞質。Derlin-2形成多聚體化的現象，並不是藉由蛋白質的cysteine胺基酸形成雙硫鍵而形成的。對於Derlin-2上的哪一區域是主要參與形成多聚化的，將藉由已建構好的各種Derlin-2 mutants去探討。

誌謝
Abstract 3
中文摘要 4
Contents 5
Introduction 8
The mechanisms involved in endoplasmic reticulum protein quality control 8
ER quality control (ERQC) 9
Signal integration in the ER unfolded protein response 11
ER-associated protein degradation 12
Components of the ERAD complex 13
Sonic hedgehog (Shh): The first endogenous luminal substrate identified by ERAD 16
Derlin family 18
Figure 1.UPR mechanism in mammalian. 21
Figure 2.Time-dependent phase shift in mammalian 21
Figure 3.Mechanism of protein degradation in yeast and mammalian cells 22
Figure 4.The processing of human Sonic hedgehog. 23
Table 1.Select components required for ERAD 24
Materials and Methods 25
Materials 25
Mammalian cell Culture and generation of stable cell lines 25
Immunoprecipitation 26
Immunoblotting 27
Immunofluorescence 28
Cycloheximide Chase Assays 28
Chemical cross-linking 29
Plasmid construction and site-directed mutagenesis 29
Result 31
Derlin-2 is required for degradation of HShh 31
Derlin-2 is an ER-resident protein 32
Derlin-2-GFP exerts dominant negative effect on HShh degradation 32
Derlin-2-Flag stable cells are functional for HShh degradation 33
Derlin-2 interacts with HShh 34
Derlin-2 interacts with each other 34
Derlin-2 forms homo-oligomers 35
Dimeric Derlin-2 is not mediated by disulfide bond 37
Construction of Derlin-2 mutants 38
Discussion 40
Reference 43
Figure 49
Figure 1. Derlin-2 is required for HShh degradation. 50
Figure 2. Subcellular localization of Derlin-2 51
Figure 3. Derlin-2-GFP fusion is dominant negative on HShh degradation 53
Figure 4. Derlin-2-Flag stable cells were functional for substrates
degradation 55
Figure 5. Derlin-2 is specifically associated with HShh. 56
Figure 6. Derlin-2 interacts with each other. 58
Figure 7. Derlin-2 forms homo-oligomers 60
Figure 8. Disulfide bond formation doesn’t contribute to the formation of dimeric Derlin-2. 62
Figure 9. Expression of Derlin-2 mutants. 65
Table S1. The list of antibodies. 66
Table S2. Primers used for constructs 66

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