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|dc.description.abstract||FAF1은 한 단백질 내에 여러 개의 Ubiquitin 관련 도메인을 가지고 있는 Ubiquitin receptor 로 각 domain들이 각각의 역할을 수행하여 그 생물학적 기능을 나타낼 것이라 생각된다. FAF1 는 ubiquitin associated (UBA) domain을 통해 polyubiquitination된 단백질을 recruit하며, Ubiquitin 과 구조가 비슷한 ubiquitin like 1,2 (UBL1,2) domain을 통해 Heat shock protein 70 (Hsp70)과의 상호작용을 통해 Hsp70의 chaperone activity를 저해한다고 보고되었다. 또한 ubiquitin regulatory X (UBX) domain을 통해 valosin containing protein (VCP) 라는 proteasome system의 chaperone으로 알려진 단백질과 상호작용한다. 이 논문에서는, FAF1이 한 단백질 내에 여러 개의 ubiquitin 관련 domain을 가지고 있는 것에 관심을 갖고, ubiquitin 관련 domain간의 상호작용과 그 domain의 binding partner를 proteomic tool을 이용해 밝힘으로써 FAF1의 ubiquitin receptor 로서의 세포내의 기능을 연구하였다. 이 논문에서는 VCP가 결합이 FAF1의 UBA domain의 polyubiquitinated protein 결합에 중요하다는 것을 밝히고 이것은 UBX domain과 UBA domain의 직접적인 상호작용 없이 cooperative regulation 에 의한 것임을 밝혔다. 또한 FAF1의 새로운 결합 단백질로서 Npl4-Ufd1을 찾고 이 npl4-Ufd1의 binding이 VCP와 FAF1의 결합에 중요하다는 것을 보였다. VCP-Npl4-Ufd1 complex는 ERAD 의 중요한 요소인데, 이 논문에서 ERAD의 substrate인 Cd3δ 의 degradation을 관찰함으로써 FAF1이 VCP-Npl4-Ufd1과 함께 ERAD를 조절하는 것을 보였다. 또한 새로운 결합 단백질로 ATF6와 VAPB를 발견하였다. ATF6는 ER stress에 반응하는 receptor로서 FAF1의 결합단백질인 VCP를 통해 붙는 것으로 확인되었고 reporter assay를 통해 FAF1이 ATF6의 activation을 증가시키는 것을 관찰 하였다. 또한 FAF1은 VAPB와 결합함으로써 ATF6 가 p50으로 activation 되기 위해 processing 되는 과정을 조절한다. 이 결합을 통해 FAF1이 ATF6의 활성화 단계를 조절하여 ER stress 에 의한 apoptosis를 촉진한다. FAF1은 UBA domain을 통해 polyubiquitinated protein과 결합하는데 이 UBA domain은 FAF1의 세포 내 기능에 중요한 역할을 한다. UBA domain은 FAF1이 cancer cell을 colonization 시키는데 중요한 역할을 하며 UBA domain이 deletion된 FAF1은 그 역할을 하지 못한다. 이 논문에서는 UBA specific tandem immunoprecipitation을 이용해 UBA domain에 결합하는 polyubiquitinated protein을 규명하였다. Polyubiquitinated substrate로 Hsp70가 mass spectrometry를 이용해 확인 되었으며 polyubiquitinated Hsp70가 FAF1에 결합하는 것을 확인하였다. FAF1은 이 결합을 통해 Hsp70의 half life를 조절하였고, poly-ub가 붙지 못하는 UBA deletion mutant나 UBA defect mutant는 그러한 기능을 잃는 것을 관찰 하였다. FAF1은 Hsp70의 조절을 통하여 cancer 의 형성을 억제하는 것으로 생각된다. 이 논문에서는 FAF1의 결합 단백질을 기초로 하여, FAF1이 ERAD와 ER stress 를 경유한 apoptosis, UBA domain을 통한 tumor suppression에 기여한다는 것을 증명하였다. ;Fas-associated factor 1 (FAF1) is an ubiquitin receptor protein having multiple ubiquitin related domains including ubiquitin associated (UBA), ubiquitin like 1, 2 (UBL1, UBL2), and ubiquitin regulatory X (UBX) domain. We previously showed that N-terminal UBA domain recognizes K48-ubiquitin linkage to recruit polyubiquitinated proteins, UBL1 domain interacts with Hsp70 and regulates its chaperone activity, and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). In the first, this study observed VCP association to C-terminal UBX domain is required to recruit polyubiquitinated proteins to N-terminal UBA domain without direct interaction between UBA and UBX domain. FAF1 interacts with only VCP complexed with Npl4-Ufd1 heterodimer, which stabilizes the recruitment of polyubiquitinated proteins to UBA domain. These interactions are well characterized by structural analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation (ERAD). This study shows that FAF1 promotes ERAD in a VCP-Npl4-Ufd1-dependent manner by cooperative regulation of UBA-UBX domains. Next, FAF1 was observed to increase ER stress mediated cell death. It turned out that FAF1 interacts with the ER transmembrane transcription factor, activating transcription factor 6 alpha (ATF6α), via VCP binding through UBX domain. ATF6α is known to function in a regulated transcription pathway involved in ER homeostasis and the unfolded protein response (UPR). ATF6 is one of the ER stress marker protein and it is cleaved for activation when ER stress occurred. FAF1 was observed to potentiate ATF6α activation in ER stress induced by tunicamycin by up-regulating activated ATF6α p50 with FAF1 overexpression. Simultaneously, FAF1 interacts with VAPB through UBL1 domain. VAPB was reported to interact with ATF6 and attenuates the activity of ATF6 regulated transcription. this study shows that FAF1 increases ER stress mediated cell death by potentiating ATF6α activation through VAPB interaction. In the last, this study shows that FAF1 serves as an inhibitor of tumor proliferation by regulating degradation of Hsp70 mediated via its UBA domain. To identify moiety in the UBA domain of FAF1 causing tumor cell death, UBA-specific tandem immunoprecipitation was employed combining mass spectrometry to identify. Polyubiquitinated Hsp70 was identified to be associated with FAF1 UBA domain. FAF1 overexpression augments or promotes Hsp70 degradation via the proteasome. This was confirmed that mutations of the FAF1 UBA domain as well as knocking it down with RNAi abolish its ability to promote Hsp70 proteasomal degradation. This was verified in clinical cervical cancer tissues that the expressions of FAF1 are discernibly reduced and Hsp70 increased in cancer tissues compared to normal tissue. These studies establish the multiple biological functions and their regulation mechanism of FAF1 by acting as a scaffolding protein. Intriguingly, FAF1 as an ubiquitin receptor forms complex with various proteins through ubiquitin related domains such as UBA, UBL2 and UBX domain and their interactions are tightly regulated by domain-domain cooperative way. FAF1 is a multi-player protein to have various populations inside cells to accomplish its various biological functions including promoting ERAD, tumor suppression, ER stress mediated apoptosis. Well established proteomics tools make it possible to understand these functions and regulations.||-|
|dc.description.tableofcontents||1. INTRODUCTION 1 2. MATERIALS AND METHODS 8 2.1 Reagents 8 2.2 Expression Constructs 8 2.3 Site directed mutagenesis 9 2.4 Cell cultures and Transfection 9 2.5 siRNA transfection 9 2.6 Immunoprecipitation from cell extracts 10 2.7 UBA-specific tandem immunoprecipitation 10 2.8 GST pull down 11 2.9 Antibodies and Western analysis 11 2.10 Luciferase reporter assay 12 2.11 Protein expression and purification 13 2.12 Protein identification by Protein identification by nanoUPLC-ESI-q-TOF tandem mass spectrometry 13 2.13 Hydrogen/Deuterium exchange using MS employing pepsin digestion 14 2.14 H/D exchange analyzed by nanoUPLC-ESI-q-TOF tandem mass spectrometry 15 2.15 Cell viability: MTT assay and Cell growth analysis (xCELLigence) 15 2.16 Confocal microscopy 16 2.17 FACS analysis 17 2.18 In vitro soft agar colony-formation assay 17 2.19 Sample preparation of cancer tissues for SDS PAGE 18 3. RESULTS 19 Part I. FAF1 Promotes ERAD in a VCP-Npl4-Ufd1-Dependent Manner by Cooperative Regulation of UBA-UBX domains 19 I-1 Binding of VCP to UBX domain of FAF1 is necessary for the recruitment of polyubiquitinated proteins to UBA domain 19 I-2 UBX domain regulates UBA domain through VCP binding without direct interactions 21 I-3 FAF1 complexes with the substrate recruiting cofactor, Npl4-Ufd1 heterodimer, through VCP. 25 I-4 Npl4-Ufd1 heterodimer is required for VCP-FAF1 interaction 27 I-5 FAF1 promotes ER-associated degradation via ubiquitin receptor function 27 I-6 FAF1 promotes ER-associated degradation in a VCP-Npl4-Ufd1 dependent manner 32 Part II. FAF1 increase ER stress mediated apoptosis by promoting ATF6 activation. 36 II-1 FAF1 localizes ER membrane and increases ER stress induced cell death. 36 II-2 FAF1 interacts with ATF6 through VCP complex 37 II-3 FAF1 promotes ATF6 activation induced by tunicamycin 42 II-4 VAPB associated with FAF1 via UBL1 domain and plays role for ATF6 activation by TM 44 II-5 FAF1 promotes ATF6 activation by promoting of processing p50 ATF6 death 47 Part III. Ubiquitin-Associated (UBA) Domain in Human Fas Associated Factor 1 Inhibits Tumor Proliferation by Promoting Hsp70 Degradation 51 III-1 Overexpression of FAF1 suppresses colony formation via its UBA domain 51 III-2 Overexpression of FAF1 induces cell death through its UBA domain 54 III-3 Identification of polyubiquitinated proteins interacting with UBA domain in FAF1 55 III-4 Polyubiquitinated Hsp70 binds to FAF1 through its UBA domain. 64 III-5 FAF1 promotes Hsp70 degradation. 64 III-6 Expression of FAF1 is reduced in cervical cancer tissues. 67 4. DISCUSSION 72 Part I FAF1 Promotes Endoplasmic Reticulum-Associated Degradation (ERAD) in a VCP-Npl4-Ufd1-Dependent Manner by Cooperative Regulation of UBA-UBX domains. 72 Part II. FAF1 increases ER stress mediated apoptosis by potentiating of activating ATF6α 75 Part III Identification of polyubiquitinated substrates for FAF1 UBA domain 77 APPENDIX : Application of proteomics 82 A-1 Posttranslational modification of FAF1; Heat shock stress regulates the interaction between FAF1 and VCP by modifying the FAF1 82 A-2 Identification of PTMS of N-end rule substrates, RGS4 and FABP4 lead to predict degradation pathways. 88 A-3 Structure activity relationship and click chemistry 98 A-4 IV-4 Structural analysis of inhibitor binding to UCH-L1 using Hydrogen/Deuterium exchange (HDX) 105 5. REFERENCES 107 한글초록 118 감사의 글 120||-|
|dc.title||Proteomic Analysis of Fas-Associated Factor 1 as a Scaffolding Protein||-|
|dc.format.page||x, 122 p.||-|
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