초록 ▼

본 연구개발의 최종 목표는 (1) 유비퀴톰 관련효소 및 표적 단백질의 생리적 기능조절 기구를 규명하고, (2) TIMP-1 의 MMP 저해 회피 기전과 MMP 표적 단백질의 기능 분석을 통하여, Ub/Ubl 변형효소 및 MMP의 기질 단백질간의 네트워크를 구축함으로써, (3) 이들과 관련된 질환의 진단 및 치료를 위한 자료를 확보코자 한다. (4) 인간과 포유동물세포에서 SUMO 및 ISG15 등의 유비퀴틴 (Ub)유사단백질 (Ubiquitin-like protein, Ubl)에 의해 변형되는 표적 단백질들을 발굴하고, (5) 이들

본 연구개발의 최종 목표는 (1) 유비퀴톰 관련효소 및 표적 단백질의 생리적 기능조절 기구를 규명하고, (2) TIMP-1 의 MMP 저해 회피 기전과 MMP 표적 단백질의 기능 분석을 통하여, Ub/Ubl 변형효소 및 MMP의 기질 단백질간의 네트워크를 구축함으로써, (3) 이들과 관련된 질환의 진단 및 치료를 위한 자료를 확보코자 한다. (4) 인간과 포유동물세포에서 SUMO 및 ISG15 등의 유비퀴틴 (Ub)유사단백질 (Ubiquitin-like protein, Ubl)에 의해 변형되는 표적 단백질들을 발굴하고, (5) 이들의 결합효소와 유리효소를 규명하며, (6) Ubl 관련 효소들의 기질인식과 활성조절의 작용 기전 을 규명하고, (7) 이들의 상호작용과 구조를 연구함으로서, 궁극적으로 Ubl 관련 생체기능조절의 원리 규명 및 질병의 발생에 대한 원인을 이해하고 관련 원전기술을 개발하는 것이다. (8) 프로테아좀 활성 조절을
연구하고, (9) polyubiquitin chain 형성 메카니즘과 신규 조절 유전자의 세포기능을 규명, (10) E3 ligase의 기질 발굴 및 기능 규명을 하여, 조절 단백질의 기능 변이에 의한 암, 뇌 질환 등의 질병 관련 작용기작을 밝히고자 한다. 이러한 목표를 위해 아래의 연구를 3 단계에 걸쳐 수행함.
1단계 (2005-2008):
(1) Ub 결합, 유리효소와 MMP의 표적단백질의 발굴 및 세포 내 기능 분석
(2) SUMO, SUMO 유리효소, ISG15 의 표적 단백질 발굴 및 기질 인식 기전 연구
(3) 유비퀴틴-프로테아좀 (UPS) 활성 조절 유전자와 Rbx의 분자기전 연구
2단계 (2008-2010):
(1) Ub 외 Ubl(SUMO,lSG 15, Ufml), 결합효소, 유리효소, MMP 의 기능 및 작용기작 연구
(2) Leukemia, HCMV 감염 시 Ubl 표적단백질의 및 DUB의 기능 분석과 세포 신호전달 조절 연구
(3) Zfand family,UPS, E2-25K, Prsx , FADD, 기능규명
3단계 (2011- 2013):
(1) Ub 와 Ubl에 의한 katanin p60, aPix, DBCl, PCNA, ASC1 타껏의 생리적 기능 연구
(2) Leukemia, HCMV 감염시 SUMO/ISG15 시스템과 관련된 동물모델 분석 및 Ubl 유리 효소의 저해제 탐색
(3) Prsx 외. AK2, ZFAND family 와 ERAD pathway, E2-5K, FADD의 각 매커니즘 규명
(출처: 보고서 요약서 5p)

Abstract ▼

Protein modification is a reversible process that is catalyzed by two functionally antagonistic enzyme groups, called Ub ligases (E3) and deubiquitinating enzymes (DUBs). Until present, nearly 500 potential Ub E3 ligases have been identified and more than 100 DUBs have been predicted to present in c

Protein modification is a reversible process that is catalyzed by two functionally antagonistic enzyme groups, called Ub ligases (E3) and deubiquitinating enzymes (DUBs). Until present, nearly 500 potential Ub E3 ligases have been identified and more than 100 DUBs have been predicted to present in cells. Furthermore, cellular proteins are also reversibly modified by nearly ten other Ub-like proteins (Ubls) including SUMO, ISG15 and Ufml, in a way similar to Ub modification (i.e., through the action of Ubl E3 ligases and Ubl-specific proteases, ULPs). As their diversity reflects, these enzymes play an essential role in the regulation of numerous important cellular processes, such as abnormal protein degradation, cell cycle, signal transduction, transcription, and immune response. Critically, abnormalities in the protein modification system lead to various human diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, and infectious diseases. Therefore, this study aims to determine the molecular mechanism for the involvement of Ub/Ubl modification system in the control of key cellullar processes and thereby maintain homeostasis against human diseases. Aiming this goal, we identified a number of Ub E3 ligases, DUBs, ULPs, and their target proteins for functional analysis during the last 3 years. Results obtained are summarized as shown below:
The aims of this study are (ⅰ) to identify substrate proteins that are covalently modified by ubiquitin (Ub)-like proteins (Ubls) such as SUMO and ISG15, (ⅱ) to identify Ubl conjugating and deconjugating enzymes, (ⅲ) to understand the action mechanisms of conjugating and deconjugating enzymes, and (iv) to investigate the protein-protein interactions and tertiary structures of ubiquitome-related proteins, for understaning of the principle of the Ubl-related biological processes and the mechanisms for diseases, and for development of the related source technology. The research goals for three consecutive research periods are shown below.

• Ub E3 system: (1) Identification of SPOP as a key component of Cul3 Ub ligase for Daxx; ubiquitination (Role of SPOP/Cul3/Rocl E3 ligase complex in the control of anti-apoptotic function of Daxx; (2) Identification of HDAC1 as a target for Chfr Ub E3 ligase (Role of Chfr in cell cycle arrest at G1 phase by HDAC1 degradation).
• DUB system: (1) Identification of katanin-p60 as a target for USP47 (Role of USP47 in the control of neuronal differentiation by p60 stabilization); (2) Identification of HDAC1 as a target of USP7 (Role of USP7 in the control of AR -mediated transcription by HDAC1 stabilization).
• SUMO system: (1) Identification of Mdm2 as a target of SUSP4 (Role of SUSP4 in the control of p53 stability); (2) Identification of RXRalpha as a target of SUSP1 (Role of SUSP1 in the control of RXRalpha transactivity).
• ISG15 system: Identification of filamin B as a target for ISG15 modification (Role of ISG15 conjugation as a negative regulatory gate in interferon-induced JNK signaling pathway).
• Ufml system (1) Development of a new method for assaying DUBs and ULPs; (2) Identification of novel Ufml-processing proteases, UfSP1 and UfSP2.
• MMP system: (1) Identification of MMP target proteins by degradomic technology; (2) Modification of TIMP1 by N-glycan; (3) Functional analysis of MMPs and TIMP1 under tumorigenic conditions
• The active site of HslV protease was shown to play a critical role in the interaction of HslV with HslU A TPase and thereby in the formation of ATP-dependent HslVU protease complex. This finding contributes to the action mechanism of proteasome inhibitors, of which some are used as anti-cancer drugs • USP2 deubiquitinating enzyme and CHIP ubiquitin E3 ligase wre shown to play an antagonistinc function in the control of apoptosis inducing factor (AIF)-mediated caspase-independent ceU death. This finding raises a possibility for the use of the inbitors against USP2 and/or CHIP in AIF-mediated diseases.
• SUMO modification of hnRNP K, which can be induced by UV-mediated DNA damage, was shown to be promoted by the increased interaction of hnRNA K with Ubc9 as well as with SUMO-specific protease SENP2. This finding suggests that hnRNP K might serve as a tumor suppressor protein by acting as a co-activator of p53.
• Doxorubicin, the most widely used anti-cancer drug, was shown to induce ISG15 modification of the oncoprotein ∆ Np63. Furthermore, caspase-2 was found to preferentialIy cleave ISGylated ∆ Np63, thereby abrogating the oncogenic potential of ∆ Np63. These findings establish the nobel mechanism for the involvement of ISG15 modification in prevention of ∆ Np63-mediated tumorigenesis and the new mechanism for the anti-cancer action of doxorubicin .
• The deacetylating enzyme SIRTl was shown to be a target for ISG15 modification. Moreover, this modification was found to inhibit the deacetylation function of SIRTl.
• The transcription co-activator ASCl was found to be a target for modification by a new ubiquitin-like protein, Ufml. The Ufml acceptor site in ASCl was identified, which should clarify the role of Ufml modification in the control of ASCl function.
• Chfr was shown to control the physiological in vivo function of SIRTl.
• ATR was shown to regulated the cellular function Chfr by phosphorylation.
• SUMO modification was found to control the stability of Chfr.
• The Molecular basis for the MMP-mediated diseases were clarified by identification of target proteins of the metalloprotease.
• N-glycation of TIMP-l was shown to reduce its ability to inhibit MMPs in cancer cells.
• Identification of SUMO target proteins and their cellular functions
• Cell비. ar distribution and substrate recognition of SUMO proteases
• Identification of cellular ISG15 target proteins and roles of ISGylation
• Purification and interaction studies of ubiquitome-related proteins
• Functions of leukemia-associated Ubl targets and their regulation of cellular signaling pathways
• Functions of Ubl targets and a DUB in HCMV infection and their interactions with the interferon signaling pathway
• Regulation of the SUMO conjugating and deconjugating enzyme activities
• Structures, interactions, and inhibitors of ubiquitome-related proteases
• Interaction between HCMV and the cellular ISGylation system
• HIPK2 mutations and biomakers in leukemia development
• Animal models associated with SUMO/ISG15
• Novel functions and inhibitors of Ubl proteases
• We have demonstrated that kinases phosphorylate and inhibits the proteasome. We have characterized the human E3 ubiquitin ligase TRIP12 which ubiquitinates beta-adaptin and APP-BPl.
• We have identified 10 new genes modulating the UPS activity using cell-based assays and have characterized the function of these genes in regualation of UPS by knockdown and over-expression assays.
• We have demonstrated the association of these genes in human diseases such as tumorigenesis (in case of AK2), inflammation (Prsx), and oxidative stress (Atg)
• We have characterized ZFAND family proteins and have found that ZFAND2a specifically interacts with the proteasome whereas ZF AND2b interacts with VCP/p97.
• We have found that E2-25K is capable of synthesizing K48-linked anchored or unanchored polyubiquitin not only by one-by-one but also by en bloc transfer of a polyubiquitin chain in addition to self-ubiquitination and cyclization.
• Prsx was induced by cytokine to inhibit proteasome assembly. Prsx knockdown blocks cytokine-mediated proteasome inhibition, whereas tissues of transgenic mouse over-expressing Prsx shows enhanced proteasome activity, providing a new pathway implicated in cytokine-mediated proteasome regulation
• We have found that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysisand have demonstrated that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP.
• We have developed a novel method to identify substrates of specific E3 ubiquitin ligases using BirA biotin ligase. We have found by this procedure that RNF167 ubiquitinates Arl8b and Vamp2/3 to regulate endocytosis
• We have elucidated the mechanism of polyubiquitin formation by E2-25K and have identified amino acid residlles important for isopeptide bond formation kinetics and K48 likage specificity.
(출처: Summary 23p)

목차 Contents

• 표지 ... 1
• 제출문 ... 4
• 보고서 요약서 ... 5
• 요약문 ... 6
• SUMMARY ... 23
• CONTENTS ... 26
• 목차 ... 27
• 제 1 장 연구개발과제의 개요 ... 28
• 제1절 연구개발의 필요성 ... 28
• 제2절 연구개발의 최종 목표 및 연구 내용 ... 36
• 제 2 장 국내외 기술개발 현황 ... 41
• 제1절 국내외 타기관 연구개발 현황 ... 41
• 제 3 장 연구개발수행 내용 및 결과 ... 59
• 제1절 연구범위 및 연구수행 방법 ... 59
• 제2절 연구 결과 ... 113
• 제3절 표 및 그림 ... 271
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 679
• 제 1 절. 연도별 연구개발 목표의 달성도 ... 679
• 제 5 장 연구개발결과의 활용계획 ... 708
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 713
• 제 7 장 연구시설 • 장비 현황 ... 720
• 제 8 장 참고문헌 ... 721
• Ub 변형효소 및 표적단백질 발굴과 이들의 작용기전 ... 742
• 제출문 ... 743
• 보고서 요약서 ... 744
• 요약문 ... 745
• SUMMARY ... 752
• CONTENTS ... 755
• 목차 ... 756
• 제 1 장 연구개발과제의 개요 ... 757
• 제 2 장 국내외 기술개발 현황 ... 765
• 제 3 장 연구개발수행 내용 및 결과 ... 775
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 990
• 제 5 장 연구개발결과의 활용계획 ... 997
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 999
• 제 7 장 참고문헌 ... 1003
• Ubl 표적발굴 및 결합효소， 유리효소， 표적단백질의 작용기전 ... 1015
• 제출문 ... 1016
• 보고서 요약서 ... 1017
• 요약문 ... 1018
• SUMMARY ... 1024
• CONTENTS ... 1025
• 목차 ... 1026
• 제 1 장 연구개발과제의 개요 ... 1027
• 제1절 연구개발의 필요성 ... 1027
• 제2절 연구개발의 최종 목표 및 연구 내용 ... 1028
• 제 2 장 국내외 기술개발 현황 ... 1031
• 제1절 국내외 타기관 연구개발 현황 ... 1031
• 제 3 장 연구개발수행 내용 및 결과 ... 1037
• 제1절 연 구범위 및 연구수행 방법 ... 1037
• 제2절 연구 결과 ... 1058
• 제3절 표 및 그림 ... 1131
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 1303
• 제 5 장 연구개발결과의 활용계획 ... 1319
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 1322
• 제 7 장 연구시설 • 장비 현황 ... 1324
• 제 8 장 참고문헌 ... 1325
• 유비퀴틴 의존성 단백질 분해의 작용기전 연구 ... 1330
• 제출문 ... 1331
• 보고서 요약서 ... 1332
• 요약문 ... 1333
• SUMMARY ... 1342
• CONTENTS ... 1343
• 목차 ... 1344
• 제 1 장 연구개발과제의 개요 ... 1345
• 제 2 장 국내외 기술개발 현황 ... 1349
• 제 3 장 연구개발수행 내용 및 결과 ... 1356
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 1604
• 제 5 장 연구개발결과의 활용계획 ... 1612
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 1614
• 제 7 장 참고문헌 ... 1615
• 끝페이지 ... 1628