E3 유비퀴틴 리가아제 LNX1의 MAGE 결합 모티프가 기질 안정성에 미치는 영향

다국어 초록 (Multilingual Abstract)

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during development. It possesses an amino-terminal phosphotyrosine binding domain (PTB) domain and a proline-rich carboxyl-terminal region (PRR). LNX is a RING finger and multiple PDZ domain-containing E3 ubiquitin ligase that interacts with Numb. LNX has been reported to bind Numb directly for its proteasome dependent protein destruction. While the LNX1 dependent Numb degradation pathway has been well documented elsewhere, LNX2 mediated post-translational modification of Numb has not been analyzed so far. Even though LNX2 showed high sequence similarity with LNX1 and also bound to Numb for poly-ubiquitylation, Numb was not destabilized by LNX2. To identify key domains of LNX for Numb degradation, we shuffled the arbitrarily defined domains of LNX1 and LNX2. The domains swapped were individually tested for their significance toward Numb degradation, and we found that a small fragment, known to MAGE B18 binding motif, located between the RING finger and the first PDZ domain of LNX was critical for demarcating the molecular function of LNX on the Numb stability. Collectively our data suggested that on the contrary to the generally accepted roles for ubiquitination tightly associated with the function of RING domain which serves as a binding platform for E2 conjugation enzymes, the neighboring sequence of the RING domain of LNX family is critical determinant for Numb stability regardless of the origins of RING domain.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 재 료 및 방 법 8
• 1. 세포 배양 8
• 2. Western blot analysis 8
• 3. 면역 침강법 (Immunoprecipitation) 9
• 4. 단백질분해효소복합체 억제제 (MG132) 처리 10
• 5. Ubiquitylation assay 10
• 6. Competition assay 11
• 7. 플라스미드 및 돌연변이 제작 11
• Ⅲ. 결 과 14
• 1. LNX1과 LNX2는 높은 1차원 구조적 유사성을 지님에도 불구하고, 기질인 Numb 단백질의 안정성 (stability)에는 서로 다른 기능을 보임 14
• 2. Numb 단백질은 LNX의 기질로서 작용하며, Numb 단백질의 안정성은 E3 리가아제 기능을 통하여 조절됨 15
• 3. LNX1과 LNX2 RING 도메인 아미노 염기서열 차이는 Numb 단백질 안정성 조절에 있어 중요하지 않음 17
• 4. LNX1의 MAGE 결합 모티프는 Numb 단백질 분해에 중요함 19
• 5. LNX1의 MAGE 결합 모티프에 의해 Numb 단백질에 이루어지는 유비퀴틴화 양상이 달라짐 22
• 6. MAGE-B18은 LNX1과 상호작용을 통해 Numb 단백질 분해를 억제함 24
• Ⅳ. 고 찰 42
• Ⅴ. 결 론 48
• Ⅵ. 참 고 문 헌 49
• ABSTRACT 55