N-end rule 작용에 의한 p62 단백질의 알로스테릭 조절

국문 초록 (Abstract)

N-end rule (N-말단 법칙) 경로란 단백질에 존재하는 아미노 말단 (N-말단)에 특정 불안정화 잔기 즉 N-degron을 갖는 단백질을 분해하는 단백질 분해 경로를 일컫는 것이다. N-말단 법칙 경로의 기질은 N-degron 결합영역을 갖는 N-recognin이라는 단백질에 의해 인식된다. 대표적으로는 UBR 박스라고 불리는 N-recognin이 존재한다. 이러한 UBR 박스는 기질의 처음 두 개 잔기와의 상호작용을 통해 기질을 인식한다. 이번 연구를 통해서 p62가 N-end rule 경로에 관여하는 신규의 N-recognin임을 밝혀냈으며, p62 내에 존재하는 ZZ 영역을 통해 기존에 알려진 N-recognin보다 높은 결합력으로 Arg/N-degron과 결합한다. N-degron과 p62의 결합으로 인해, p62가 불활성한 상태에서 활성을 띤 상태로 구조적 변화가 야기된다. 결합에 의한 p62 올리고머화가 N-말단 법칙의 알지닌화 경로에 특이적이며, p62 올리고화를 유도한다. 이는 자가포식의 활성화로 이어져 변성된 단백질이 제거된다. N-degron과 p62내 ZZ 영역과의 결합 단백질 제거 기전을 통해 변성된 단백질의 비정상적인 축적으로 인한 다양한 질환의 치료 또는 예방을 위한 치료 방법 개발에 유용하게 사용될 것으로 예측된다.

다국어 초록 (Multilingual Abstract)

The aim of this study is a novel molecular mechanism by which p62/SQSTM1 functions as an autophagic N-recognin of the Arg/N-end rule pathway and the result is that the binding of N-end rule ligands to p62 ZZ domain induces an conformational change that leads to the acceleration of p62 self oligomerization/aggregation. The N-end rule pathway is the part of the ubiquitin-proteasome system (UPS) in which short-lived proteins are degraded through the recognition of N-terminal residues (N-degrons) by recognition components (N-recognins), a class of E3 ligases. So far, known N-recognins included UBR1, UBR2, UBR4 and UBR5, which possess an evolutionally conserved 72-residue UBR box that is required for recognizing N-degrons. It has been predicted that more N-recognins are present in mammalian cells. Our genome-wide proteomic screen for N-degron binding proteins identified p62/SQSTM1 as a new recognition component of the N-end rule pathway. p62 binds various N-degrons but exhibits a stronger affinity (30 nM) for Arg/N-degrons. The ZZ domain of p62 is very similar to the UBR box of known UBR E3 ligases and is required for the binding of p62 to N-degrons. We found that p62 interaction with Arg/N-degrons induces a ZZ domain-dependent allosteric modulation which facilitates p62 oligomerization/aggregation and thereby, enhances p62-LC3 interaction. The Arg/N-degron-induced p62 oligomerization/aggregation requires intermolecular disulfide bond formation likely mediated by cysteine residue(s) present in the ZZ domain. ZZ domain binding synthetic molecules that mimic the effect exerted by Arg/N-degrons also facilitate p62 oligomerization/aggregation leading to stimulated p62 puncta formation which results in enhanced autophagosome formation and autophagy flux. This study has yielded a novel molecular mechanism by which p62/SQSTM1 functions as an autophagic N-recognin of the Arg/N-end rule pathway, providing a new potential therapeutic target for disease associated with cytosolic accumulation of protein aggregates.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• 1. Ubiquitin Proteasome System (UPS) 1
• 1-1. Ubiquitin 1
• 1-2. The ubiquitin proteasome system 2
• 1-3. Proteasome 3
• 2. Autophagy-Lysosome System 3
• 2-1. Autophagy 3
• 2-2. Process of autophagy 4
• 2-3. p62/SQSTM1 5
• 3. N-end rule pathway 6
• 3-1. The N-end rule pathway 6
• 3-2. Substrates of the N-end rule pathway 7
• Ⅱ. MATERIALS AND METHODS 18
• Ⅲ. RESULTS 26
• Ⅳ. DISCUSSION 44
• Ⅴ. CONCLUSION 48
• REFERENCE 49
• 국문초록 54