윤, 수한 류, 명이 2011-01-31T08:21:45Z 2011-01-31T08:21:45Z 2007 http://repository.ajou.ac.kr/handle/201003/1410 α-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Synphilin-1, interaction partner of α-Synuclein is a major component of inclusion bodies, but it is unknown how synphiln-1 contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein interaction partners of synphilin-1, we performed a yeast two-hybrid screen. We identified a new interacting protein LIM domain only 7 protein, LMO7. This protein localized in the nucleus, cytoplasm and cell surface, particularly adhesion junctions and contains a PDZ and LIM domain, both of which mediate protein-protein interactions. In this study, LMO7 interacts with α-Synuclein interacting protein, synphilin-1 and revealed that the co-expression with synphilin-1 results in the formation of cytoplasmic inclusions in cultured HEK293 and SY5Y cells. Synphilin-1 interacts preferentially with the C-terminal LIM domain of LMO7 and LMO7 interacts with the ankyrin domain of synphilin-1. These findings have important implications for understanding the molecular mechanism by which Lewy-body-associated proteins interact through synphilin-1. We immunostained sections of brains from patients with Parkinson's disease and demonstrated that LMO7, as well as synphilin-1, accumulates in the inclusion bodies. To define the role of LMO7 in the formation of these inclusion bodies, we performed a co-transfection with synphilin-1 and LMO7 using cultured HEK293 cells. This assay showed that LMO7 in the formation of these inclusion bodies promotes the formation of cytoplasmic inclusions. α-Synuclein, synphilin-1 and its interacting partner LMO7 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated LMO7 in immunoprecipitation /immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for LMO7, ubiquitin and synphilin-1. These observations indicate that LMO7 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated LMO7 due to impaired clearance results in its aggregation as peri-nuclear inclusions. These results suggest that LMO7 could serve as a neuropathological marker in patients with α-Synucleinopathies because it is strongly accumulated with synphilin-1 in the inclusions of their brain cells. They also suggest that LMO7 could be a potential therapeutic target for α-Synucleinopathies. 세포 내 응집체인 α-synuclein은 다양한 퇴행성 신경질환의 병리학적 표식자로 보고되어져 있으며, 결합 단백질로 잘 알려져 있는 synphilin-1은 파킨슨씨병을 포함한 신경퇴행성 질환에서 관찰되는 세포 내 응집체의 중요한 구성성분이다. 그러나 이러한 질환에서 synphilin-1 단백질의 세포학적, 생화학적 메커니즘과 세포 내에서의 역할은 여전히 규명되어 있지 않다. 따라서 본 연구에서는 synphilin-1의 기능을 알기 위해 synphilin-1과 결합하는 유전자를 yeast two-hybrid screen을 통하여 탐색한 결과, LMO7이라는 새로운 단백질을 얻을 수 있었다. LMO7은 핵과 세포질, 그리고 세포 표면과 세포연접 부위에서도 발견되는 단백질이며, 서로 다른 단백질과 단백질 사이의 결합 도매인인 PDZ와 LIM 도매인을 가진 단백질로서, 연접한 세포들끼리의 결합에 관여하여 많은 생화학적 반응에 관여하는 것으로 알려져 있다. Yeast two-hybrid screening과 mammalian cell에서의 binding assay를 통하여, synphilin-1의 ankyrin-like repeats와 coiled-coil domain이 LMO7의 LIM domain을 포함한 C-terminal에 결합한다는 것을 알았고, HEK293 세포에 synphilin-1과 LMO7을 같이 과발현한 후 이중세포염색을 통하여 두 단백질이 형성된 응집체 내에 같이 존재함을 알 수 있었다. LMO7 단백질의 발현은 α-synuclein 과 synhilin-1으로 형성된 응집체의 수의 증가를 유도하였으나, LMO7단백질의 과발현이 세포에게 toxic하게 작용하지는 않았다. 파킨슨씨병을 가진 환자의 중뇌조직에서도 synphilin-1과 LMO7이 같은 응집체 내에서 관찰되는 것을 확인함으로써, LMO7은 synphilin-1과 결합하여 퇴행성 신경질환에 관여하는 중요한 단백질일 것이라 시사되어진다. 퇴행성 신경질환에 형성되는 α-synuclein 과 synphilin-1을 비롯한 응집체의 구성성분의 분해는 유비퀴틴 (ubiquitin)을 통한 단백질 분해경로 (protein degradation pathway) 를 통해 이루어진다고 알려져 있다. 따라서 synphilin-1과 결합하여 루이소체의 응집체에서 발견되는 LMO7 단백질 또한 유비퀴틴에 의해 단백질의 분해과정과 연관이 있을 것이며, proteasome inhibitor의 처리 후 LMO7단백질을 유비퀴틴으로 확인을 한 결과, LMO7 단백질도 proteasom에 의해 분해되고 유비퀴틴화 (ubiquitination) 되는 것을 확인할 수 있었다. 이로써 LMO7 단백질은 신경퇴행성 신경질환에 형성되는 응집체의 새로운 물질이며, 이 단백질의 연구는 퇴행성 신경질환의 새로운 접근 방법을 제시하였다. "Ⅰ. INTRODUCTION = 1 Ⅱ. MATERIALS AND METHODS = 14 A. MATERIALS = 14 B. METHODS = 14 1. Yeast two-hybrid screening = 14 2. X-gal assay and ONPG assay = 15 3. Generation of LMO7 complementary DNA = 16 4. Cell culture and transfection = 16 5. In vitro binding assay = 17 6. Immunocytochemistry = 18 7. Preparation of LMO7 polyclonal antibodies = 18 8. Western immunoblot analysis = 19 9. Hematoxylin and eosin staining = 19 10. Primary culture of rat brain cortex = 20 11. LMO7-siRNA treatment = 20 12. Quantitation of cells containing inclusions = 21 13. Immunohistochemistry on human brain tissues = 21 14. Statistical analysis = 23 Ⅲ. RESULTS = 24 Ⅳ. DISCUSSION = 42 Ⅴ. CONCLUSION = 45 REFERENCES = 46 Ⅰ. INTRODUCTION = 55 Ⅱ. MATERIALS AND METHODS = 60 A. MATERIALS = 60 B. METHODS = 60 1. Cell culture and generation of stable cell line = 60 2. Inhibition of LMO7 protein synyhesis = 60 3. Immunoprecipitation and immunoblot analysis = 61 4. Immunostaining of LMO7-293 cell treated protease inhibitor = 61 Ⅲ. RESULTS = 63 Ⅳ. DISCUSSION = 68 Ⅴ. CONCLUSION = 70 REFERENCES = 71 Ⅰ. INTRODUCTION = 77 Ⅱ. MATERIALS AND METHODS = 79 1. Case material = 79 2. Immunohistochemical analysis of human brain tissues = 79 Ⅲ. RESULTS = 80 Ⅳ. DISCUSSION = 82 Ⅴ. CONCLUSION = 83 REFERENCES = 84 국문요약 = 87" text/plain en Studies on the Cytoplasmic Inclusions Detected in Parkinson's disease 파킨슨씨병에서 형성된 세포내 응집체에 관한 연구 Thesis http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000002190 Parkinson's disease Lewy body Cytoplasmic Inclusions PDZ LIM Doctor 대학원 의학과 류, 명이 2007 Theses 2007 9999-12-31 9999-12-31