β-아밀로이드 정제방법 개발과 nuclear scaffold 단백질분해효소의 활성화에 의한 lamin의 절단에 관한 연구

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비제이 산카
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Aβ의 응집과 축적은 알쯔하이머병의 중요한 병인이다. 여러 구조체 중 Aβ oligomer가 가장 세포독성이 강하다. 이 세포독성의 기전을 이해하기 위해서는 다량의 Aβ의 준비가 필요하다. 또한 여러 변이체가 필요한데 그 중 flag등에 의해 tag된 Aβ등은 Aβ에 결합하는 단백질의 확인등에 매우 유용할 것이다. 따라서 본 연구에서는 이 펩티드를 정제하기 위한 방법을 고안하였다. 이 펩티드는 inclusion body의 형태로 발현시켜 urea에 녹인 fusion 단백질 상태로 준비하고 이어 deubiquilinating enzyme으로 절단시켰다. fusion 단백질로부터 절단된 Aβ를 Ni-NTA column과 reverse phase-HPLC chromatography로 순수한 Aβ를 얻었다. 이를 이용하여 Aβ의 세포독성을 연구하였다. HeLa 세포에 Aβ oligomer를 처리하고 그 독성과 apoptosis의 중요효소인 caspase 활성화, 그 기질의 processing을 immunoblotting과 enzyme 활성조사를 통해 분석하였다. 오랫동안 세포에 Aβ를 처리하면 농도 의존적으로 caspase 활성과 그 기질의 processing이 증가하였다. 그러나 짧은 시간을 처리하면 그렇지 않았다. 그러나 세포독성은 이 짧은 시간에 관찰되었는데 이는 Suc-AAPF-CMK에 의해 저해가 되어 이 세포독성이 caspase와는 상관없이 일어나며 아마도 nuclear serine protease와 연관있으리라 생각되었다. 이 연관성은 이 효소의 기질인 lamin A와 B가 절단되는 것으로 확인되었으며 confocal imaging등의 후속 실험에 의해 재차 확인되었다. 이는 Aβ가 caspase활성화와 상관없이 nuclear scaffold protease의 활성화에 의해 그 세포독성을 나타냄을 의미한다.|The aggregation and accumulation of Aβ plays a significant role in the pathogenesis of Alzheimer’s disease. Aβ oligomeric aggregates are believed to be the main toxic species underlying the pathogenicity. Understanding the mechanistic details of Aβ-induced cell death is crucial to developing effective strategies to prevent and control AD. As evidenced by a number of cell-based experiments and animal model studies, there are good correlations between the pathogenesis and structural species of Aβ. In this study, an efficient system was constructed to purify the peptides in soluble form. The target peptides were expressed as inclusion bodies. Aβs were purified after solubilization of fusion protein in urea and cleavage by a deubiquilinating enzyme. The cleaved peptide solution were then subjected to Ni-NTA column and Reverse phase–HPLC chromatography to obtain homogeneous Aβ peptide. Further, to study the Aβ binding or interacting proteins, a FLAG tagged Ab42 vector system was developed. The fusion protein was overexpressed, purified and their binding efficiency was analyzed by using Immunoprecipitation and confocal imaging methods.
. Next, we made an attempt to correlate specific protease(s) to selected cell death-inducing agent. Particularly, non-caspase proteases functioning in the cell death were interested, because roles of the enzymes in the death process are relatively less understood. β-amyloid (Aβ), a hallmark peptide of Alzheimer’s disease (AD), was chosen as a death inducer, because it is important in pathogenesis of the disease, and its cell death inducing mechanism is not well understood. Cytotoxicity, Caspases and their substrates processing were analyzed using immunoblotting and enzyme assays. Our results show that lamin A and B reduction and consequent nuclear morphological change occur in cells treated by Aβ42 before caspase activation. Data of our study indicate that the reduction of lamin proteins may be due to NS protease function. Suppression of NS protease by its inhibitor, AAPF-CMK, effectively inhibited the cell death induced by Aβ42.This study provides an idea that inhibition of NS protease might be an effective way to control pathological process of AD.
Alternative Title
Development of β-amyloid purification method and its identification as an inducer of nuclear scaffold protease-mediated lamin cleavage which occurs independently of caspase activation
Alternative Author(s)
Vijaysankar Ramasamy
일반대학원 생물신소재학과
일반대학원 생물신소재학과
Awarded Date
Table Of Contents
I.Introduction 6- 17
I.1.Amyloid beta cascade hypothesis 6
I.2.Amyloid beta aggregation and toxicity 7
I.3. Amyloid beta: Interaction with membranes and intra-Cellularproteins 9
I.4.Caspase and caspase independent mechanism in apoptosis 12
l.5.Amyloid beta and apoptosis 14
I.6.Outline of this thesis 15
ll. Purification of recombinant β-amyloid peptide expressed with inclusion body-forming or solubilizationfacilitating fusion partner proteins 18- 40
ll.1. Introduction 18
ll.2. Previous results 20
ll.3. Experimental design 26
ll.4. Materials and Methods 29
ll.4.1. Reagents 29
ll.4.2. Equipments 29
ll.4.3. Equipment setup 30
ll.5. Procedure 31
II-5.1. Preparation of large scale culture 31
II-5. 2. Preparation of Inclusion bodies, solubilization and Ultra-Filtration of soluble protein 32
II-5. 3. Solubilization 33
II-5. 4. Purification of Inclusion body using Ni2+-NTA column chromatography 33
II-5. 5. Digestion of GroES-Ubiquitin-Aβ (1-42) fusion protein with Usp2cc enzyme 34
II-5. 6. Purification of Aβ (1-42) using Ni2+-NTA column chromatography 35
II-5. 7. HPLC: Separation of amyloid beta peptide on Polymer based column 36
II-5. 8. Purification of Aβ (1-42) fused with Trigger factor 37
II-5. 9. Aliquoting and Storage 38
II-5.10.Aggregation kinetics using thioflavin (ThT) binding assay 39
II-6. Results and Discussion 40
lll. Construction and purification of FLAG-Aβ42 45- 48
lll-1. Introduction 45
lll-2. Materials and Methods 46
lll-2. 1. Construction and purification of FLAG tagged amyloid beta peptides 46
lll-2. 2. Immunoprecipitation 47
lll-2. 3. Immunocytochemistry 48
lll-3. Results and Discussion 48
IV. Amyloid beta 42 induces nuclear scaffold mediated lamin cleavage independently of Caspase activation 53- 86
lV-1. Introduction 53
lV-2. Materials and Methods 58
lV-2. 1. Materials 58
lV-2. 2. Preparation of Aβ42 59
lV-2. 3. Cell Culture and Cytotoxicity Assay 59
lV-2. 4. Measurement of Caspase activity 60
lV-2. 5. Protease inhibitor screening (PIS) 61
lV-2. 6. Measurement of Nuclear Scaffold protease assay 62
lV-2. 7. Western blot analysis 63
lV-2. 8. Immunocytochemistry 63
IV-3.Results and Discussion 65
lV-3. Searching for condition in which cell death occurs without caspase activation in Aβ42-treated cells 65
lV-4. Activation of NS protease in Aβ42-induced cell death 72
lV-5. Differential processing of lamin A/C and B in STS- and Aβ42-treated cells 75
lV-6. NS protease inhibitor but not caspase inhibitor reduces Aβ42-induced lamin A and B cleavage 83
lV-7. Aβ42 induces nuclear deformation which is suppressed by NS protease inhibitor 86
V-1.Introduction 93
V-2. Materials and Methods 93
V-2.1. Production of recombinant caspases 93
V-2.2. Measurement of purified caspase activity using synthetic substrates 94
V-2.3. Immunocytochemistry 95
V-3. Results and Discussion 96
조선대학교 대학원
비제이 산카. (2013). β-아밀로이드 정제방법 개발과 nuclear scaffold 단백질분해효소의 활성화에 의한 lamin의 절단에 관한 연구.
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