The role of tau-interacting protein in regulation of tau protein aggregations
- 주제(키워드) Aggregation , p62 , Seeding , Sequestosome-1 , Tau
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 박선아
- 발행년도 2021
- 학위수여년월 2021. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031199
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The accumulation and aggregation of tau are hallmark pathology of Alzheimer’s disease (AD). In physiological condition tau binds to microtubules and supports their functions. Tau is released from microtubules after pathologic transformation, which renders it to interact easily with other proteins. Sequestosome-1 (SQSTM1, also known as p62) involves in tau clearance through trafficking tau into degradation machinery, which contribute to diminishing tau toxicity. The interaction between tau and p62 has been thought indirect through polyubiquitin chains which were attached to tau, however we recently identified that polyubiquitinations are not essential for interaction between two proteins. We were curious whether the binding of p62 with tau give an impact on pathogenic process of tau itself like aggregation and seeding. To elucidate it we set-up ex vitro and in vitro model using recombinant proteins and genetically modified cell lines. In response to exogenous applications of tau fibrils tau phosphorylation and aggregations were induced in tau overexpressed p62 knock-out HEK293T cell lines, but not in wildtype and p62 overexpression cells. On ex vitro assay using Thioflavin T the recombinant protein of p62 was effective in inhibiting heparin-induced K18 tau aggregations. The aggregation inhibiting effect was similar to that of methylene blue when compared at the same concentration of 0.6 μM. However, the comparable effect became different at higher concentration, 2.5 μM to show lower efficacy in p62. Next, we explored the domain-specific effect of p62 on tau aggregation. We produced another recombinant protein, p62ΔTB in which TB domain was deleted. Ex vitro aggregation test demonstrated that decreased efficiency of p62 in inhibiting K18 tau aggregations in the absence of TB domain. In conclusion, our study demonstrates that p62 prevents the pathologic conversion of tau proteins to insoluble aggregates by tau fibril seeder and heparin-induced tau aggregations. It suggests that to modulate p62 could be a possible therapeutic strategy in neurodegenerative disorders with predominant tau pathology.
more목차
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 4
Ⅱ.A. Cloning for protein purification 4
Ⅱ.B. Expression of recombinant protein 8
Ⅱ.C. Purification of recombinant protein 8
Ⅱ.D. Heparin induced tau aggregation (assembly of tau fibrils) 9
Ⅱ.E. Transmission Electron Microscopy imaging 9
Ⅱ.F. Thioflavin T assay 9
Ⅱ.G. Cell culture and generation of p62 knock-out and overexpression cell line 10
Ⅱ.H. Transfection and tau seeding assay 10
Ⅱ.I. Cell preparation for SDS-PAGE 11
Ⅱ.J. Western blotting 12
Ⅱ.K. LDH assay 12
Ⅱ.L. MTT assay 13
Ⅱ.M. Statistical analysis 13
Ⅲ. RESULT 15
Ⅲ.1. Induction of tau aggregation using poly anions 15
Ⅲ.2. The role of p62 in tau protein seeding 22
Ⅲ.3. Effect of p62 in heparin-induced tau aggregation 31
Ⅳ. DISCUSSION 36
Ⅵ. REFERENCE 38
