Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody
Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody
- 주제(키워드) Anti-DNA antibody , TRIM21 , β-TrCP2 , Tandem affinity purification (TAP)
- 발행기관 아주대학교
- 지도교수 권명희
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029530
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
In the pathogenesis of autoimmune diseases, autoantibodies cause inflammatory response through the formation of immune complexes. In human monocyte THP-1 cells, it has been recently reported that cell-internalizable 3D8 IgG, anti-DNA antibody derived from an autoimmune-prone MRL-lpr/lpr mouse, induce the release of proinflammatory cytokines, interleukin (IL)-8 and tumor necrosis factor (TNF)-α without the formation of immune complexes. However, the mechanisms of Fc-mediated signaling in the cytosol still remained unclear. Here, the novel Fc-interacting proteins were identified using three types of tandem affinity purification (TAP) assays coupled with mass-spectrometry analysis. Among them, β-TrCP2, F-box protein of SCF E3 ubiquitin ligase complex was intensively studied. The down-regulation of β-TrCP2 reduced the production of IL-8 and TNF-α by treatment with 3D8 IgG in THP-1 cells. In Fc-mediated cytokine signaling, 3D8 IgG was ubiquitinated by β-TrCP2 in THP-1 cells, and ubiquitination of 3D8 IgG was increased in TRIM21-knockdown THP-1 cells. All these findings suggest that β-TrCP2 plays a pivotal role in the ubiquitination of 3D8 IgG in HEK293T and THP-1 cells and in the cytokine production after treatment with 3D8 IgG in THP-1 cells. Thus, it can be speculated that a novel Fc-interacting protein β-TrCP2 is a potential therapeutic target for autoimmune diseases.
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ABSTRACT i
TABLE OF CONTENTS ii
LIST OF FIGURES iii
LIST OF TABLES iv
I. INTRODUCTION 1
II. MATERIALS AND METHODS 8
A. Cell culture 8
B. Construction of expression vectors and peptide 8
C. Protein preparation using HEK293F cell 9
D. Antibodies 9
E. Lentiviral vector construction 10
F. Establishment of stable cell lines 11
G. Tandem affinity purification (TAP) 11
H. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel tryptic digestion 12
I. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis 13
J. Protein Microarray 13
K. Enzyme-Linked Immunosorbent assay (ELISA) 14
L. Confocal microscopy 14
M. Western blotting 15
N. Immunoprecipitation (IP) 15
O. Quantitative RT-PCR (qRT-PCR) 16
P. In vivo ubiquitination assay 17
Q. Statistical analysis 17
III. RESULTS 18
A. Production of IL-8 and TNF-α induced by cell-internalizable anti-DNA IgG is not mediated by TRIM21-signaling pathway. 18
B. Tandem affinity purification (TAP) is used as a method for the identification of novel Fc-interacting proteins in cell cytosol. 23
C. Extracellular tandem affinity purification (ExTAP) is designed to preserve the structural integrity of IgG-Fc used for identification of novel Fc-receptors in cell cytosol. 28
D. Human IgG Fc-interacting proteins are identified by three independent TAP assays coupled with mass-spectrometry analysis. 33
E. The binding between Fc-interacting proteins and purified IgCw-γκ is confirmed in HEK293T cells. 37
F. The binding between Fc-interacting proteins and internalizing 3D8 IgG is confirmed in HEK293T cells. 41
G. Production of IL-8 and TNF-α response to treatment of 3D8 IgG is reduced by knockdown of Fc-interacting proteins in THP-1 cells. 44
H. Ubiquitination of cell-internalizable 3D8 IgG is not mediated by TRIM21 in THP-1 cells. 48
I. Ubiquitination of 3D8 IgG is increased by TRIM21 knockdown in THP-1 cells. 53
J. β-TrCP2 ubiquitinates 3D8 IgG. 57
IV. DISCUSSION 60
V. CONCLUSION 64
REFERENCES 65
국문요약 72
