A TIRAP-derived decoy peptide hinders the TLR4-mediated down-stream signaling
A TIRAP-derived decoy peptide hinders the TLR4-mediated down-stream signaling
- 주제(키워드) Toll-like receptors , TIRAP , PBM , LBP , CD14 , Inflammation
- 발행기관 아주대학교
- 지도교수 최상돈
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029276
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Toll-like Receptor (TLR) signaling cascade proceed with the help of several co-receptors and adaptor proteins resulting in the activation of a protective immune response against the invading pathogens. TLR signaling pathway can be activated through the recruitment of either Myeloid Differentiation factor 88(MyD88) or Toll-Interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF). TLR4, on the other hand, is endowed with the ability of activating both. During the recognition of Lipopolysaccharide (LPS) by TLR4, LPS binding protein (LBP) detaches the monomeric LPS and transfer it to CD14, which in turn exposes the captured LPS to the TLR4/MD2 complex, leading to the subsequent activation of the downstream signaling cascade and the secretion of pro-inflammatory cytokines. However, a dysregulated TLR4 response is often a hallmark of various inflammatory and autoimmune diseases, for this reason, TLRs are being actively pursued as drug targets in the treatment of immune-related disorders. Herein, we report a TIRAP-derived decoy peptide, Ajou5, which displays significant inhibitory effect on TLR4-mediated pathway. In RAW267.4 and THP-1 cells, Ajou5 inhibits the NF-κB activation monitored by SEAP assay, hinders the LPS-induced secretion of inflammatory cytokines such as TNF-α and IL-6, and inhibits the activation of Mitogen-activated protein kinases (MAPKs) and Interferon regulatory factor 3 (IRF3). Ajou5 was unable to inhibit inflammatory cytokines induced by other TLR ligands. In silico data suggested that the interaction of LBP and CD14 is similar to the interaction between Ajou5 and CD14 suggesting that Ajou5 can interrupt ligand transfer to CD14 by delaying the CD14-mediated signaling pathway.
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TABLE OF CONTENTS
INTRODUCTION 1
RESULTS 4
The design and validation of the peptides derived from TIRAP, Ajou5 4
Ajou5 inhibits the LPS-induced cytokine secretion and oxidative stress 5
Ajou5 hinders the TLR4-mediated TNF-α cytokine and mRNA level in RAW264.7 7
Ajou5 suppresses the pro-inflammatory cytokines through interruption of the CD14-LBP-TLR4 complex formation 7
DISCUSSION 21
MATERIALS AND METHODS 26
Cell culture 26
Cell viability test 26
Secreted Embryonic Alkaline Phosphatase (SEAP) assay 26
Cytokine detection by the Enzyme-Linked Immunosorbent Assay (ELISA) 27
Western blot analysis 27
Real Time-Polymerase Chain Reaction (RT-PCR) 28
Secreted Nitric Oxide (NO) detection 29
Intracellular Reactive Oxygen Species (ROS) and Intracellular NO (DAF-FM, DCF-DA) 29
Protein-peptide docking 29
Statistical analysis 30
REFERENCES 31
