A novel phage-displayed peptide (PAP5) potentiates TLR2-mediated cytokine expression and innate immune response
- 주제(키워드) Toll-like receptor 2 , agonist , phage display , Pam3CSK 4 , vaccine adjuvant , peptide , ischemic stroke
- 발행기관 아주대학교
- 지도교수 Sangdun Choi
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030854
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Toll-like receptor 2 (TLR2), together with its two co-receptors TLR1 and TLR6, plays a significant role in the protection of host immunity through the secretion of pro-inflammatory cytokines. Occasionally, the traditional activation of TLR2 is inefficient in neutralizing the surge of pathogenic invasion; thus, the utilization of therapeutic activators for a sustained and balanced immune response becomes necessary. Here, employing a library of phage-displayed 12-mer random peptides, we identified a completely unique sequence (herein mentioned as PAP5) that specifically invokes TLR1/2-mediated downstream signaling, which might be blocked by pretreatment with a TLR1/2 antagonist. PAP5 remarkably induced the phosphorylation of mitogen-activated protein kinases (MAPKs) and activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in RAW 264.7 cells. We showed that PAP5 upregulated NF-κB-mediated expression of TNF-α and IL-6 in both mouse and human THP-1 macrophages. Altogether, our results suggest that PAP5 binds to the extracellular domains of both TLR1 and TLR2, thus facilitating TLR1/2 heterodimerization and downstream signal transduction. The peptide showed markedly specificity towards the activation of TLR1/2-mediated downstream signaling without affecting other TLR signaling pathways. We envision that PAP5 might be an appropriate candidate for the development of a potent vaccine adjuvant and antitumor agent or a drug to stop ischemic stroke from white matter. Our data demonstrate the usefulness of a phage-displayed peptide library-based high-throughput screening in the development of novel peptides as anticancer agents, vaccine adjuvants or monotherapy drugs to treat various TLR2-associated diseases.
more목차
Introduction 1
Results 3
Identification of PAP5 as a novel TLR1/2 agonist 3
PAP5 triggers TLR1/2-mediated NF-κB and mitogen-activated protein kinase (MAPK) activation
in mouse macrophages 3
PAP5 stimulates the expression of pro-inflammatory cytokines in human cell lines 4
PAP5 acts as a specific agonist of TLR1/2 4
PAP5 occupies the triacyl lipopeptide binding cavity on TLR1/2 ECD 5
Discussion 16
Materials and Methods 19
Construction of the phage-displayed 12-mer peptide library 19
Panning procedure 19
High-throughput screening 20
DNA sequencing and peptide synthesis 20
Cell culture and treatment 21
Protein extraction and quantification 21
Western blotting 22
Cell viability assay 22
Assessing cytokine secretion via ELISA 22
Confocal microscopy 23
Quantitative RT-PCR 23
PAP5 model construction, protein-protein docking, and MD simulation 23
References 25
