| Summary: | 博士 === 國立中興大學 === 分子生物學研究所 === 100 === SARS coronavirus (SARS-CoV) papain-like protease (PLpro) recognizes a consensus motif LXGG as consensus cleavage sequence of cellular deubiquitinating enzymes and demonstrates inactivation of IRF3 and NF-κB, reduction of interferon (IFN) induction and suppression of type I IFN signaling pathway. This study investigates type I IFN antagonist mechanism, cytokine expression and proteomic change induced by PLpro in human promonocyte cells using proteomic analysis. PLpro significantly inhibited IFN-mediated promoter (ISRE, AP-1) and genes (PKR, 2’-5’-OAS, IL-6 and IL-8). 2-D electrophoresis, mass spectrometry, Western blotting and quantitative real-time PCR assays indicated PLpro decreased ERK1 expression and activated the ubiquitin proteasome pathway via up-regulation of ubiquitin-conjugating enzyme E2-25k and proteasome subunit alpha type 5. In addition, proteasome inhibitor and ERK1/2 inhibitor significantly reduced ERK1 expression and regulated STAT1 and c-Jun phosphorylation levels via activation of ERK1/2. Moreover, PLpro strongly increased the mRNA and protein expression of TGF-β1. 2DE/MS, Western blotting, ELISA and quantitative real-time PCR assays indicated PLpro up-regulating many TGF-β1-associated genes, including heat-shock protein 27, protein disulfide-isomerase A3 precursor, vimentin, retinal dehydrogenase 2, glial fibrillary acidic protein, and glutathione transferase Ω-1. ERK1/2 inhibitor and proteasome inhibitor significantly regulated the expression of TGF-β1 and vimentin in PLpro-expressing cells. Furthermore, PLpro up-regulated heat shock protein 27, linking with the activation of p38 MAPK and ERK1/2 signaling pathways. The treatment with p38 MAPK and ERK1/2 inhibitors significantly reduced PLpro-induced expression of TGF-β1, vimentin and type I collagen. Results demonstrated SARS PLpro promotes TGF-β1 expression through up-regulating ubiquitin proteasomal system and activation of p38 MAPK and ERK1/2 signaling pathway, and down-regulates ERK1 expression thus inhibiting the interaction between type I IFN activation of ERK1 and STAT1 via up-regulating of ubiquitin-proteasomal system.
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