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The Human Lysyl-tRNA Synthetase Secreted by Shiga Toxins Triggers Proinflammatory Response to the Toxin-sensitive Cells : 시가 독소로 인해 분비되는 인간유래 리실-tRNA 합성효소가 독소-민감성 세포에 염증 전 반응을 유발함
DC Field | Value | Language |
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dc.contributor.advisor | 최상호 | - |
dc.contributor.author | 권해냄 | - |
dc.date.accessioned | 2017-07-14T06:44:14Z | - |
dc.date.available | 2017-07-14T06:44:14Z | - |
dc.date.issued | 2015-08 | - |
dc.identifier.other | 000000028707 | - |
dc.identifier.uri | https://hdl.handle.net/10371/125901 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2015. 7. 최상호. | - |
dc.description.abstract | Shiga toxin producing Escherichia coli (STEC) strains are foodborne pathogens that cause fatal systemic complications such as hemolytic uremic syndrome and central nervous system (CNS). In patients infected with STEC, inflammatory responses are involved in reducing or exacerbating disease severity upon infections with the pathogen and require the participation of cytokine/chemokine in concert of leukocytes on Stxs-induced intestinal, renal, and CNS vascular lesions. To date, although numerous studies have been reported defining the proinflammatory response to Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) in in vivo and in vitro, no coherent mechanism has been proposed to explain the dynamic immune regulation involving cytokines or unknown mediators of inflammation during intoxication.
A complex of aminoacyl-tRNA synthetases, known as Multi-aminoacyl-tRNA synthetase complex (MSC), is only founded in higher organisms such as yeast, zebra-fish, mouse, and human. In human, MSC is composed of eight aminoacyl-tRNA synthetases and three axillary proteins. It is well known that each aminoacyl-tRNA synthetases has noncanonical functions such as immune response, tumorigenesis, angiogenesis, and inflammatory response. Classical role of human lysyl-tRNA synthetase (KRS) within MSC is to support translational regulation. Otherwise, KRS also has been known that cytosolic KRS can regulate transcriptional activity of transcription factor MITF and USF2. Furthermore, the form of secreted KRS engages in secondary cellular function triggering inflammatory response. However, the function of KRS against infection of human pathogenic bacteria was poorly understood. This study identified the release of KRS to the extracellular space from the Stx1 or Stx2-treated human cervical cancer cells (HeLa) or macrophage-like THP-1 cells (D-THP-1). As I predicted, incubations of the D-THP-1 cells with enzymatic deficient Stxs were not capable of secreting KRS into the media. Furthermore, neither Stx1 nor Stx2 induced transcription of KRS mRNA and nucleus translocation. Unlike KRS, aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2), known as cytosolic binding partner of KRS, remained in cytosol without extracellular secretion when it is exposed to Stx2. Furthermore, addition of the exogenous KRS or co-treatment of KRS with Stxs activated secretion of inflammatory cytokines including IL-1β or TNF-α and chemokines including IL-8 or MIP1α in autocrine and paracrine manner. To support the pathogenesis mechanisms of Stxs associated with KRS, mouse model following the administration with Stx2 LD50 (10 ng/kg) demonstrated that the survival rate from the intoxicated KRS-hetero knockdown mice was significantly higher than that of the wild-type mice. In conclusion, this study implicated that KRS may be a key player to mediate proinflammatory signal transductions in susceptible host infected with STEC. | - |
dc.description.tableofcontents | I. INTRODUCTION 1
II. MATERIALS AND METHODS 7 Antibodies and reagents 7 Expression vector 7 Toxins 8 Cell culture 8 Preparation of cellular lysates and western blotting 10 KRS secretion test in human HeLa cells 11 Purification of Multi-aminoacyl-tRNA synthetase complex in HeLa cells 11 KRS-expressing stable cell line and purification 12 Isolation of nuclear and cytoplasmic extracts 13 Immunoprecipitation 13 Enzyme-linked immunosorbent assay (ELISA) 14 RNA isolation 14 Real-time PCR 15 MTS-based cell cytotoxicity assay 16 Animal studies 16 Statistical analysis 17 III. RESULTS 18 Secretion of KRS in Stxs treated macrophage-like human monocytic (D-THP-1) cells 18 Identification of the origin of secreted KRS by Stxs treatment 21 Secretion of KRS in HeLa cells by Stx2 24 Dissociation of KRS from Multi-aminoacyl-tRNA synthetase complex in Stx1 intoxicated HeLa cells 26 Scheme of generating KRS-expressing stable cell line and purification 28 Cytokines and chemokines production in D-THP-1 cells by KRS 31 Enhancement of proinflammatory cytokines production via co-treatment of KRS with Stxs 34 Paracrine effects of KRS in human kidney cells 37 Cytotoxicity of KRS in D-THP-1 cells 40 In vivo experiments of Stx2 administrated KRS heterozygous knockout mice 42 IV. DISCUSSION 45 V. REFERENCES 49 VI. 국문초록 57 | - |
dc.format | application/pdf | - |
dc.format.extent | 1345072 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Shiga toxins | - |
dc.subject | Lysyl-tRNA synthetase | - |
dc.subject | Proinflammatory response | - |
dc.subject.ddc | 630 | - |
dc.title | The Human Lysyl-tRNA Synthetase Secreted by Shiga Toxins Triggers Proinflammatory Response to the Toxin-sensitive Cells | - |
dc.title.alternative | 시가 독소로 인해 분비되는 인간유래 리실-tRNA 합성효소가 독소-민감성 세포에 염증 전 반응을 유발함 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Haenaem Kwon | - |
dc.description.degree | Master | - |
dc.citation.pages | vi, 60 p | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2015-08 | - |
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