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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 江皓森(Hao-Sen Chiang) | |
| dc.contributor.author | Ling-Chia Yen | en |
| dc.contributor.author | 顏伶珈 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:16:42Z | - |
| dc.date.available | 2027-12-31 | |
| dc.date.copyright | 2018-02-26 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-09-27 | |
| dc.identifier.citation | 1. Mazzon, M. and J. Mercer, Lipid interactions during virus entry and infection. Cell Microbiol, 2014. 16(10): p. 1493-502.
2. Greseth, M.D. and P. Traktman, De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection. PLoS Pathog, 2014. 10(3): p. e1004021. 3. Heaton, N.S., et al., Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis. Proc Natl Acad Sci U S A, 2010. 107(40): p. 17345-50. 4. Herker, E. and M. Ott, Unique ties between hepatitis C virus replication and intracellular lipids. Trends Endocrinol Metab, 2011. 22(6): p. 241-8. 5. Petersen, J., et al., The major cellular sterol regulatory pathway is required for Andes virus infection. PLoS Pathog, 2014. 10(2): p. e1003911. 6. Sharookh B. Kapadia and Francis V. Chisari, Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids. Proc Natl Acad Sci U S A, 2005. 102(7): p. 2561-6. 7. Munger, J., et al., Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotech, 2008. 26(10): p. 1179-86. 8. Olagnier, D. and J. Hiscott, Breaking the barrier: membrane fusion triggers innate antiviral immunity. Nat Immunol, 2012. 13(8): p. 713-5. 9. Blanc, M., et al., The transcription factor STAT-1 couples macrophage synthesis of 25-Hydroxycholesterol to the interferon antiviral response. Immunity, 2013. 38(1): p. 106-18. 10. Liu, S.Y., et al., Interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol. Immunity, 2013. 38(1): p. 92-105. 11. Cyster, J.G., et al., 25-Hydroxycholesterols in innate and adaptive immunity. Nat Rev Immunol, 2014. 14(11): p. 731-43. 12. Chukkapalli, V., N.S. Heaton, and G. Randall, Lipids at the interface of virus-host interactions. Curr Opin Microbiol, 2012. 15(4): p. 512-8. 13. Reboldi, A., et al., Inflammation. 25-Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I interferon. Science, 2014. 345(6197): p. 679-84. 14. Gold, E.S., et al., 25-Hydroxycholesterol acts as an amplifier of inflammatory signaling. Proc Natl Acad Sci U S A, 2014. 111(29): p. 10666-71. 15. Koarai, A., et al., 25-Hydroxycholesterol enhances cytokine release and Toll-like receptor 3 response in airway epithelial cells. Respir Res, 2012. 13: p. 63. 16. Brown, M.S. and J.L. Goldstein, The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell, 1997. 89(3): p. 331-40. 17. Radhakrishnan, A., et al., Switch-like control of SREBP-2 transport triggered by small changes in ER cholesterol: a delicate balance. Cell Metab, 2008. 8(6): p. 512-21. 18. Duncan, E.A., et al., Cleavage site for sterol-regulated protease localized to a leu-Ser bond in the lumenal loop of sterol regulatory element-binding protein-2. J Biol Chem, 1997. 272(19): p. 12778-85. 19. Horton, J.D., J.L. Goldstein, and M.S. Brown, SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest, 2002. 109(9): p. 1125-31. 20. Michael S. Brown and Joseph L. Goldstein, The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell, 1997. 89(3): p. 331-40. 21. Robertson, K.A. and P. Ghazal, Interferon control of the sterol metabolic network: bidirectional molecular circuitry-mediating host protection. Frontiers in Immunology, 2016. 7: p. 634. 22. Blanc, M., et al., Host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis. PLoS Biol, 2011. p. e1000598. 23. York, A.G., et al., Limiting cholesterol biosynthetic flux spontaneously engages type I IFN signaling. Cell, 2015. 163(7): p. 1716-29. 24. Gorenshteyn, D., et al., Interactive big data resource to elucidate human immune pathways and diseases. Immunity, 2015. 43(3): p. 605-14. 25. Guan, Y., R.M. Torres, and J.M. Hartney, The influence of Arhgef1 on pulmonary leukocyte function. Immunologic Research, 2013. 55(1): p. 162-6. 26. Girkontaite, I., et al., Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses. Nat Immunol, 2001. 2(9): p. 855-62. 27. Kozasa, T., et al., p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. Science, 1998. 280(5372): p. 2109-11. 28. Etienne-Manneville, S. and A. Hall, Rho GTPases in cell biology. Nature, 2002. 420(6916): p. 629-35. 29. Hart, M.J., et al., Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Scienc, 1998. 280(5372): p. 2112-4. 30. Neubig, R.R. and D.P. Siderovski, Regulators of G-Protein signalling as new central nervous system drug targets. Nat Rev Drug Discov, 2002. 1(3): p. 187-97. 31. Liu, C., et al., Oxysterols direct B-cell migration through EBI2. Nature, 2011. 475(7357): p. 519-23. 32. Husted, A.S., et al., GPCR-mediated signaling of metabolites. Cell Metab, 2017. 25(4): p. 777-96. 33. Oh, D.Y., et al., GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects. Cell. 142(5): p. 687-98. 34. Hirasawa, A., et al., Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. Nat Med, 2005. 11(1): p. 90-4. 35. de Oliveira, M.C., et al., Bile acid receptor agonists INT747 and INT777 decrease oestrogen deficiency-related postmenopausal obesity and hepatic steatosis in mice. Biochim Biophys Acta, 2016. 1862(11): p. 2054-62. 36. Yoneno, K., et al., TGR5 signalling inhibits the production of pro-inflammatory cytokines by in vitro differentiated inflammatory and intestinal macrophages in Crohn's disease. Immunology, 2013. 139(1): p. 19-29. 37. Reboldi, A., et al., Inflammation. 25-Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I interferon. Science, 2014. 345(6197): p. 679-84. 38. Bauman, D.R., et al., 25-Hydroxycholesterol secreted by macrophages in response to Toll-like receptor activation suppresses immunoglobulin A production. Proc Natl Acad Sci U S A, 2009. 106(39): p. 16764-9. 39. Diczfalusy, U., et al., Marked upregulation of cholesterol 25-hydroxylase expression by lipopolysaccharide. J Lipid Res, 2009. 50(11): p. 2258-64. 40. Fessler, M.B. and J.S. Parks, Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling. J Immunol, 2011. 187(4): p. 1529-35. 41. Ouellet, H., J.B. Johnston, and P.R.O.d. Montellano, Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis. Trends in Microbiology, 2011. 19(11): p. 530-9. 42. Parihar, S.P., et al., Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation. J Infect Dis, 2014. 209(5): p. 754-63. 43. Park, K. and A.L. Scott, Cholesterol 25-hydroxylase production by dendritic cells and macrophages is regulated by type I interferons. J Leukoc Biol, 2010. 88(6): p. 1081-7. 44. Spann, N.J. and C.K. Glass, Sterols and oxysterols in immune cell function. Nat Immunol, 2013. 14(9): p. 893-900. 45. Tall, A.R. and L. Yvan-Charvet, Cholesterol, inflammation and innate immunity. Nat Rev Immunol, 2015. 15(2): p. 104-16. 46. Yvan-Charvet, L., et al., Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions. Circulation, 2008. 118(18): p. 1837-47. 47. Zhu, X., et al., Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol. J Lipid Res, 2010. 51(11): p. 3196-206. 48. Chiang, H.S., et al., GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses. Nat Immunol, 2014. 15(1): p. 63-71. 49. Zidovetzki, R. and I. Levitan, Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochimica et biophysica acta, 2007. 1768(6): p. 1311-24. 50. Nagoshi, E. and Y. Yoneda, Dimerization of sterol regulatory element-binding protein 2 via the helix-loop-helix-leucine zipper domain is a prerequisite for its nuclear localization mediated by importin beta. Mol Cell Biol, 2001. 21(8): p. 2779-89. 51. Nagoshi, E., et al., Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor, occurs through the direct interaction of importin beta with HLH-Zip. Mol Biol Cell, 1999. 10(7): p. 2221-33. 52. Zhang, X., et al., Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Molecular Cell, 2013. 51(2): p. 226-35. 53. Clarke, P.R. and C. Zhang, Spatial and temporal coordination of mitosis by Ran GTPase. Nat Rev Mol Cell Biol, 2008. 9(6): p. 464-77. 54. Fukuhara, S, et al., RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho? Oncogene, 2001. 20(13): p. 1661-8. 55. Bos, J.L., H. Rehmann, and A. Wittinghofer, GEFs and GAPs: critical elements in the control of small G proteins. Cell, 2007. 129(5): p. 865-77. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68288 | - |
| dc.description.abstract | 細胞內的膽固醇在免疫系統中扮演著重要的角色，研究已證實膽固醇可調控宿主抗病毒能力及多種發炎反應。第二型固醇調節元件結合蛋白 (sterol regulatory element-binding protein 2, SREBP2)是個已知調控膽固醇合成的轉錄因子，其活化時會經由固醇調節元件結合蛋白切割活化蛋白 (SREBP cleavage activating protein, SCAP)的運送離開內質網，並在高基氏體內被水解後進入細胞核。近期研究指出第一型干擾素 (type I interferon)會藉由調控SREBP2的活化去影響膽固醇代謝，但其中的機制尚未明瞭。透過大數據資料庫網站ImmuNet分析SREBP2後顯示，其和Rho型鳥嘌呤核苷酸交換因子 (Rho guanine nucleotide exchange factor 1, ARHGEF1)具有功能上的相關性。ARHGEF1主要表現在免疫細胞中，其參與調控的G蛋白偶聯受體 (G protein–coupled receptor, GPCR)被證明可作為細胞代謝物的受體，再加上同樣身為鳥嘌呤核苷酸交換因子蛋白的GEF-H1已被知道可影響第一型干擾素生成，因此我欲探討ARHGEF1是否可和藉由和SREBP2交互作用，進而調控膽固醇與第一型干擾素之間的反應。
在轉染SREBP2的猴子腎臟細胞COS-7中過表現ARHGEF1，可減少SREBP2前導蛋白 (precursor SREBP2)表現並使細胞內膽固醇含量維持在恆定值。在ARHGEF1基因減弱的人類單核球細胞THP-1中，第一型干擾素IFNβ1、其下游基因與促發炎因子表現皆上升；給予THP-1分化後的巨噬細胞干擾素基因(stimulator of interferon gene, STING)受體3’3’-cGAMP的刺激後，IFNβ1與膽固醇、脂肪酸合成相關基因的表現大幅上升，有趣的是，額外加入膽固醇培養可消除此上升表現。以CRISPR方式在人類肝癌細胞株Huh-7中減弱ARHGEF1的表現後亦可觀察到SREBP2前導蛋白與細胞內膽固醇含量增加的現象。在過表現ARHGEF1的Huh-7細胞中有胞內膽固醇含量上升、SREBP2前導蛋白表現下降的現象，過表現ARHGEF1亦可消除3’3’-cGAMP所引起IFNβ1與膽固醇、脂肪酸合成相關基因的高表現量。由以上結果推測ARHGEF1藉由改變SREBP2活化狀態影響細胞內膽固醇含量，進而調控第一型干擾素訊息途徑。可惜的是，實驗結果證明ARHGEF1與SREBP2之間並沒有直接或間接的蛋白交互作用，推測ARHGEF1並非藉由調控SREBP2而參與膽固醇與第一型干擾素之間的反應，或是此兩蛋白可能在特定環境條件下才會互相作用。總括來說，本研究揭露ARHGEF1可藉由改變細胞內膽固醇合成與含量影響第一型干擾素反應，藉此影響細胞抗病毒能力，至於其詳細作用機制仍有待探討。 | zh_TW |
| dc.description.abstract | Cellular cholesterol homeostasis plays a critical role in immunity that affects antiviral and inflammatory responses. The sterol regulatory element-binding protein 2 (SREBP2) is a transcriptional factor with a well-defined function in the regulation of cholesterol synthesis. SREBP cleavage activating protein (SCAP) is a sterol-regulated chaperone protein that escorts SREBP2 from the ER membrane to the Golgi apparatus where the active domains of SREBP2 are released proteolytically to enter the nucleus. Recent study has indicated that type I interferon (IFN) pathway reprograms cholesterol homeostasis by regulating SREBP2 activation. However, it remains unclear how cholesterol metabolism and type I IFN responses are co-regulated. In order to identify functionally important interactions with SREBP2, I first utilized an interactive big data resource (ImmuNet, http://immunet.princeton.edu) to generate a data-driven hypothesis that Rho Guanine Nucleotide Exchange Factor 1 (ARHGEF1), a GEF that is highly expressed in lymphocytes, is functionally related to SREBP2. ARHGEF1 is well known for its role in Gα12/13-ARHGEF1-RhoA signaling pathway. Previous studies also indicated that G protein–coupled receptors (GPCRs) act as receptors for metabolites including fatty acids and bile acids. Moreover, another GEF protein, GEF-H1, is demonstrated to regulate IFNβ expression. Therefore, I hypothesized that ARHGEF1 is involved in the interaction of cholesterol homeostasis and type I IFN responses via modulating SREBP2 maturation.
Here I showed that overexpression of ARHGEF1 in SREBP2-transfected COS-7 cells reduced the levels of SREBP2 precursor form and maintained total cellular cholesterol levels. Gene expression studies on unstimulated THP-1 cells revealed that knockdown of ARHGEF-1 resulted in the spontaneous induction of IFNβ1, interferon-stimulated genes (ISGs), and pro-inflammatory cytokines. The expression of IFNβ1 and cholesterol and fatty acid synthesis genes were further increased in ARHGEF1-reduced THP-1 macrophages in response to STING ligand 3’3’-cGAMP. Importantly, the addition of cholesterol significantly diminished this upregulation. It was also observed in Huh-7 cells that knockdown of ARHGEF1 reduced the level of precursor SREBP2 and cellular cholesterol. Overexpression of ARHGEF1 in Huh-7 cells increased cellular cholesterol level and decreased precursor SREBP2 expression. 3’3’-cGAMP-induced IFNβ and cholesterol and fatty acid synthesis genes expression were diminished in ARHGEF1-overexpressed Huh-7 cells. From the above results, it was inferred that ARHGEF1 affects the type I IFN pathway by regulating the activation of SREBP2. However, the results of co-immunoprecipitation assay and yeast-two hybrid assay indicated that there is no protein interaction between ARHGEF1 and SREBP2. ARHGEF1 may take part in the cholesterol homeostasis and type I IFN responses through other pathways, or ARHGEF1 interacts with SREBP2 only under specific conditions. Taken together, this study elucidate that ARHGEF1 is involved in the regulatory loop of cholesterol homeostasis and type I IFN responses, therefore regulates the antiviral responses. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:16:42Z (GMT). No. of bitstreams: 1 ntu-106-R04b21008-1.pdf: 3539988 bytes, checksum: 64945a745b5003e07fddff0e9746e749 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 中文摘要 iv Abstract vi Contents ix List of figures xii Chapter 1 Introduction 1 1. Type I IFN responses and lipid homeostasis 1 1.1 Type I IFN signaling 1 1.2 Cholesterol 25-hydroxylase (CH25H) is identified as an interferon stimulated gene (ISG) 1 1.3 Sterol Regulatory Element-Binding Protein (SREBP) 3 1.3.1 Activation mechanism 3 1.3.2 Function 3 1.3.3 Isoforms 4 1.4 A regulatory loop of type I IFN signaling and cholesterol homeostasis 4 1.5 ImmuNet 5 2. ARHGEF1 (Rho Guanine Nucleotide Exchange Factor 1) 6 2.1 Guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) function 6 2.2 ARHGEF1, cholesterol homeostasis and immunity 6 3. Aim 7 Chapter 2 Materials and Methods 10 1. Cell Culture 10 2. Preparation of drug solution 11 3. Expressing plasmids 11 4. Lentivirus production, titration and transduction 12 5. CRISPR-Cas9 system 13 6. Western blotting 13 7. Co-immunoprecipitation (Co-IP) 15 8. RNA extraction and real time PCR analysis 16 9. Measurement of total cellular cholesterol concentration by enzymatic assay……. 17 10. Yeast two-hybrid assay 18 Chapter 3 Results 19 1. Overexpression of ARHGEF1 and SREBP2 maintained cholesterol homeostasis 19 2. Loss of ARHGEF1 primed type I IFN expression and inflammatory responses 19 3. ARHGEF1 regulates type I IFN immune responses and cholesterol and fatty acid synthesis genes 20 4. Overexpression of ARHGEF1 in Huh-7 cells reprogramed total cellular cholesterol, precursor SREBP2 and type I IFN responses 22 5. There is no protein-protein interactions between ARHGEF1 and SREBP2 23 Chapter 4 Discussion 25 References 30 Figure 38 Table 60 Appendix 61 | |
| dc.language.iso | en | |
| dc.subject | 膽固醇 | zh_TW |
| dc.subject | 第一型干擾素 | zh_TW |
| dc.subject | Rho型鳥嘌呤核?酸交換因子；第二型固醇調節元件結合蛋白 | zh_TW |
| dc.subject | 干擾素基因途徑 | zh_TW |
| dc.subject | SREBP2 | en |
| dc.subject | ARHGEF1 | en |
| dc.subject | cholesterol | en |
| dc.subject | type I interferon | en |
| dc.subject | STING pathway | en |
| dc.title | 探討膽固醇體內平衡與第一型干擾素免疫反應的相互關係 | zh_TW |
| dc.title | Identifying the link between cholesterol homeostasis and type I interferon responses | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 丁詩同(Shih-Torng Ding),林甫容(Fu-Jung Lin),劉旻禕(Min-Yi Liu) | |
| dc.subject.keyword | Rho型鳥嘌呤核?酸交換因子；第二型固醇調節元件結合蛋白,膽固醇,第一型干擾素,干擾素基因途徑, | zh_TW |
| dc.subject.keyword | ARHGEF1,SREBP2,cholesterol,type I interferon,STING pathway, | en |
| dc.relation.page | 63 | |
| dc.identifier.doi | 10.6342/NTU201704237 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-09-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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