臺灣博碩士論文加值系統

當細胞受到內質網壓力時，C/EBP homologous protein (CHOP)會被轉譯出來，而CHOP的產生可以決定細胞的存活與凋亡。在chop mRNA的5’ UTR 上具有一段upstream open reading frame (uORFchop)會抑制下游coding sequence (CDS)轉譯成CHOP 。當逆境來臨時，uORFchop才會失去抑制轉譯下游CDS的功能，進而轉譯出CHOP蛋白質。然而是以何種分子機制調控uORFchop抑制轉譯的能力至今仍不甚清楚。因此，本實驗室構築DNA片段其中含有CMV promoter驅動人類uORFchop(huORFchop)來抑制轉譯下游以綠色螢光蛋白(green fluorescent protein, GFP) cDNA 當CDS的表現載體，並轉殖到斑馬魚胚胎而得到含有huORFchop-gfp mRNA的轉殖品系斑馬魚- huORFZ。當受到內質網壓力時huORFchop便會失去抑制轉譯出GFP蛋白質的功能。於是我們利用雷射顯微切割(Laser microdissection)的方式，各別收集於熱逆境之下huORFZ的胚胎會表現GFP(+)的腦細胞作為實驗組而以GFP(−)的腦細胞作為對照組，利用microarray分析篩選出一個A值7.2 (A值大於7表示可信度高)及M值2.4 (M值大於0表示強度高)的up-regulated基因－endonuclease poly(U)-specific C (endouc)。若直接注射endouc mRNA於斑馬魚胚胎時，縱使在不給予stress的情況下也會轉譯出下游CDS的GFP，即是endouc會促使huORFchop抑制轉譯能力的喪失；因而在沒有內質網壓力的huORFZ 胚胎也會表現GFP。進一步地發現不管在in vivo或in vitro，Endouc的overexpression都會造成eIF2α的磷酸化上升以及CHOP蛋白質的表現量上升。這些證據說明了Endouc具有破壞huORFchop抑制轉譯下游CDS的能力。同時，為了排除過量加入Endouc所得的結果乃是因stress所造成，我們透過西方浸漬法證實了過量加入Endouc對細胞內ER stress markers如，Bip和p58IPK蛋白質的表現量都沒有明顯的影響。另一方面，為了進一步了解Endouc是如何破壞huORFchop抑制轉譯的能力，首先進行Endouc的domain mapping，發現轉染移除137到299之間氨基酸的突變endouc cDNA到在HEK293T細胞株中，CDS所含的 luciferase其活性是下降的，這表示這段序列的移除會導致Endouc無法破壞huORFchop抑制轉譯的能力。再利用西方浸漬法得知eIF2α磷酸化和CHOP的蛋白質的表現量卻都會下降。上述這些證據表示Endouc上137到299之間的氨基酸片段與破壞huORFchop抑制轉譯的能力極為相關。更進一步地，我們過量表現單點突變具有RNase活性的Endouc-H181A或Endouc-K242A到斑馬魚胚胎(in vivo)和 HEK293T及Hela細胞株(in vitro)時，都得到下游CDS的luciferase活性下降；且利用西方浸漬法也得知eIF2α磷酸化和CHOP蛋白質的表現量也都下降。這data說明了H181和K242這兩個位點是破壞huORFchop抑制轉譯能力的關鍵氨基酸。另外，利用RNase activity實驗我們得知Endouc確實能夠截切huORFchop RNA序列，而突變型的Endouc-K242A可以有效地破壞Endouc RNase的活性。最後，我們藉由polysome profiling assay 來探討Endouc參與在translation的過程，並了解其在整個translation過程中可能存在的位置。結果我們發現不同於突變型的Endouc-K242A坐落在polysomes上，正常型的Endouc卻只存在於free protein到40s-80s的fractions之間。這個可能是由於Endouc的RNase活性在進行對huORFchop mRNA切割後從polysomes上脫離，故在polysomes的區間並無法看見Endouc的訊號。綜合上述實驗結果，我們認為Endouc藉由RNase活性對huORFchop mRNA進行切割，被切割後的mRNA可能透過不清楚的機制重新reinitiation下游CDS的轉譯。這是一個新發現的機制藉由切割5’端的uORF序列，進而促進下游chop基因的轉譯。另一方面，Endouc也會促進eIF2α進行磷酸化，而已知eIF2α的磷酸化上升又能夠更加促進huORFchop轉譯抑制能力的失去，藉由此方式形成一個正回饋。

The C/EBP homologous protein (CHOP), which determines that cells undergo apoptosis or survival, starts to be translated when cells encounter endoplasmic reticulum (ER) stress. It has been reported that the 5’UTR of chop mRNA contains an inhibitory upstream open reading frame (uORFchop) which inhibits the translation of the downstream coding sequence (CDS) such as chop during normal condition. When cells encounter ER stress, this uORFchop–mediated translational inhibition (uORF-MTI) is repressed, resulting that the chop located at CDS in mRNA is translated. However, the molecular mechanism underlying uORFchop-MTI is still controversial. To understand the plausible mechanism involved in uORFchop-MTI, I employed the zebrafish transgenic line huORFZ, which harbors an exogenous DNA fragment that the CDS GFP cDNA fused with human uORFchop (huORFchop) and driven by a cytomegalovirus promoter. When huORFZ embryos were treated with heat-shock stress, GFP was exclusively expressed in the central nerve system. Employing laser-capture microdissection combined with microarray to compare the gene expression levels between GFP(+) brain cells and GFP(-) brain cells, we found that the endonuclease poly(U)-specific C gene (endouc) of zebrafish was significantly up-regulated in GFP(+) cells. Overexpression of endouc mRNA was able to repress uORF-MTI, resulting that GFP was expressed in the non-stressed huORFZ embryos. Moreover, the phosphorylation of eIF2α (p-eIF2α) and CHOP proteins were increased greatly in the endouc-overexpressive embryos (in vivo) and HEK293T cells (in vitro), indicating that endouc is involved in the repression of uORF-MTI. We proved that levels of many stress factors including Bip, p58IPK, and pPERK were not ectopic expression, suggesting that endouc overexpression per se did not induce ER stress intracellularly. To identify which domain of Endouc is involved in repression of uORF-MTI, I performed domain mapping and found a domain that contains amino acid residues between 137 and 299 is necessary for repression of uORF-MTI. Furthermore, I generated a single mutation of Endouc at H181 (EndoucH181A) and K242 (EndoucK242A), which disturb the endonuclease activity of Endouc, and found that overexpression of either endoucH181A or endoucK242A led to reduce the protein levels of p-eIF2α and CHOP. I also demonstrated that Endouc was able to enzymatically digest the huORF RNA fragment, while the mutant EndoucK242A was not, suggesting that the function of Endouc to disrupt uORF-MTI is depend on its endoribonuclease activity. Finally, using polysome profiling assay, I clearly demonstrated that, under stress condition, Endouc presented as a free protein and a 40S-80S associated complex. On contrast, the mutant EndoucK242A was always associated with polysomes, suggesting that Endouc was able to be released from polysomes under stress due to its RNase activity. Taken together, I hypothesize a model to explicit how Endouc plays role on the suppression of uORF-MTI: During ER stress, the RNase activity of Endouc might be triggered to digest the huORFchop motif located at 5''UTR of mRNA, resulting in a cap-independent mRNA, which in turn, the downstream CDS of CHOP cDNA is therefore reinitiated to translate through bypassing the hindrance of huORFchop structure. Furthermore, I found a positive feedback involved in suppression of uORF-MTI: the ectopic expression of Endouc enhances the eIF2α phosphorylation, which helps to disrupt the uORF-MTI, resulting in the increase of downstream CDS translation.

中文摘要(1)
英文摘要(3)
文獻回顧(5)
前言(14)
實驗材料與方法(16)
結果(23)
討論(33)
參考文獻(38)
圖說(43)
附錄(54)

黃薇臻 (2013) 碩士論文:利用有對熱逆境敏銳反應的腦細胞來探討轉譯抑制的機制。台灣大學分子與細胞生物研究所
胡家榕 (2014) 碩士論文: dkey參與內質網壓力中去除由人類uORFchop所主導的轉譯抑制。台灣大學分子與細胞生物研究所
Andersen MH, Becker JC, Straten Pt. (2005) Regulators of apoptosis: suitable targets for immune therapy of cancer. Nat Rev Drug Discov., 4(5):399-409
Averous J, Bruhat A, Jousse C, Carraro V, Thiel G, Fafournoux P. (2004) Induction of CHOP expression by amino acid limitation requires both ATF4 expression and ATF2 phosphorylation. J Biol Chem., 279(7):5288-97
Barbosa C, Peixeiro I, Romão L. (2013) Gene expression regulation by upstream open reading frames and human disease. PLoS Genet., 9(8):e1003529
Brush MH, Weiser DC, Shenolikar S. (2003) Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Mol Cell Biol., 23(4):1292-303
Calvo SE, Pagliarini DJ, Mootha VK. (2009) Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc Natl Acad Sci U S A, 106(18):7507-12
Chakrabarti A, Chen AW, Varner JD. (2011) A review of the mammalian unfolded protein response. Biotechnol Bioeng, 108(12):2777-93
Clarke HJ, Chambers JE, Liniker E, Marciniak SJ. (2014) Endoplasmic reticulum stress in malignancy. Cancer Cell, 25(5):563-73
Chen Y, Brandizzi F. (2013) IRE1: ER stress sensor and cell fate executor, Trends Cell Biol., 23(11):547-55
Coelho DS, Domingos PM. (2014) Physiological roles of regulated Ire1 dependent decay. Front Genet., 5:76
Cullinan SB, Zhang D, Hannink M, Arvisais E, Kaufman RJ, Diehl JA. (2003) Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Mol Cell Biol., 23(20):7198-209
Dai RY, Chen SK, Yan DM, Chen R, Lui YP, Duan CY, Li J, He T, Li H. (2010) PI3K/Akt promotes GRP78 accumulation and inhibits endoplasmic reticulum stress-induced apoptosis in HEK293 cells. Folia Biol., 56(2):37-46
Gioia U, Laneve P, Dlakic M, Arceci M, Bozzoni I, Caffarelli E. (2005) Functional characterization of XendoU, the endoribonuclease involved in small nucleolar RNA biosynthesis. J Biol Chem., 280 :18996-9002
Hetz C. (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. , 13(2):89-102
Hetz C, Martinon F, Rodriguez D, Glimcher LH. (2011) The unfolded protein response: integrating stress signals through the stress sensor IRE1α. Physiol Rev., 91(4):1219-43
Ivanov KA, Hertzig T, Rozanov M, Bayer S, Thiel V, Gorbalenya AE, Ziebuhr J. (2004) Major genetic marker of nidoviruses encodes a replicative endoribonuclease. Proc Natl Acad Sci U S A., 101(34):12694-9
Jackson RJ, Hellen CU, Pestova TV. (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol. 11(2):113-27
Jousse C, Bruhat A, Carraro V, Urano F, Ferrara M, Ron D, Fafournouz P. (2001)Inhibition of CHOP translation by a peptide encoded by anopen reading frame localized in the chop 5’UTR. Nucleic Acid Res., 29: 4341-51
Kim I, Xu W, Reed JC. (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov., 7:1013-30
Kim R, Ohi Y, Inoue H, Aogi K, Toge T. (1999) Introduction of gadd153 gene into gastric cancer cells can modulate sensitivity to anticancer agents in association with apoptosis. Anticancer Res., 19(3A):1779-83
Kudchodkar SB, Yu Y, Maguire TG, Alwine JC. (2004) Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase. J Virol., 78(20):11030-9
Lee HC, Chen YJ, Liu YW, Lin KY, Chen SW, Lin CY, Lu YC, Hsu PC, Lee SC, Tsai HJ. (2011) Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene. Nucleic Acid Res., 39: e139
Laneve P, Altieri F, Fiori ME, Scaloni A, Bozzoni I, Caffarelli E. (2003) Purification, cloning, and characterization of XendoU, a novel endoribonuclease involved in processing of intron-encoded small nucleolar RNAs in Xenopus laevis. J Biol Chem., 278(15):13026-32
Lee KP, Dey M, Neculai D, Cao C, Dever TE, Sicheri F. (2008) Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing. Cell, 132(1):89-100
Liu Z, Lv Y, Zhao N, Guan G, Wang J. (2015) Protein kinase R-like ER kinase and its role in endoplasmic reticulum stress-decided cell fate. Cell Death Dis., 6:e1822
Lu Y, Liang FX, Wang X. (2014) A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB. Mol Cell, 55(5):758-70
Mayer M, Kies U, Kammermeier R, Buchner J. (2000) BiP and PDI cooperate in the oxidative folding of antibodies in vitro. J Biol Chem., 275(38):29421-5
Mehmet H. (2000) Caspases find a new place to hide. Nature, 403(6765):29-30
Mendlovic F, Conconi M. (2010) Calreticulin: a multifaceted protein. Nature edu.,4:1
Meijer HA, Thomas A AM. (2002) Control of eukaryotic protein synthesis by upstream open reading frames in the 5’-untranslated region of an mRNA. Biochem. J., 367, 1–11
Mounir Z, Krishnamoorthy JL, Wang S, Papadopoulou B, Campbell S, Muller WJ, Hatzoglou M, Koromilas AE. (2013) Akt determines cell fate through inhibition of the PERK-eIF2α phosphorylation pathway. Sci Signal, 4(192):62
Oyadomari S, Mori M. (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ., 11:381-89
Palam LR, Baird TD, Wek RC. (2011) Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation. J Biol Chem., 286(13):10939-49
Perfettini JL, Kroemer RT, Kroemer G. (2004) Fatal liaisons of p53 with Bax and Bak. Nat Cell Biol., 6(5):386-8
Price J, Zaidi AK, Bohensky J, Srinivas V, Shapiro IM, Ali H. (2009) Akt-1 mediates survival of chondrocytes from endoplasmic reticulum-induced stress. J Cell Physiol., 222(3):502-8
Ramji DP, Foka P. (2002) CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J., 365(Pt 3):561-75
Renzi F, Caffarelli E, Laneve P, Bozzoni I, Brunori M, Vallone B. (2006) The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication. Proc Natl Acad Sci U.S.A.,103 :12365-70
Ricagno S, Egloff MP, Ulferts R, Coutard B, Nurizzo D, Campanacci V, Cambillau C, Ziebuhr J, Canard B. (2006) Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family. Proc Natl Acad Sci U S A., 103(32):11892-7
Schwarz DS, Blower MD. (2014) The calcium-dependent ribonuclease XendoU promotes ER network formation through local RNA degradation. J Cell Biol., 207(1):41-57
Shen J, Chen X, Hendershot L, Prywes R. (2002) ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev Cell, 3(1):99-111
Silvera D, Formenti SC, Schneider RJ. (2010) Translational control in cancer. Nat Rev Cancer, 10(4):254-66
Szegezdi E, Logue SE, Gorman AM, Samali A. (2006) Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep., 7(9):880-5
Tabas I, Ron D. (2011) Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol., 13(3):184-90
Tsang KY, Chan D, Bateman JF, Cheah KS. (2010) In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences. J Cell Sci., 123:2145-54
Walter P, Ron D. (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science, 334(6059): 1081-6
Wang K, Yin XM, Chao DT, Milliman CL, Korsmeyer SJ. (1996) BID: a novel BH3 domain-only death agonist. Genes Dev., 10(22):2859-69
Wang M, Kaufman RJ. (2014) The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat Rev Cancer, 14(9):581-97
Wang S, Kaufman RJ. (2012) The impact of the unfolded protein response
on human disease. J Cell Biol.,197: 857-67
Wang XZ, Lawson B, Brewer JW, Zinszner H, Sanjay A, Mi LJ, Boorstein R, Kreibich G, Hendershot LM, Ron D. (1996) Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153). Mol Cell Biol., 16(8):4273-80
Merrick WC. (2004) Cap-dependent and cap-independent translation in eukaryotic systems. Gene, 332:1-11
Ubeda M, Habener JF. (2000) CHOP gene expression in response to endoplasmic-reticular stress requires NFY interaction with different domains of a conserved DNA-binding element. Nucleic Acid Res., 28(24):4987-97
Ubeda M, Wang Xz, Zinszner H, Wu I, Habener JF, Ron D. (1996) Stress-induced binding of the transcriptional factor CHOP to a novel element. Mol cell boil., 16(4): 1479-89
Yanagitani K, Imagawa Y, Iwawaki T, Hosoda A, Saito M, Kimata Y, Kohno K. (2009) Cotranslational targeting of XBP1 protein to the membrane promotes cytoplasmic splicing of its own mRNA. Mol Cell, 34(2):191-200
Yang XJ, Seto E. (2008) Lysine acetylation: codified crosstalk with other posttranslational modifications. Mol Cell, 31(4):449-61
Young SK, Palam LR, Wu C, Sachs MS, Wek RC. (2016) Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response. J Biol Chem., 291(12):6546-58
Zhang X, Tang N, Hadden TJ, Rishi AK. (2011) Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta, 1813(11):1978-86
Zhou W, Jeyaraman K, Yusoff P, Shenolikar S. (2013) Phosphorylation at tyrosine 262 promotes GADD34 protein turnover. J Biol Chem., 288(46):33146-55