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  本文關鍵詞：肌醇需求酶-1抑制劑發(fā)現及其脂代謝調控研究　出處：《華東師范大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 內質網應激 肌醇需求酶－1α 昆蟲表達 高通量篩選 脂代謝調節(jié)

【摘要】：內質網是真核細胞中負責蛋白折疊和裝配的場所,對細胞內的蛋白質穩(wěn)態(tài)平衡非常重要。內質網應激是由于Ca2+穩(wěn)態(tài)平衡的紊亂、分泌蛋白合成的增加、錯誤折疊蛋白質的表達、葡萄糖饑餓、蛋白質糖基化的抑制或膽固醇合成超載等脅迫條件,干擾蛋白質的合成,導致內質網內積累大量的未折疊蛋白質,細胞所做出的應激反應,主要激發(fā)包括PERK-elF2α,IRE1α-XBP1及ATF6等3條未折疊蛋白反應(UPR)信號通路,通過激活CHOP、ATF4、XBP1以及ATF6等多個b-zip轉錄因子活性,調控相關基因的表達調控。 肌醇需求酶-1(inositol-requiring enzyme-1α, IRE-lα)是最保守的UPR信號通路感應器,單次跨膜定位在內質網膜上,具備絲氨酸/蘇氨酸激酶和核酸酶雙重功能。IRE1α通過剪切底物XBP1mRNA,激活一系列UPR相關基因的轉錄,從而緩解內質網壓力；IRE1還可以通過不依賴XBP1的途徑,激活JNK (c-junk N-terminal kinase),引發(fā)細胞的凋亡。IRE1α-XBP1通路參與眾多病理和生理過程：IRElα激酶促進突變性亨廷頓蛋白的聚集,從而加劇亨廷頓綜合癥；IRE1α是腫瘤血管生成的關鍵因子；脂肪生成依賴于IRE1α/XBP1通路的激活,等等。但目前IRE1α激酶與核酸酶之間的相互調控關系及其與疾病關系尚不明晰。發(fā)現IRE1α激酶以及核酸酶的抑制劑,對IRE1α激酶與核酸酶之間的關系研究、IRE1α信號通路調控機制以及相關藥物開發(fā)具有重要意義。 我們在體外運用昆蟲表達體系成功地表達純化出人源重組IRE1α蛋白,通過對底物濃度、DMSO濃度的優(yōu)化,我們建立了合格的IRE1α激酶抑制劑高通量篩選模型,該模型的Z’因子為0.58,CV值為7.5%,并對包含天然產物在內的12683個化合物進行了高通量篩選,最終獲得了20個有效的IRE1α激酶抑制劑。隨后,通過對底物濃度、DMSO濃度以及線性時間等條件進行優(yōu)化,我們建立了分子水平的IRE1α核酸酶抑制劑篩選模型,并對包括細胞水平上的XBP1剪切抑制劑、NFκB-Luciferase抑制劑和分子水平上的IRE1α激酶抑制劑共810個化合物進行篩選,最終獲得5個IRE1核酸酶直接抑制劑。 IRElα-XBP1通路與脂代謝調控有著密切關系,但目前相關化合物應用主要集中在對多發(fā)性骨髓瘤的治療評價,在脂代謝調控方面尚未見報道。本文首次對在XBP1剪切細胞模型上發(fā)現的活性化合物進行代謝調控的生物學活性評價,發(fā)現多個化合物具有抑制脂肪細胞分化和抑制肝細胞甘油三酯含量。綜合考慮化合物對IRE1α核酸酶、激酶以及細胞水平對XBP1剪切細胞模型抑制活性,我們重點考察17#化合物對于脂代謝紊亂的調節(jié)作用。該化合物在分子水平上抑制IRE1核酸酶的活性,其IC50為14.92μM,在細胞水平上也顯著抑制XBP1剪切,IC50約在10μM左右。功功能實驗結果顯示,17#化合物能明顯抑制脂肪細胞3T3-L1的分化,而且抑制XBP1s以及FAS、ACC1、SCD1等相關基因表達：此外顯著降低HepG2細胞中甘油三酯含量,而且抑制XBP1s、FAS、 ACC1、SCD1以及sREBP-c1等相關基因表達。 綜上所述,本論文的研究工作建立了IREl a激酶抑制劑分子模型以及IRE1α核酸酶的高通量篩選模型。IRE1α激酶以及核酸酶小分子抑制劑的發(fā)現為研究IRE1α激酶與核酸酶的關系提供了工具,也為IRE1α相關信號通路研究打下了基礎；17#化合物的發(fā)現也為IRE1α/XBP1抑制劑在抗脂肪異常代謝類藥物的開發(fā)上提供了新思路。
[Abstract]:The endoplasmic reticulum is responsible for protein folding and assembly sites in eukaryotic cells, the protein homeostasis in cells is very important. The endoplasmic reticulum stress is due to Ca2+ homeostasis disorder, secretory protein synthesis increased expression of misfolded proteins, glucose starvation, protein glycosylation or inhibition of cholesterol synthesis overload stress condition, disturbance of protein synthesis, leading to accumulation of unfolded proteins in the endoplasmic reticulum stress reaction of cells, mainly including the excitation of PERK-elF2 alpha, alpha -XBP1 and IRE1 ATF6 3 unfolded protein response (UPR) signaling pathway through the activation of CHOP, ATF4, XBP1, ATF6 and other B-ZIP transcription factors activity, expression and regulation of genes.
-1 (inositol-requiring enzyme-1 inositol requiring enzyme alpha, alpha IRE-l) UPR signaling pathway sensor is the most conservative, a single transmembrane localization in the endoplasmic reticulum, a serine / threonine kinase functions and nuclease.IRE1 alpha by shear substrate XBP1mRNA, transcriptional activation of a series of UPR related genes, to alleviate ER stress; IRE1 but also through the way which is not dependent on XBP1, activation of JNK (c-junk N-terminal kinase), triggering apoptosis of.IRE1 alpha -XBP1 pathway is involved in many physiological and pathological processes: IREl alpha kinase promotes aggregation of mutant Huntington egg white, thus exacerbating the Huntington syndrome; IRE1 alpha is a key factor in tumor angiogenesis; adipogenesis is dependent on IRE1 alpha activation of the /XBP1 pathway, and so on. But at present between IRE1 alpha kinase and nuclease regulatory interaction and its relationship with diseases is still not clear. IRE1 to alpha kinase And inhibitors of nuclease are of great significance for the study of the relationship between IRE1 alpha kinase and nuclease, the regulation mechanism of IRE1 alpha signaling pathway and the development of related drugs.
We use the insect expression system in vitro successfully expressed and purified recombinant IRE1 protein, the optimal substrate concentration, DMSO concentration, we established IRE1 alpha kinase inhibitor high-throughput screening qualified model, the model Z factor was 0.58, CV was 7.5%, and the 12683 compounds containing natural the product, a high throughput screening, finally obtained 20 effective IRE1 alpha kinase inhibitors. Then, based on the substrate concentration, DMSO concentration and linear time were optimized, we established a screening model of IRE1 nucleic acid enzyme inhibitor at the molecular level, and to include the XBP1 shear cell level IRE1 inhibitors. Alpha kappa B-Luciferase kinase inhibitor NF and inhibitor at the molecular level, a total of 810 compounds were screened, obtained 5 IRE1 nuclease inhibitors directly.
The IREl alpha -XBP1 pathway and lipid metabolism are closely related, but the related compounds mainly used in the evaluation of treatment for multiple myeloma, has not been reported in the regulation of lipid metabolism. To evaluate the biological activity for the first time on the metabolism regulation of active compounds found in XBP1 cells on the shear model, found that more than one compounds can inhibit adipocyte differentiation and inhibit liver cell triglyceride content. Considering the compounds of IRE1 alpha nuclease, inhibit the activity of kinase and cell level on the XBP1 shear cell model, we study the 17# compounds in the regulation of lipid metabolism. The compound inhibits IRE1 nuclease activity at the molecular level, which is 14.92 IC50. M, at the cellular level also significantly inhibited XBP1 shear, about IC50 at about 10 M. The function of the experimental results show that 17# can obviously inhibit fatty compounds Cell 3T3-L1 differentiation, and inhibit XBP1s and FAS, ACC1, SCD1 and other related gene expression: in addition, significantly reduce the content of triglyceride in HepG2 cells, and inhibit XBP1s, FAS, ACC1, SCD1 and sREBP-c1 and other related gene expression.
In summary, this paper established the IREl model and IRE1 molecular a kinase inhibitor alpha nuclease high-throughput screening model of.IRE1 alpha kinase and nuclease inhibitor discovery to study the relationship between IRE1 kinase and alpha nuclease provides tools for IRE1 a study related signal pathway foundation; 17# for compounds found in IRE1 alpha /XBP1 inhibitors in development of anti fat abnormal metabolism of drugs provides new ideas.

【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R341

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