本文選題：USP10 + AMPK��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2016年博士論文

【摘要】：第一部分AMPK(5’AMP-activated protein kinase)即AMP依賴的蛋白激酶,是生物能量代謝調(diào)節(jié)的關(guān)鍵分子。AMPK作為細胞能量感受器,其激活后可通過開啟ATP的補償機制和關(guān)閉ATP的消耗進程,恢復細胞內(nèi)的能量平衡。AMPK參與調(diào)控多種細胞代謝進程,因此,其信號途徑是研究肥胖、2型糖尿病等代謝性疾病的核心。AMPK的活性受到嚴格調(diào)控。當外界能量應激造成ATP減少,AMP/ATP比值增加,AMP可通過結(jié)合AMPKγ亞基,促進AMPK上游激酶(AMPKK)對AMPK第172位點的蘇氨酸進行磷酸化,進而激活AMPK。當前,對AMPK激活調(diào)控多集中在對該位點的磷酸化修飾研究上,而其它類型的翻譯后修飾是否參與AMPK的激活還未見報道。本實驗室前期研究發(fā)現(xiàn)去泛素化修飾在AMPK激活過程中發(fā)揮重要作用,且鑒定了首個AMPK去泛素化酶USP10,同時發(fā)現(xiàn)USP10-AMPK通路受到精細的調(diào)控。因此,對USP10-AMPK信號通路的研究將為闡明AMPK信號通路的作用機制提供一條新的線索,具有非常重要的意義。本文的研究內(nèi)容及結(jié)論主要包括以下兩個方面:(一)USP10調(diào)控AMPKα泛素化及其活性。1)在AMPK激活劑AICAR刺激條件下,我們發(fā)現(xiàn)AMPK的激活伴隨著其自身泛素化修飾水平的下降;2)通過生物信息學的方法分析USP10序列,我們鑒定了AMPKα上4個潛在泛素化位點。點突變實驗證實這些位點的泛素化受到USP10調(diào)控;4)通過免疫印跡檢測AMPK下游底物ACC1,Raptor的磷酸化,我們發(fā)現(xiàn)USP10的敲除抑制了AMPK的激活及其功能;油紅O染色證明,USP10敲除導致脂滴形成顯著提高;5)對USP10序列分析,我們發(fā)現(xiàn)USP10存在AMPK的潛在磷酸化位點Ser76。利用AMPK磷酸化通用底物抗體檢測USP10磷酸化,我們發(fā)現(xiàn)能量應激上調(diào)胞內(nèi)USP10的磷酸化;體外激酶實驗進一步證明,AMPK可特異性磷酸化USP10;6)USP10敲除細胞中回補USP10野生型與S76突變體,實驗證明USP10 S76位點的磷酸化促進其激活AMPK。(二)小鼠肝臟特異性敲除USP10導致多種代謝損傷。1)我們利用CRISPRCas9技術(shù)構(gòu)建USP10肝臟特異性敲除小鼠模型,T7EN1酶切及免疫印跡檢測USP10肝臟敲除效率;2)對小鼠表型檢測,我們發(fā)現(xiàn)在肝臟內(nèi)USP10的特異性敲除降低了AMPK的活性及其下游底物磷酸化。3)此外,USP10肝臟敲除小鼠表現(xiàn)出多種代謝缺陷,如甘油三酯、膽固醇及血糖含量明顯升高。高胰島素-正血糖鉗夾實驗證實,USP10敲除小鼠的葡萄糖灌注速率明顯下調(diào)。綜上所述,我們的研究發(fā)現(xiàn)了在能量應激下,一種放大AMPK激活信號的關(guān)鍵分子機制。我們發(fā)現(xiàn)AMPKα的泛素化會抑制其激活,而去泛素化酶USP10可以特異性的去泛素化AMPKα激活AMPK。在能量應激下,AMPK反過來磷酸化USP10絲氨酸76位點,提高USP10的活性。因此,AMPK與USP10之間形成一種反饋調(diào)控回路,確保能量應激下,AMPK激活信號的放大。小鼠敲除模型證明,對這種反饋調(diào)控回路的干擾,會導致AMPK的激活異常及多種代謝缺陷的發(fā)生。我們的研究揭示了泛素化修飾在AMPK活性調(diào)控中的新功能,進一步加深了我們對AMPK活性調(diào)控機制的理解,同時擴展了我們對于AMPK翻譯后修飾新功能的認識。AMPK由于其重要功能已經(jīng)成為肥胖,胰島素抵抗,2型糖尿病,代謝綜合征,甚至腫瘤等多種疾病的理想治療靶點。AMPK激活劑如二甲雙胍已經(jīng)應用于胰島素抵抗,2型糖尿病的臨床治療。鑒于USP10在AMPK激活中的重要作用,其有望為成為上述代謝性疾病的治療靶標。第二部分c-ABL(Abelson tyrosine kinase)是一種非受體酪氨酸激酶,其可與bcr(breakpoint cluster region)基因發(fā)生融合突變,形成具有高度酪氨酸激酶活性的BCR/ABL融合蛋白。這種突變是造成慢性髓質(zhì)白血病(CML)的根本原因。BCR/ABL酪氨酸激酶抑制劑(TKI)已經(jīng)作為治療慢性髓質(zhì)白血病(CML)的一線藥物。然而在臨床治療中,患者對imatinib及其它TKI的耐藥性已成為CML治療面臨的新問題。當前,對imatinib耐藥性潛在的分子機制還不十分清楚。本實驗室前期研究發(fā)現(xiàn)有絲分裂調(diào)控蛋白PLK1(Polo-like kinase-1)是cABL的一個重要的激酶底物。c-ABL可以與PLK1相互作用并磷酸化PLK1,促進細胞周期進程及細胞增殖。同時,c-ABL介導的PLK1過表達與白血病患者的imatinib耐藥性正相關(guān)。因此,對c-ABL-PLK1信號通路的研究不但可以進一步拓展人們對細胞周期調(diào)控機制的認識,而且可能為CML及其它腫瘤的治療提供新的組合用藥策略。本文的研究內(nèi)容及結(jié)論主要包括以下四個方面:(一)c-ABL調(diào)控PLK1蛋白降解及其活性。我們發(fā)現(xiàn)c-ABL磷酸化PLK1,抑制其泛素化降解并提高其活性。1)利用免疫共沉淀與GST-pull down分析,我們證明c-ABL可以與PLK1在體內(nèi)體外發(fā)生直接相互作用;2)通過找尋二者相互作用區(qū)域,我們發(fā)現(xiàn)PLK1通過PBD結(jié)構(gòu)域與c-ABL結(jié)合;c-ABL通過SH2/SH3,PTKs結(jié)構(gòu)域與PLK1相互作用;3)激酶磷酸化實驗證明,c-ABL可以體內(nèi)體外磷酸化PLK1;通過質(zhì)譜鑒定,我們發(fā)現(xiàn)c-ABL磷酸化PLK1 Y217,Y425,Y445位點4)過表達c-ABL促進PLK1蛋白水平表達;敲除c-ABL抑制PLK1蛋白水平表達;利用放線菌酮阻抑證明c-ABL抑制PLK1降解;蛋白酶體抑制劑及泛素化實驗證明,c-ABL抑制PLK1通過泛素化降解;點突變實驗證明,c-ABL通過Y425位點調(diào)控PLK1蛋白穩(wěn)定性;5)同步化細胞于有絲分裂期,我們證明c-ABL通過磷酸化PLK1 Y425位點促進PLK1的激活。體外磷酸化反應證明,PLK1 Y425位點的磷酸化促進Aurora A對其激活。(二)c-ABL磷酸化PLK1調(diào)控有絲分裂進程。1)流失細胞儀檢測c-ABL敲低He La細胞,我們發(fā)現(xiàn)c-ABL影響細胞周期進程;2)免疫熒光及流式細胞儀檢測PLK1野生型與Y425突變株細胞周期,發(fā)現(xiàn)Y425磷酸化影響細胞G2/M轉(zhuǎn)換;3)激光共聚焦顯微鏡實時觀察細胞,進一步確定c-ABL介導的PLK1 Y425磷酸化促進細胞進入有絲分裂。(三)c-ABL-PLK1軸影響CML化療應答。1)Real-Time PCR及Western Blot檢測臨床CML患者外周血樣本,發(fā)現(xiàn)BCR/ABL與PLK1在CML中高表達;與imatinib敏感患者相比,PLK1在imatinib抗性患者體內(nèi)表達更高;2)imatinib有效降低臨床患者CML中PLK1蛋白表達水平;3)K562細胞過表達PLK1抑制其對imatinib的化療應答;4)c-ABL與PLK1抑制劑聯(lián)合用藥顯著提高CML細胞的化療應答;5)構(gòu)建imatinib抗性CML小鼠模型,證實聯(lián)合用藥延長CML腫瘤模型小鼠存活時間。(四)c-ABL-PLK1軸影響宮頸癌腫瘤增殖及病人存活率。1)通過收集臨床宮頸癌組織樣本,發(fā)現(xiàn)c-ABL與PLK1在宮頸癌中高表達,同時在腫瘤組織中PLK1具有高度酪氨酸磷酸化修飾;2)裸鼠成瘤實驗證明,PLK1 Y425位點突變抑制腫瘤增殖;3)臨床隨訪數(shù)據(jù)證明,PLK1酪氨酸磷酸化與不良預后正相關(guān);4)聯(lián)合用藥實驗證明,c-ABL,PLK1抑制劑促進宮頸癌細胞的化療應答。綜上所述,我們的研究揭示了c-ABL-PLK1軸在有絲分裂進程及CML化療應答中的重要功能。c-ABL通過直接磷酸化PLK1調(diào)控PLK1的蛋白穩(wěn)定性和活性。過表達PLK1抑制了CML細胞對imatinib的化療應答。同時imatinib與PLK1抑制劑的聯(lián)合用藥有助于imatinib抗性CML及宮頸癌細胞的治療。我們的研究有望為imatinib抗性CML患者提供新的治療靶標。此外由于PLK1的酪氨酸磷酸化與宮頸癌患者的不良預后相關(guān),我們的研究同樣為宮頸癌的診斷和治療提供了新的線索。
[Abstract]:The first part, AMPK (5 'AMP-activated protein kinase), a protein kinase dependent on AMP, is a key molecule of bioenergy metabolism, the key molecule.AMPK as a cell energy receptor, which can be activated by opening the compensatory mechanism of ATP and closing the consumption process of ATP to restore the intracellular energy balance.AMPK to participate in the regulation of various cell metabolic processes. Therefore, the signal pathway is to study the core.AMPK activity of obesity, type 2 diabetes and other metabolic diseases. When the external energy stress causes the decrease of ATP and the increase of the AMP/ATP ratio, AMP can promote the phosphorylation of the AMPK upstream kinase (AMPKK) to the threonine at the AMPK 172nd site by combining the AMPK gamma subunit, and then activates the AMPK. current, AMPK activation regulation is mostly focused on the phosphorylation of the site, while other types of post-translational modification have not been reported to participate in the activation of AMPK. Previous studies in this laboratory found that ubiquitination modification plays an important role in the activation of AMPK, and the first AMPK de ubiquitination enzyme USP10 is identified, and USP10-AMPK has been found. Therefore, the study of the USP10-AMPK signaling pathway will provide a new clue to clarify the mechanism of the AMPK signaling pathway, which is of great significance. The contents and conclusions of this study include the following two aspects: (1) USP10 regulation of AMPK alpha ubiquitination and its active.1 in AMPK activator AICAR stimulation Under the conditions, we found that the activation of AMPK was accompanied by a decline in its own ubiquitination modification level; 2) we identified 4 potential ubiquitination sites on AMPK alpha by bioinformatics analysis. The point mutation experiments confirmed that the ubiquitination of these sites was regulated by USP10; 4) the downstream substrate ACC1, Rapto was detected by immunoblotting, Rapto. R phosphorylation, we found that USP10 knockout inhibited the activation and function of AMPK; oil red O staining showed that USP10 knockout led to a significant increase in lipid droplets; 5) we found that USP10 presence AMPK's potential phosphorylation site Ser76. using AMPK phosphorylation general substrate antibody test USP10 phosphorylation, we found energy stress Phosphorylation of intracellular USP10; in vitro kinase experiments further demonstrated that AMPK can be specifically phosphorylated USP10; 6) USP10 knockout cells recharge USP10 wild type and S76 mutants. Experiments show that the phosphorylation of USP10 S76 site promotes its activation of AMPK. (two) mice liver specific knockout USP10 leads to a variety of metabolic damage.1) we use CRISPRCas9 Technical construction of USP10 liver specific knockout mouse model, T7EN1 digestion and immunoblotting to detect USP10 liver knockout efficiency; 2) we found that the specific knockout of USP10 in the liver decreased the AMPK activity and the downstream substrate phosphorylated.3) in the liver, and the USP10 liver knockout mice showed a variety of metabolic defects, such as glycerol. Three ester, cholesterol and blood sugar content increased significantly. High insulin positive glucose clamp experiments confirmed that the glucose perfusion rate of USP10 knockout mice was obviously down. In summary, our study found a key molecular mechanism to amplify the AMPK activation signal under energy stress. We found that the ubiquitination of AMPK alpha would inhibit its activation, The ubiquitininase USP10 can specifically activate the ubiquitinated AMPK alpha to activate AMPK. under energy stress, and AMPK in turn phosphorylates the USP10 serine 76 site, which improves the activity of USP10. Therefore, AMPK and USP10 form a feedback loop to ensure the amplification of the AMPK activation signal under energy stress. The interference of the control loop leads to the activation abnormality of AMPK and the occurrence of a variety of metabolic defects. Our study reveals the new functions of ubiquitination in the regulation of AMPK activity, further deepening our understanding of the regulatory mechanism of AMPK activity, and expanding our understanding of the new function of post-translational modification of AMPK because of its important work. The target.AMPK activator, such as metformin, has been applied to insulin resistance and in the clinical treatment of type 2 diabetes, which can become a target for obesity, insulin resistance, type 2 diabetes, metabolic syndrome, and even tumor. In view of the important use of USP10 in AMPK activation, it is expected to be the target for the treatment of these metabolic diseases. The second part of c-ABL (Abelson tyrosine kinase) is a non receptor tyrosine kinase which can be fused with the BCR (breakpoint cluster region) gene to form a BCR/ABL fusion protein with a high tyrosine kinase activity. This mutation is the root cause of chronic medullary leukaemia (CML),.BCR/ABL tyrosine kinase inhibition TKI has been used as a first-line drug for the treatment of chronic medullary leukemia (CML). However, in clinical treatment, drug resistance to imatinib and other TKI has become a new problem in CML therapy. Currently, the potential molecular mechanism of imatinib resistance is not yet very clear. Earlier studies in this laboratory found the mitosis regulatory protein PLK1 (Po). Lo-like kinase-1) is an important kinase substrate for cABL,.C-ABL can interact with PLK1 and phosphorylate PLK1 to promote cell cycle process and cell proliferation. Meanwhile, c-ABL mediated PLK1 overexpression is positively related to imatinib resistance in leukemia patients. Therefore, the study of c-ABL-PLK1 signaling pathway can not only further expand people 's pathway. The understanding of cell cycle regulation mechanism and may provide a new combination strategy for the treatment of CML and other tumors. The contents and conclusions of this paper mainly include the following four aspects: (1) c-ABL regulation of PLK1 protein degradation and its activity. We find c-ABL phosphorylation PLK1, inhibit its ubiquitination degradation and improve its active.1) utilization Immunoprecipitation and GST-pull down analysis, we have shown that c-ABL can interact directly with PLK1 in vivo and in vitro; 2) by searching for the two interacting regions, we found that PLK1 is combined with c-ABL in the PBD domain; c-ABL through SH2/SH3, PTKs domain and PLK1 interaction; 3) kinase phosphorylation experiments show that c-ABL can be in vivo Exogenous phosphorylation of PLK1; through mass spectrometry identification, we found that c-ABL phosphorylation PLK1 Y217, Y425, Y445 site 4) expressed c-ABL to promote the expression of PLK1 protein; knocking out c-ABL inhibition PLK1 protein level expression; using actinomycone inhibition to prove c-ABL inhibition of PLK1 degradation; proteasome inhibition and ubiquitination experiments proved to inhibit through Ubiquitin The point mutation experiment shows that c-ABL regulates the stability of PLK1 protein through the Y425 site; 5) the synchrotron cells are in mitotic phase, and we prove that c-ABL promotes PLK1 activation through the phosphorylation PLK1 Y425 site. In vitro phosphorylation shows that the phosphorylation of PLK1 Y425 loci promotes Aurora A to activate it. (two) c-ABL phosphorylation regulates the filament The split process.1) the loss cell instrument detected c-ABL knock down He La cells, we found that c-ABL affected the cell cycle process; 2) immunofluorescence and flow cytometry detected the cell cycle of PLK1 wild type and Y425 mutant, and found Y425 phosphorylation affected the cell G2/M conversion; 3) laser confocal microscopy observed the cells in real time, and further determined c-ABL mediated PL K1 Y425 phosphorylation promotes cell entry into mitosis. (three) c-ABL-PLK1 axis affects CML chemotherapy response.1) Real-Time PCR and Western Blot detection of peripheral blood samples from clinical CML patients, and it is found that BCR/ABL and PLK1 are high in expression; compared with sensitive patients, the expression is higher in the anti sexual patients; 2) effectively reduce the clinical value. The expression level of PLK1 protein in patients with CML; 3) K562 cells overexpressed PLK1 to inhibit the response to chemotherapy of imatinib; 4) the combination of c-ABL and PLK1 inhibitors significantly increased the response to chemotherapy in CML cells; 5) construct imatinib resistant CML mouse model, and confirmed the combination of drugs to prolong the survival time of CML tumor model mice. (four) the c-ABL-PLK1 axis affects cervical cancer. Tumor proliferation and patient survival rate.1) by collecting clinical cervical cancer tissue samples, it was found that c-ABL and PLK1 were highly expressed in cervical cancer, and PLK1 had high tyrosine phosphorylation in the tumor tissue; 2) nude mice tumor experiment proved that PLK1 Y425 site mutation inhibited tumor proliferation; 3) clinical follow-up data showed that PLK1 tyrosine phosphorylation A positive correlation with bad prognosis; 4) combined use of drug experiments proved that c-ABL, PLK1 inhibitor promoted the chemotherapy response of cervical cancer cells. In summary, our study revealed that the important function of the c-ABL-PLK1 axis in the mitosis process and the CML chemotherapy response,.C-ABL regulates the stability and activity of the protein in PLK1 through direct phosphorylation of PLK1. Over express PLK1 Inhibition of the chemotherapeutic response of CML cells to imatinib. Combined use of imatinib and PLK1 inhibitors contributes to the treatment of imatinib resistant CML and cervical cancer cells. Our study is expected to provide a new therapeutic target for patients with imatinib resistant CML. In addition, the tyrosine phosphorylation of PLK1 is associated with poor prognosis in patients with cervical cancer. The study also provides new clues for the diagnosis and treatment of cervical cancer.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R363
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本文編號：1835164