去泛素化酶USP10调控AMPK的功能和作用机制研究

发布时间：2018-05-02 19:22

  本文选题：USP10 + AMPK　； 参考：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：第一部分AMPK(5’AMP-activated protein kinase)即AMP依赖的蛋白激酶,是生物能量代谢调节的关键分子。AMPK作为细胞能量感受器,其激活后可通过开启ATP的补偿机制和关闭ATP的消耗进程,恢复细胞内的能量平衡。AMPK参与调控多种细胞代谢进程,因此,其信号途径是研究肥胖、2型糖尿病等代谢性疾病的核心。AMPK的活性受到严格调控。当外界能量应激造成ATP减少,AMP/ATP比值增加,AMP可通过结合AMPKγ亚基,促进AMPK上游激酶(AMPKK)对AMPK第172位点的苏氨酸进行磷酸化,进而激活AMPK。当前,对AMPK激活调控多集中在对该位点的磷酸化修饰研究上,而其它类型的翻译后修饰是否参与AMPK的激活还未见报道。本实验室前期研究发现去泛素化修饰在AMPK激活过程中发挥重要作用,且鉴定了首个AMPK去泛素化酶USP10,同时发现USP10-AMPK通路受到精细的调控。因此,对USP10-AMPK信号通路的研究将为阐明AMPK信号通路的作用机制提供一条新的线索,具有非常重要的意义。本文的研究内容及结论主要包括以下两个方面:(一)USP10调控AMPKα泛素化及其活性。1)在AMPK激活剂AICAR刺激条件下,我们发现AMPK的激活伴随着其自身泛素化修饰水平的下降;2)通过生物信息学的方法分析USP10序列,我们鉴定了AMPKα上4个潜在泛素化位点。点突变实验证实这些位点的泛素化受到USP10调控;4)通过免疫印迹检测AMPK下游底物ACC1,Raptor的磷酸化,我们发现USP10的敲除抑制了AMPK的激活及其功能;油红O染色证明,USP10敲除导致脂滴形成显著提高;5)对USP10序列分析,我们发现USP10存在AMPK的潜在磷酸化位点Ser76。利用AMPK磷酸化通用底物抗体检测USP10磷酸化,我们发现能量应激上调胞内USP10的磷酸化;体外激酶实验进一步证明,AMPK可特异性磷酸化USP10;6)USP10敲除细胞中回补USP10野生型与S76突变体,实验证明USP10 S76位点的磷酸化促进其激活AMPK。(二)小鼠肝脏特异性敲除USP10导致多种代谢损伤。1)我们利用CRISPRCas9技术构建USP10肝脏特异性敲除小鼠模型,T7EN1酶切及免疫印迹检测USP10肝脏敲除效率;2)对小鼠表型检测,我们发现在肝脏内USP10的特异性敲除降低了AMPK的活性及其下游底物磷酸化。3)此外,USP10肝脏敲除小鼠表现出多种代谢缺陷,如甘油三酯、胆固醇及血糖含量明显升高。高胰岛素-正血糖钳夹实验证实,USP10敲除小鼠的葡萄糖灌注速率明显下调。综上所述,我们的研究发现了在能量应激下,一种放大AMPK激活信号的关键分子机制。我们发现AMPKα的泛素化会抑制其激活,而去泛素化酶USP10可以特异性的去泛素化AMPKα激活AMPK。在能量应激下,AMPK反过来磷酸化USP10丝氨酸76位点,提高USP10的活性。因此,AMPK与USP10之间形成一种反馈调控回路,确保能量应激下,AMPK激活信号的放大。小鼠敲除模型证明,对这种反馈调控回路的干扰,会导致AMPK的激活异常及多种代谢缺陷的发生。我们的研究揭示了泛素化修饰在AMPK活性调控中的新功能,进一步加深了我们对AMPK活性调控机制的理解,同时扩展了我们对于AMPK翻译后修饰新功能的认识。AMPK由于其重要功能已经成为肥胖,胰岛素抵抗,2型糖尿病,代谢综合征,甚至肿瘤等多种疾病的理想治疗靶点。AMPK激活剂如二甲双胍已经应用于胰岛素抵抗,2型糖尿病的临床治疗。鉴于USP10在AMPK激活中的重要作用,其有望为成为上述代谢性疾病的治疗靶标。第二部分c-ABL(Abelson tyrosine kinase)是一种非受体酪氨酸激酶,其可与bcr(breakpoint cluster region)基因发生融合突变,形成具有高度酪氨酸激酶活性的BCR/ABL融合蛋白。这种突变是造成慢性髓质白血病(CML)的根本原因。BCR/ABL酪氨酸激酶抑制剂(TKI)已经作为治疗慢性髓质白血病(CML)的一线药物。然而在临床治疗中,患者对imatinib及其它TKI的耐药性已成为CML治疗面临的新问题。当前,对imatinib耐药性潜在的分子机制还不十分清楚。本实验室前期研究发现有丝分裂调控蛋白PLK1(Polo-like kinase-1)是cABL的一个重要的激酶底物。c-ABL可以与PLK1相互作用并磷酸化PLK1,促进细胞周期进程及细胞增殖。同时,c-ABL介导的PLK1过表达与白血病患者的imatinib耐药性正相关。因此,对c-ABL-PLK1信号通路的研究不但可以进一步拓展人们对细胞周期调控机制的认识,而且可能为CML及其它肿瘤的治疗提供新的组合用药策略。本文的研究内容及结论主要包括以下四个方面:(一)c-ABL调控PLK1蛋白降解及其活性。我们发现c-ABL磷酸化PLK1,抑制其泛素化降解并提高其活性。1)利用免疫共沉淀与GST-pull down分析,我们证明c-ABL可以与PLK1在体内体外发生直接相互作用;2)通过找寻二者相互作用区域,我们发现PLK1通过PBD结构域与c-ABL结合;c-ABL通过SH2/SH3,PTKs结构域与PLK1相互作用;3)激酶磷酸化实验证明,c-ABL可以体内体外磷酸化PLK1;通过质谱鉴定,我们发现c-ABL磷酸化PLK1 Y217,Y425,Y445位点4)过表达c-ABL促进PLK1蛋白水平表达;敲除c-ABL抑制PLK1蛋白水平表达;利用放线菌酮阻抑证明c-ABL抑制PLK1降解;蛋白酶体抑制剂及泛素化实验证明,c-ABL抑制PLK1通过泛素化降解;点突变实验证明,c-ABL通过Y425位点调控PLK1蛋白稳定性;5)同步化细胞于有丝分裂期,我们证明c-ABL通过磷酸化PLK1 Y425位点促进PLK1的激活。体外磷酸化反应证明,PLK1 Y425位点的磷酸化促进Aurora A对其激活。(二)c-ABL磷酸化PLK1调控有丝分裂进程。1)流失细胞仪检测c-ABL敲低He La细胞,我们发现c-ABL影响细胞周期进程;2)免疫荧光及流式细胞仪检测PLK1野生型与Y425突变株细胞周期,发现Y425磷酸化影响细胞G2/M转换;3)激光共聚焦显微镜实时观察细胞,进一步确定c-ABL介导的PLK1 Y425磷酸化促进细胞进入有丝分裂。(三)c-ABL-PLK1轴影响CML化疗应答。1)Real-Time PCR及Western Blot检测临床CML患者外周血样本,发现BCR/ABL与PLK1在CML中高表达;与imatinib敏感患者相比,PLK1在imatinib抗性患者体内表达更高;2)imatinib有效降低临床患者CML中PLK1蛋白表达水平;3)K562细胞过表达PLK1抑制其对imatinib的化疗应答;4)c-ABL与PLK1抑制剂联合用药显著提高CML细胞的化疗应答;5)构建imatinib抗性CML小鼠模型,证实联合用药延长CML肿瘤模型小鼠存活时间。(四)c-ABL-PLK1轴影响宫颈癌肿瘤增殖及病人存活率。1)通过收集临床宫颈癌组织样本,发现c-ABL与PLK1在宫颈癌中高表达,同时在肿瘤组织中PLK1具有高度酪氨酸磷酸化修饰;2)裸鼠成瘤实验证明,PLK1 Y425位点突变抑制肿瘤增殖;3)临床随访数据证明,PLK1酪氨酸磷酸化与不良预后正相关;4)联合用药实验证明,c-ABL,PLK1抑制剂促进宫颈癌细胞的化疗应答。综上所述,我们的研究揭示了c-ABL-PLK1轴在有丝分裂进程及CML化疗应答中的重要功能。c-ABL通过直接磷酸化PLK1调控PLK1的蛋白稳定性和活性。过表达PLK1抑制了CML细胞对imatinib的化疗应答。同时imatinib与PLK1抑制剂的联合用药有助于imatinib抗性CML及宫颈癌细胞的治疗。我们的研究有望为imatinib抗性CML患者提供新的治疗靶标。此外由于PLK1的酪氨酸磷酸化与宫颈癌患者的不良预后相关,我们的研究同样为宫颈癌的诊断和治疗提供了新的线索。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R363
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本文编号：1835164