AMP激活蛋白激酶α亚基C末端影响激酶活性
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摘要
研究背景：AMP激活蛋白激酶(AMP activated protein kinase, AMPK)是丝氨酸／苏氨酸蛋白激酶,活化后通过磷酸化下游蛋白的某些丝氨酸或苏氨酸残基增强或抑制该蛋白的生物学活性从而调节细胞能量代谢。各种导致细胞内ATP消耗增加合成减少从而导致AMP／ATP比值升高的因素均可引起AMPK活化。AMPK活化后能够抑制各耗能过程如胆固醇合成、甘油三酯和蛋白质合成,抑制脂肪细胞内脂质水解,减少骨骼肌细胞脂肪氧化,抑制细胞生长分化,从而维持细胞能量平衡,保护细胞免受应激损伤。多种应激条件如缺血、缺氧、饥饿均能激活AMPK。越来越多的证据表明,AMPK活化对于心血管系统具有保护作用。AMPK活化不仅能够调节胆固醇代谢、改善血脂异常,还能够通过改善内皮功能发挥抗动脉粥样硬化的有益作用。
AMPK以三聚体形式存在,包括一个催化亚基α及两个调节亚基β和γ,哺乳动物细胞内三亚基共表达实验证实,AMPK最大活性需要三个亚基同时存在。鉴于AMPK在维持机体和细胞能量代谢、调节细胞生长、调节心血管系统功能中的重要作用,AMPK被认为是未来治疗代谢性疾病和心血管疾病最有希望的靶蛋白之一,了解AMPK蛋白结构和活性调节机制对寻找新的AMPK活性调节药物具有重要意义。
目前,因为AMPKα基和β亚基无法形成蛋白结晶,AMPK三聚体的完整晶体结构尚不完全清楚。蛋白结构稳定是各生物酶发挥生物活性的基础,蛋白结构改变会影响酶活性。既往关于AMPKα亚基结构的研究均限于α亚基N末端催化结构域,而对于C末端的认识仅局限于C末端提供β亚基结合位点,对于C末端在调节AMPK激酶活性中的作用仍缺乏了解。Towley等对α亚基C末端氨基酸序列进行分析后提出,α亚基C末端在二级结构上和MARK3 C末端高度相似,均为α螺旋和β片层相间的“三明治”结构,具有形成1型激酶相关结构域(kinase-associated domain 1, KA1)的基础。研究AMPKα亚基C末端的蛋白结构对于了解AMPK活性调节机制,寻找研发调节AMPK活性的新药物具有重要意义。
目的：研究AMPK各亚基间相互作用及其活性调节机制。
方法：(1)分离AMPKα2及α1基因敲除小鼠骨骼肌、心脏、肾脏及脑组织,通过蛋白印渍技术检测AMPKα及β亚基蛋白表达水平并通过荧光实时定量PCR检测其mRNA水平。(2)通过RNA干扰技术分别减少AMPKα1、α2、β1、β2表达后检测AMPKα及β亚基蛋白表达水平。(3)放线菌酮追踪实验(Cycloheximide Pulse Chase)分别检测野生型和AMPKα亚基敲除小鼠成纤维细胞内AMPKβ亚基蛋白降解速率。(4)构建了一系列表达逐渐缺失α亚基C末端的GST融合蛋白质粒,分别表达全长α亚基WTα2、a2(1～540,a2△12)α2(1～476)、α2(1～426)和α2(1～412),利用GST beads提取并纯化后,加入α亚基敲除成纤维细胞的蛋白裂解液,检测不同长度α亚基与β亚基的结合情况,判断α2亚基上β亚基的结合位点。(5)利用PDB蛋白域数据库(Protein Domains Database)中已有的AMPK晶体蛋白结构数据,在计算机上进行分子动力学模拟,使用Root Mean Square Disturbance (RMSD)计算软件,计算β亚基在α亚基存在或缺失时的RMSD改变,以此反映β亚基蛋白空间构象在液相中的改变程度；使用GROMACS-4.0显示研究β亚基在α亚基存在或缺失时原子空间轨迹变化；RMSD计算正常α亚基及其突变体在液相中的构象变化,以及在其构象变化中起关键作用的氨基酸残基。(6)利用AMPKα亚基敲除小鼠成纤维细胞(Murine Embryonic Fibroblasts, MEFs)作为AMPK酶活性检测系统,瞬时转染后过表达AMPKα2正常全长蛋白(WTα2)和突变蛋白,通过检测AMPK下游蛋白乙酰辅酶A脱羧酶(acetyl CoA Carboxylase, ACC)的磷酸化水平反应AMPK激酶活性。
结果：(1)AMPKα、β亚基间的相互作用对维持彼此的蛋白稳定性具有重要作用,α亚基缺失时β亚基蛋白水平降低,同样,β亚基缺失时α亚基的蛋白水平亦降低。
(2)与α亚基存在时相比,α亚基缺失后,β1及β2亚基mRNA水平不变,蛋白稳定性下降,蛋白降解加速。放线菌酮处理3小时后,α亚基敲除细胞中β1剩余约21%,而野生型细胞中,β1剩余约58%(p<0.05)。
(3)小鼠AMPKα2亚基上第412～426位氨基酸在α、β相互作用中具有关键意义。第412～426位氨基酸缺失严重影响α2亚基与β亚基的结合。
(4)α2亚基第540～552位氨基酸缺失后(α2△12)不影响其与β亚基的结合,但AMPK激酶活性降低。α2△12能够作为无功能突变体阻断内源性AMPK的作用。在HEK 293细胞中分别过表达和WTα2和α2△12,AMPK特异活化剂AICAR刺激后,磷酸化AMPK (pAMPK)及pACC水平均明显增高,与空质粒对照组相比,WTα2过表达时,细胞内基础水平pACC相应增加,AICAR刺激后pACC进一步增高；相反,过表达α2△12时,虽然pAMPK明显增加,pACC水平并未相应增加,提示AMPK活性下降。(5)在已有的AMPK晶体结构基础上进行计算机三维结构模拟后认为,AMPKα亚基C末端三维结构与MARK3的C末端高度吻合,有可能形成类似KA1结构域的能够调节AMPK活性的结构域,第540～552位氨基酸可能在α2亚基C末端特殊αβ疏水结构域形成中具有重要作用。位于该结构域疏水中心的第550位和546位亮氨酸突变为极性亲水性谷氨酸后,AMPK激酶活性亦明显降低。
结论：AMPKα、β亚基间相互作用对于维持亚基稳定性具有重要意义。α2亚基C末端由α螺旋和β片层形成的具有疏水中心的特殊结构域在维持AMPK激酶活性中具有重要意义。α2亚基C末端a螺旋(a.a 540～552)缺失或者第550位和546位亮氨酸发生突变后均可能因为破坏该结构域的空间结构从而降低AMPK激酶活性。
研究意义：单核苷酸多态性(Single Nucleotide Polymorphisms, SNP),是指在基因组上单个核苷酸的变异,形成的遗传标记,其数量众多,现在普遍认为SNP研究是人类基因组走向应用的重要步骤。SNP将为高危群体的发现、疾病相关基因的鉴定、药物的设计和测试以及生物学的基础研究提供一个强有力的工具。近年来,关于AMPK基因多态性与代谢性疾病如脂代谢异常及2型糖尿病相关性的研究也在逐步开展。迄今已报导多个位于AMPK各亚基基因外显子内能够改变AMPK氨基酸序列的SNP。其中,γ2点突变与WPW综合征及家族性肥厚型心肌病密切相关,携带γ3 R200Q突变的猪表现为糖原沉积性疾病。我们的研究提示,在人群中,特别是存在代谢异常的人群中是否存在L550Q、L546Q的突变从而影响AMPK激酶活性及其与代谢性疾病的相关性值得进一步研究。
血流切应力是血液流经血管时作用于血管内膜的摩擦力,生理状态下血管直段的层流切应力(Laminar Shear Stress)对于维持血管正常功能具有重要意义,参与调节血管紧张度、血管内皮细胞增殖、抑制炎症反应等。本实验主要通过细胞实验和离体动物实验研究了层流切应力对内皮细胞线粒体生物合成、氧化应激状态及血管张力的调节及其相关机制。方法：(1)将脐静脉内皮细胞(HUVEC, ECs)暴露于12dyns／cm2层流切应力下12小时后,应用MitoTracker探针对ECs内线粒体染色,共聚焦显微镜下观察细胞线粒体的数量和体积；MTT试剂盒检测线粒体抗氧化活性(2)实时定量荧光PCR检测线粒体生物合成相关基因如PGC1a、COX4和NRF1的mRNA水平。(3)免疫印渍技术检测层流切应力刺激后ECs中AMP激活蛋白激酶(AMPK)水平及其磷酸化水平及PGC1α蛋白表达水平。(4)应用RNA干扰技术抑制细胞内SIRT1表达后,观察层流切应力刺激后PGC1a、COX4及NRF1的mRNA水平的改变。(5)应用RNA干扰技术抑制ECs内CaMKKβ表达后,检测Nrf2及SOD2的mRNA水平的改变。(7)分离野生型C57BL／6小鼠大脑中动脉,连接至SoftEdge Acquisiton系统,分别应用CaMKKβ抑制剂STO-609和AMPK抑制剂Compound C或者二甲基亚砜(DMSO)预处理半小时后,检测层流诱导的动脉舒张情况。(7)分离CaMKKβ基因敲除小鼠、AMPKα2基因敲除小鼠及其野生对照组小鼠的大脑中动脉,通过SoftEdge Acquisiton系统进行离体血管内灌注,检测CaMKKβ或AMPKα2基因敲除时对层流诱导的血管舒张的影响。
结果：(1)层流切应力增加线粒体生物合成：内皮细胞线粒体体积增大。(2)层流切应力能够诱导调节线粒体生物合成的主要基因表达PGC1a,NRF及COX4的表达。(3)层流切应力通过SIRT1调节线粒体生物合成：应用RNA干扰技术降低细胞内SIRT1表达水平后,层流切应力诱导的线粒体生物合成被显著抑制,证明层流切应力通过SIRT1调节内皮细胞线粒体生物合成。(4)层流切应力能够诱导AMPK活化,层流切应力刺激后,ECs的AMPK磷酸化水平升高,应用RNA干扰技术抑制内皮细胞内CaMKKβ的表达后,AMPK磷酸化被抑制。(5)应用CaMKKβ抑制剂STO-609和AMPK抑制剂Compound C预处理后,与DMSO对照组相比,层流诱导的大脑中动脉舒张显著被抑制(p<0.05)。(6)与野生对照组相比,CaMKKβ基因敲除小鼠、AMPKα2基因敲除小鼠的大脑中动脉被层流诱导的舒张程度显著降低(p<0.05)。
结论：层流切应力能够通过调节SIRT1增加细胞线粒体生物合成；层流切应力通过CaMKKβ调节AMPK活化并诱导动脉舒张功能。
研究意义：层流切应力是公认的血管系统保护性因素,生理条件下层流切应力的存在对于维持血管内皮细胞正常功能,抑制动脉粥样硬化的发生和发展具有重要意义。
Comprising a catalytic a subunit and two regulatoryβand y subunits, AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis as well as cell proliferation and polarity. To date, the mechanism underlying the autoregulation of AMPK kinase activity remains elusive. Using tissues collected from mice ablated with AMPK a2, we showed that the stability ofβsubunit depended on the presence of a. In the absence ofα1 or a2 subunit, the decrease inβwas due to an accelerated protein degradation, which could be rescued by the a complementation. Similarly, siRNA knocking down ofβdecreased a expression in cultured cells. Experiments with a2 truncations revealed that residues 412-426 of murine a2 were critical for the binding ofβsubunit. Exhibited little effect onαβinteraction, a 12-a.a truncation at the C-terminal (aa.541-552α2△12) of a2 resulted in decreased kinase activity. This truncation could act as a domain negative mutant to abate endogenous AMPK activity in phosphorylating the downstream targets such as acetyl-CoA carboxylase. The C-terminus of AMPKα2 subunit forms a specialαβdomain with a hydrophobic center similar to that of MARK3 C-terminus. The deletion of the a helix possibly disrupts the conformation of this domain therefore affects the kinase activity. The mutations of highly conserved Leu550 and Leu546 that involved in the formation of hydrophobic center to glutamate similarly decreased kinase activity. Taken together, the interaction ofαβis important in maintaining the AMPK stability, whereas the conformation integrity of C-terminal tail of the a is necessary for the kinase activity of AMPK.
Endothelial dysfunction is closely associated with vascular diseases such as atherosclerosis and hypertension. Physiological range of shear stress is critical in maintaining endothelial functions such as anti-inflammation, anti-proliferation and regulating vessel tones. Multiple mechanisms have been elucidated to be involved in the vascular protective effects mediated by shear stress. However, whether shear stress can regulate the redox state of endothelial cells and the underlying mechanism is still unknown. In the present study, we proved that laminar shear can induce the endothelial cell (ECs) mitochondrial biogenesis and antioxidant availability which is mediated by SIRT1, a well known anti-aging molecule. Another pivotal molecule involved in the shear stress benefits is CaMKKβ, an AMP-activated protein kinase (AMPK) kinase ablation of which resulted in impaired anti-oxidant activity in ECs as genomically knockdown of CaMKKP led to decreased SOD2 and Nrf2 expression, two major genes in the antioxidant system. Loss of CaMKKP also attenuated flow-induced activation of AMPK, therefore blocked eNOS activation, which is critical in endothelial-dependent vessel dilation. This is proved in the ex vivo experiment that arteries dissected from CaMKKP and AMPK knockout mice responsed poorly to flow compared to there wild type littermates. In conclusion, shear stress can induce SIRT1 mediated ECs mitochondrial biogenesis; the vascular protective effect of shear stress is also mediated through CaMKKβ-AMPK pathway, blocking of which leads to decreased antioxidant activity and impared response to flow induced vessel dilation.
AMPK以三聚体形式存在,包括一个催化亚基α及两个调节亚基β和γ,哺乳动物细胞内三亚基共表达实验证实,AMPK最大活性需要三个亚基同时存在。鉴于AMPK在维持机体和细胞能量代谢、调节细胞生长、调节心血管系统功能中的重要作用,AMPK被认为是未来治疗代谢性疾病和心血管疾病最有希望的靶蛋白之一,了解AMPK蛋白结构和活性调节机制对寻找新的AMPK活性调节药物具有重要意义。
目前,因为AMPKα基和β亚基无法形成蛋白结晶,AMPK三聚体的完整晶体结构尚不完全清楚。蛋白结构稳定是各生物酶发挥生物活性的基础,蛋白结构改变会影响酶活性。既往关于AMPKα亚基结构的研究均限于α亚基N末端催化结构域,而对于C末端的认识仅局限于C末端提供β亚基结合位点,对于C末端在调节AMPK激酶活性中的作用仍缺乏了解。Towley等对α亚基C末端氨基酸序列进行分析后提出,α亚基C末端在二级结构上和MARK3 C末端高度相似,均为α螺旋和β片层相间的“三明治”结构,具有形成1型激酶相关结构域(kinase-associated domain 1, KA1)的基础。研究AMPKα亚基C末端的蛋白结构对于了解AMPK活性调节机制,寻找研发调节AMPK活性的新药物具有重要意义。
目的：研究AMPK各亚基间相互作用及其活性调节机制。
方法：(1)分离AMPKα2及α1基因敲除小鼠骨骼肌、心脏、肾脏及脑组织,通过蛋白印渍技术检测AMPKα及β亚基蛋白表达水平并通过荧光实时定量PCR检测其mRNA水平。(2)通过RNA干扰技术分别减少AMPKα1、α2、β1、β2表达后检测AMPKα及β亚基蛋白表达水平。(3)放线菌酮追踪实验(Cycloheximide Pulse Chase)分别检测野生型和AMPKα亚基敲除小鼠成纤维细胞内AMPKβ亚基蛋白降解速率。(4)构建了一系列表达逐渐缺失α亚基C末端的GST融合蛋白质粒,分别表达全长α亚基WTα2、a2(1～540,a2△12)α2(1～476)、α2(1～426)和α2(1～412),利用GST beads提取并纯化后,加入α亚基敲除成纤维细胞的蛋白裂解液,检测不同长度α亚基与β亚基的结合情况,判断α2亚基上β亚基的结合位点。(5)利用PDB蛋白域数据库(Protein Domains Database)中已有的AMPK晶体蛋白结构数据,在计算机上进行分子动力学模拟,使用Root Mean Square Disturbance (RMSD)计算软件,计算β亚基在α亚基存在或缺失时的RMSD改变,以此反映β亚基蛋白空间构象在液相中的改变程度；使用GROMACS-4.0显示研究β亚基在α亚基存在或缺失时原子空间轨迹变化；RMSD计算正常α亚基及其突变体在液相中的构象变化,以及在其构象变化中起关键作用的氨基酸残基。(6)利用AMPKα亚基敲除小鼠成纤维细胞(Murine Embryonic Fibroblasts, MEFs)作为AMPK酶活性检测系统,瞬时转染后过表达AMPKα2正常全长蛋白(WTα2)和突变蛋白,通过检测AMPK下游蛋白乙酰辅酶A脱羧酶(acetyl CoA Carboxylase, ACC)的磷酸化水平反应AMPK激酶活性。
结果：(1)AMPKα、β亚基间的相互作用对维持彼此的蛋白稳定性具有重要作用,α亚基缺失时β亚基蛋白水平降低,同样,β亚基缺失时α亚基的蛋白水平亦降低。
(2)与α亚基存在时相比,α亚基缺失后,β1及β2亚基mRNA水平不变,蛋白稳定性下降,蛋白降解加速。放线菌酮处理3小时后,α亚基敲除细胞中β1剩余约21%,而野生型细胞中,β1剩余约58%(p<0.05)。
(3)小鼠AMPKα2亚基上第412～426位氨基酸在α、β相互作用中具有关键意义。第412～426位氨基酸缺失严重影响α2亚基与β亚基的结合。
(4)α2亚基第540～552位氨基酸缺失后(α2△12)不影响其与β亚基的结合,但AMPK激酶活性降低。α2△12能够作为无功能突变体阻断内源性AMPK的作用。在HEK 293细胞中分别过表达和WTα2和α2△12,AMPK特异活化剂AICAR刺激后,磷酸化AMPK (pAMPK)及pACC水平均明显增高,与空质粒对照组相比,WTα2过表达时,细胞内基础水平pACC相应增加,AICAR刺激后pACC进一步增高；相反,过表达α2△12时,虽然pAMPK明显增加,pACC水平并未相应增加,提示AMPK活性下降。(5)在已有的AMPK晶体结构基础上进行计算机三维结构模拟后认为,AMPKα亚基C末端三维结构与MARK3的C末端高度吻合,有可能形成类似KA1结构域的能够调节AMPK活性的结构域,第540～552位氨基酸可能在α2亚基C末端特殊αβ疏水结构域形成中具有重要作用。位于该结构域疏水中心的第550位和546位亮氨酸突变为极性亲水性谷氨酸后,AMPK激酶活性亦明显降低。
结论：AMPKα、β亚基间相互作用对于维持亚基稳定性具有重要意义。α2亚基C末端由α螺旋和β片层形成的具有疏水中心的特殊结构域在维持AMPK激酶活性中具有重要意义。α2亚基C末端a螺旋(a.a 540～552)缺失或者第550位和546位亮氨酸发生突变后均可能因为破坏该结构域的空间结构从而降低AMPK激酶活性。
研究意义：单核苷酸多态性(Single Nucleotide Polymorphisms, SNP),是指在基因组上单个核苷酸的变异,形成的遗传标记,其数量众多,现在普遍认为SNP研究是人类基因组走向应用的重要步骤。SNP将为高危群体的发现、疾病相关基因的鉴定、药物的设计和测试以及生物学的基础研究提供一个强有力的工具。近年来,关于AMPK基因多态性与代谢性疾病如脂代谢异常及2型糖尿病相关性的研究也在逐步开展。迄今已报导多个位于AMPK各亚基基因外显子内能够改变AMPK氨基酸序列的SNP。其中,γ2点突变与WPW综合征及家族性肥厚型心肌病密切相关,携带γ3 R200Q突变的猪表现为糖原沉积性疾病。我们的研究提示,在人群中,特别是存在代谢异常的人群中是否存在L550Q、L546Q的突变从而影响AMPK激酶活性及其与代谢性疾病的相关性值得进一步研究。
血流切应力是血液流经血管时作用于血管内膜的摩擦力,生理状态下血管直段的层流切应力(Laminar Shear Stress)对于维持血管正常功能具有重要意义,参与调节血管紧张度、血管内皮细胞增殖、抑制炎症反应等。本实验主要通过细胞实验和离体动物实验研究了层流切应力对内皮细胞线粒体生物合成、氧化应激状态及血管张力的调节及其相关机制。方法：(1)将脐静脉内皮细胞(HUVEC, ECs)暴露于12dyns／cm2层流切应力下12小时后,应用MitoTracker探针对ECs内线粒体染色,共聚焦显微镜下观察细胞线粒体的数量和体积；MTT试剂盒检测线粒体抗氧化活性(2)实时定量荧光PCR检测线粒体生物合成相关基因如PGC1a、COX4和NRF1的mRNA水平。(3)免疫印渍技术检测层流切应力刺激后ECs中AMP激活蛋白激酶(AMPK)水平及其磷酸化水平及PGC1α蛋白表达水平。(4)应用RNA干扰技术抑制细胞内SIRT1表达后,观察层流切应力刺激后PGC1a、COX4及NRF1的mRNA水平的改变。(5)应用RNA干扰技术抑制ECs内CaMKKβ表达后,检测Nrf2及SOD2的mRNA水平的改变。(7)分离野生型C57BL／6小鼠大脑中动脉,连接至SoftEdge Acquisiton系统,分别应用CaMKKβ抑制剂STO-609和AMPK抑制剂Compound C或者二甲基亚砜(DMSO)预处理半小时后,检测层流诱导的动脉舒张情况。(7)分离CaMKKβ基因敲除小鼠、AMPKα2基因敲除小鼠及其野生对照组小鼠的大脑中动脉,通过SoftEdge Acquisiton系统进行离体血管内灌注,检测CaMKKβ或AMPKα2基因敲除时对层流诱导的血管舒张的影响。
结果：(1)层流切应力增加线粒体生物合成：内皮细胞线粒体体积增大。(2)层流切应力能够诱导调节线粒体生物合成的主要基因表达PGC1a,NRF及COX4的表达。(3)层流切应力通过SIRT1调节线粒体生物合成：应用RNA干扰技术降低细胞内SIRT1表达水平后,层流切应力诱导的线粒体生物合成被显著抑制,证明层流切应力通过SIRT1调节内皮细胞线粒体生物合成。(4)层流切应力能够诱导AMPK活化,层流切应力刺激后,ECs的AMPK磷酸化水平升高,应用RNA干扰技术抑制内皮细胞内CaMKKβ的表达后,AMPK磷酸化被抑制。(5)应用CaMKKβ抑制剂STO-609和AMPK抑制剂Compound C预处理后,与DMSO对照组相比,层流诱导的大脑中动脉舒张显著被抑制(p<0.05)。(6)与野生对照组相比,CaMKKβ基因敲除小鼠、AMPKα2基因敲除小鼠的大脑中动脉被层流诱导的舒张程度显著降低(p<0.05)。
结论：层流切应力能够通过调节SIRT1增加细胞线粒体生物合成；层流切应力通过CaMKKβ调节AMPK活化并诱导动脉舒张功能。
研究意义：层流切应力是公认的血管系统保护性因素,生理条件下层流切应力的存在对于维持血管内皮细胞正常功能,抑制动脉粥样硬化的发生和发展具有重要意义。
Comprising a catalytic a subunit and two regulatoryβand y subunits, AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis as well as cell proliferation and polarity. To date, the mechanism underlying the autoregulation of AMPK kinase activity remains elusive. Using tissues collected from mice ablated with AMPK a2, we showed that the stability ofβsubunit depended on the presence of a. In the absence ofα1 or a2 subunit, the decrease inβwas due to an accelerated protein degradation, which could be rescued by the a complementation. Similarly, siRNA knocking down ofβdecreased a expression in cultured cells. Experiments with a2 truncations revealed that residues 412-426 of murine a2 were critical for the binding ofβsubunit. Exhibited little effect onαβinteraction, a 12-a.a truncation at the C-terminal (aa.541-552α2△12) of a2 resulted in decreased kinase activity. This truncation could act as a domain negative mutant to abate endogenous AMPK activity in phosphorylating the downstream targets such as acetyl-CoA carboxylase. The C-terminus of AMPKα2 subunit forms a specialαβdomain with a hydrophobic center similar to that of MARK3 C-terminus. The deletion of the a helix possibly disrupts the conformation of this domain therefore affects the kinase activity. The mutations of highly conserved Leu550 and Leu546 that involved in the formation of hydrophobic center to glutamate similarly decreased kinase activity. Taken together, the interaction ofαβis important in maintaining the AMPK stability, whereas the conformation integrity of C-terminal tail of the a is necessary for the kinase activity of AMPK.
Endothelial dysfunction is closely associated with vascular diseases such as atherosclerosis and hypertension. Physiological range of shear stress is critical in maintaining endothelial functions such as anti-inflammation, anti-proliferation and regulating vessel tones. Multiple mechanisms have been elucidated to be involved in the vascular protective effects mediated by shear stress. However, whether shear stress can regulate the redox state of endothelial cells and the underlying mechanism is still unknown. In the present study, we proved that laminar shear can induce the endothelial cell (ECs) mitochondrial biogenesis and antioxidant availability which is mediated by SIRT1, a well known anti-aging molecule. Another pivotal molecule involved in the shear stress benefits is CaMKKβ, an AMP-activated protein kinase (AMPK) kinase ablation of which resulted in impaired anti-oxidant activity in ECs as genomically knockdown of CaMKKP led to decreased SOD2 and Nrf2 expression, two major genes in the antioxidant system. Loss of CaMKKP also attenuated flow-induced activation of AMPK, therefore blocked eNOS activation, which is critical in endothelial-dependent vessel dilation. This is proved in the ex vivo experiment that arteries dissected from CaMKKP and AMPK knockout mice responsed poorly to flow compared to there wild type littermates. In conclusion, shear stress can induce SIRT1 mediated ECs mitochondrial biogenesis; the vascular protective effect of shear stress is also mediated through CaMKKβ-AMPK pathway, blocking of which leads to decreased antioxidant activity and impared response to flow induced vessel dilation.
引文
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1. Chen Z, Peng IC, Sun W, Su MI, Hsu PH, Fu Y, Zhu Y, DeFea K, Pan S, Tsai MD, Shyy JY:AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase Ser633. Circ Res 104:496-505,2009
2. Cicha I, Beronov K, Ramirez EL, Osterode K, Goppelt-Struebe M, Raaz D, Yilmaz A, Daniel WG, Garlichs CD:Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-alpha and monocytic cell recruitment in a simplified model of arterial bifurcations. Atherosclerosis 207:93-102,2009
3. Pan S:Molecular mechanisms responsible for the atheroprotective effects of laminar shear stress. Antioxid Redox Signal 11:1669-1682,2009
4. Quintero M, Colombo SL, Godfrey A, Moncada S:Mitochondria as signaling organelles in the vascular endothelium. Proc Natl Acad Sci U S A 103:5379-5384,2006
5. Esposito LA, Melov S, Panov A, Cottrell BA, Wallace DC:Mitochondrial disease in mouse results in increased oxidative stress. Proc Natl Acad Sci U S A 96:4820-4825,1999
6. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM:A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92:829-839,1998
7. Bergeron R, Ren JM, Cadman KS, Moore IK, Perret P, Pypaert M, Young LH, Semenkovich CF, Shulman GI:Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis. Am J Physiol Endocrinol Metab 281:E1340-1346,2001
8. Zhang Y, Lee TS, Kolb EM, Sun K, Lu X, Sladek FM, Kassab GS, Garland T, Jr., Shyy JY:AMP-activated protein kinase is involved in endothelial NO synthase activation in response to shear stress. Arterioscler Thromb Vasc Biol 26:1281-1287,2006
9. Merlin J, Evans BA, Csikasz RI, Bengtsson T, Summers RJ, Hutchinson DS:The M(3)-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism. Cell Signal
10. Young A, Wu W, Sun W, Larman HB, Wang N, Li YS, Shyy JY, Chien S, Garcia-Cardena G:Flow activation of AMP-activated protein kinase in vascular endothelium leads to Kruppel-like factor 2 expression. Arterioscler Thromb Vasc Biol 29:1902-1908,2009
11. Schulz E, Dopheide J, Schuhmacher S, Thomas SR, Chen K, Daiber A, Wenzel P, Munzel T, Keaney JF, Jr.:Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction. Circulation 118:1347-1357,2008
12. Hock MB, Kralli A:Transcriptional control of mitochondrial biogenesis and function. Annu Rev Physiol 71:177-203,2009
13. Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M, Puigserver P, Auwerx J:Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-lalpha. Cell 127:1109-1122, 2006
14. Csiszar A, Labinskyy N, Pinto JT, Ballabh P, Zhang H, Losonczy G, Pearson K, de Cabo R, Pacher P, Zhang C, Ungvari Z:Resveratrol induces mitochondrial biogenesis in endothelial cells. Am J Physiol Heart Circ Physiol 297:H13-20, 2009
15. Canto C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, Elliott PJ, Puigserver P, Auwerx J:AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 458:1056-1060,2009
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1. Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, Orisio S, Remuzzi G, Remuzzi A:Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res 76:536-543,1995
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1. Chen Z, Peng IC, Sun W, Su MI, Hsu PH, Fu Y, Zhu Y, DeFea K, Pan S, Tsai MD, Shyy JY:AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase Ser633. Circ Res 104:496-505,2009
2. Cicha I, Beronov K, Ramirez EL, Osterode K, Goppelt-Struebe M, Raaz D, Yilmaz A, Daniel WG, Garlichs CD:Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-alpha and monocytic cell recruitment in a simplified model of arterial bifurcations. Atherosclerosis 207:93-102,2009
3. Pan S:Molecular mechanisms responsible for the atheroprotective effects of laminar shear stress. Antioxid Redox Signal 11:1669-1682,2009
4. Quintero M, Colombo SL, Godfrey A, Moncada S:Mitochondria as signaling organelles in the vascular endothelium. Proc Natl Acad Sci U S A 103:5379-5384,2006
5. Esposito LA, Melov S, Panov A, Cottrell BA, Wallace DC:Mitochondrial disease in mouse results in increased oxidative stress. Proc Natl Acad Sci U S A 96:4820-4825,1999
6. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM:A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92:829-839,1998
7. Bergeron R, Ren JM, Cadman KS, Moore IK, Perret P, Pypaert M, Young LH, Semenkovich CF, Shulman GI:Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis. Am J Physiol Endocrinol Metab 281:E1340-1346,2001
8. Zhang Y, Lee TS, Kolb EM, Sun K, Lu X, Sladek FM, Kassab GS, Garland T, Jr., Shyy JY:AMP-activated protein kinase is involved in endothelial NO synthase activation in response to shear stress. Arterioscler Thromb Vasc Biol 26:1281-1287,2006
9. Merlin J, Evans BA, Csikasz RI, Bengtsson T, Summers RJ, Hutchinson DS:The M(3)-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism. Cell Signal
10. Young A, Wu W, Sun W, Larman HB, Wang N, Li YS, Shyy JY, Chien S, Garcia-Cardena G:Flow activation of AMP-activated protein kinase in vascular endothelium leads to Kruppel-like factor 2 expression. Arterioscler Thromb Vasc Biol 29:1902-1908,2009
11. Schulz E, Dopheide J, Schuhmacher S, Thomas SR, Chen K, Daiber A, Wenzel P, Munzel T, Keaney JF, Jr.:Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction. Circulation 118:1347-1357,2008
12. Hock MB, Kralli A:Transcriptional control of mitochondrial biogenesis and function. Annu Rev Physiol 71:177-203,2009
13. Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M, Puigserver P, Auwerx J:Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-lalpha. Cell 127:1109-1122, 2006
14. Csiszar A, Labinskyy N, Pinto JT, Ballabh P, Zhang H, Losonczy G, Pearson K, de Cabo R, Pacher P, Zhang C, Ungvari Z:Resveratrol induces mitochondrial biogenesis in endothelial cells. Am J Physiol Heart Circ Physiol 297:H13-20, 2009
15. Canto C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, Elliott PJ, Puigserver P, Auwerx J:AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 458:1056-1060,2009
16. Neri S, Calvagno S, Mauceri B, Misseri M, Tsami A, Vecchio C, Mastrosimone G, Di Pino A, Maiorca D, Judica A, Romano G, Rizzotto A, Signorelli SS:Effects of antioxidants on postprandial oxidative stress and endothelial dysfunction in subjects with impaired glucose tolerance and Type 2 diabetes. Eur J Nutr
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