Regulation of Art and Mitochondrial function

艺术和线粒体功能的调节

• 批准号: 7217638
• 负责人: Joan Heller Brown
• 金额: $ 10万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217638
• 关键词: apoptosis; biological signal transduction; cardiac myocytes; cell death; enzyme activity; enzyme mechanism; fluorescence resonance energy transfer; genetically modified animals; laboratory mouse; mTOR protein; messenger RNA; myocardial ischemia /hypoxia; phosphatidylinositol 3 kinase; phosphoprotein phosphatase; phosphorylation; phosphotransferases; serine threonine protein kinase; sirolimus

Cardiomyocyte apoptosis is now accepted to be a major contributor to cell death induced by ischemia/ reperfusion (I/R) as well as to the development of dilated cardiomyopathy and heart failure. In the heart, as in isolated cardiomyocytes, the serine threonine kinase Akt protects against I/R injury and apoptosis induced by various cell stresses. Akt is activated through growth factor stimulation of PI3 kinase, an enzyme that has been well studied in the heart. The other enzymes that regulate Akt phosphorylation have not been explored in cardiomyocytes although these are likely drug targets for cardioprotection. These include the immediate upstream kinase PDK-1, and phosphatases responsible for Akt dephosphorylation amongst which is the recently discovered phosphatase, PHLPP. In Aim # 1 we address the regulation and role of PHLPP and the requirement for PDK-1 in Akt phosphorylation and activity in cardiomyocytes, using PHLPP and PDK-1 knockout mice. We further examine how loss of these enzymes affects ischemic reperfusion damage in vivo and the development of apoptosis, mitochondria! disruption and autophagy in vitro. A great many studies of Akt have used activated membrane targeted forms of the enzyme. It has also become increasingly clear, however, that Akt has actions at multiple sites including the plasma membrane, cytosol, nucleus and mitochondria. The cellular location and dynamic control of Akt by its regulatory enzymes can lead to a range of spatiotemporal signals which will in turn dictate the nature of the downstream signaling events. Thus whether Akt activation regulates cytosolic enzymes, nuclear gene expression or mitochondrial integrity may depend upon its activation kinetics and location. Accordingly in Aim # 2 we propose to examine the temporal and spatial aspects of Akt regulation using a FRET based Akt activity reporter (BKAR) and other BKARS targeted to specific cellular locations. The possibility that mTOR acts not only as a downstream target of Akt but as a feedback regulator of Akt dephosphorylation will be examined in Aim # 3, testing the hypothesis that rapamycin affects Akt dephosphorylation through PHLPP or regulates kinase mediated Akt phosphorylation. The possibility that rapamycin affects the cells decisions to undergo autophagy through regulation of Akt will also be examined. We recently demonstrated that Akt associates with mitochondria in cardiomyocytes, and in particular with components of the mitochondrial permeability transition (PT) pore including hexokinase (HKII). In Aim # 4 the regulation of the HKII expression, its association with mitochondria, and its phosphorylation by Akt are examined. The role of HKII in mediating the cardioprotective effects of Akt on the mitochondrial permeability transition pore is tested. Elucidation of novel enzymatic pathways regulating Akt phosphorylation, and information on the compartmentalized effects of this enzyme, will allow design of therapeutic interventions that can selectively target components of this pleiotropic signaling pathway.

心肌细胞凋亡现在被认为是缺血/诱导细胞死亡的主要原因 再灌注（I/R）以及扩张型心肌病和心力衰竭的发展。心里一样， 在分离的心肌细胞中，丝氨酸苏氨酸激酶 Akt 可防止 I/R 损伤和诱导的细胞凋亡 受到各种细胞应激的影响。 Akt 通过生长因子刺激 PI3 激酶而被激活，PI3 激酶是一种具有以下功能的酶： 心里已经好好研究过了。尚未探索调节 Akt 磷酸化的其他酶 尽管这些可能是心脏保护的药物靶点，但在心肌细胞中。这些包括立即 上游激酶 PDK-1 和负责 Akt 去磷酸化的磷酸酶，其中 最近发现的磷酸酶，PHLPP。在目标 1 中，我们讨论 PHLPP 的监管和作用以及 使用 PHLPP 和 PDK-1，心肌细胞中 Akt 磷酸化和活性对 PDK-1 的要求 基因敲除小鼠。我们进一步研究这些酶的损失如何影响体内缺血再灌注损伤 以及细胞凋亡、线粒体的发育！体外破坏和自噬。大量的研究 Akt 使用了活性膜靶向形式的酶。人们也越来越清楚， 然而，Akt 在多个位点都有作用，包括质膜、细胞质、细胞核和 线粒体。 Akt 的细胞定位和其调节酶的动态控制可导致一系列 时空信号的序列将反过来决定下游信号事件的性质。因此 Akt 激活是否调节胞质酶、核基因表达或线粒体完整性 取决于其激活动力学和位置。因此，在目标#2中，我们建议检查时间 使用基于 FRET 的 Akt 活动报告器 (BKAR) 和其他 BKARS 进行 Akt 调节的空间方面 针对特定的细胞位置。 mTOR 不仅作为 Akt 下游靶标的可能性 但作为 Akt 去磷酸化的反馈调节剂将在目标 # 3 中进行检查，检验以下假设： 雷帕霉素通过 PHLPP 影响 Akt 去磷酸化或调节激酶介导的 Akt 磷酸化。 雷帕霉素通过调节 Akt 影响细胞进行自噬的可能性将 也予以检查。我们最近证明 Akt 与心肌细胞中的线粒体相关，并且 特别是线粒体通透性转变 (PT) 孔的成分，包括己糖激酶 （HKII）。在目标 4 中，HKII 表达的调节、其与线粒体的关联及其与线粒体的关系。 检查 Akt 的磷酸化。 HKII 在介导 Akt 心脏保护作用中的作用 测试线粒体通透性转换孔。阐明调节 Akt 的新酶途径 磷酸化以及有关该酶的区室效应的信息将允许设计 可以选择性地针对这种多效性信号通路成分的治疗干预措施。