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.

现在接受心肌细胞凋亡是缺血/诱导的细胞死亡的主要因素 再灌注（I/R）以及扩张心肌病和心力衰竭的发展。在心中 在孤立的心肌细胞中，丝氨酸苏氨酸激酶AKT可预防I/R损伤和凋亡 通过各种细胞应力。 AKT通过PI3激酶的生长因子刺激激活，PI3激酶是一种已有的酶 在心脏中进行了很好的研究。尚未探索调节Akt磷酸化的其他酶 在心肌细胞中，尽管这些可能是心脏保护的药物靶标。这些包括即时 上游激酶PDK-1和负责Akt脱磷酸化的磷酸酶，这是 最近发现的磷酸酶PHLPP。在AIM＃1中，我们解决了PHLPP和PHLPP的角色 使用PHLPP和PDK-1，在Akt磷酸化和心肌细胞中PDK-1的需求 淘汰老鼠。我们进一步研究了这些酶的损失如何影响体内缺血再灌注损害 以及凋亡的发展，线粒体！体外破坏和自噬。很多研究 AKT使用了活化的膜靶向形式。它也变得越来越清楚， 但是，AKT在包括质膜，细胞质，核和 线粒体。通过其调节酶对Akt的细胞位置和动态控制可以导致一个范围 时空信号反过来又决定下游信号事件的性质。因此 AKT活化是否调节胞质酶，核基因表达或线粒体完整性可能 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活动记者（BKAR）和其他BKAR的AKT调节的空间方面 针对特定的细胞位置。 MTOR不仅是AKT的下游目标的可能性 但是，作为AKT去磷酸化的反馈调节剂，将在AIM＃3中检查，以检验以下假设。 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。 雷帕霉素会影响细胞通过AKT调节进行自噬的可能性 也可以检查。我们最近证明了AKT与心肌细胞中的线粒体相关联，并且 尤其是线粒体通透性过渡（PT）孔内的组件，包括己糖激酶 （HKII）。在AIM＃4中，HKII表达的调节，其与线粒体的关联及其相关性 检查了AKT的磷酸化。 HKII在介导AKT对心脏保护作用中的作用 测试了线粒体通透性过渡孔。阐明调节AKT的新型酶促途径 磷酸化以及有关该酶的分隔作用的信息，将允许设计 可以选择性地靶向此多效信号通路的组件的治疗干预措施。