The attenuation of ischemic injury by transglutaminase 2

说明（由申请人提供）：本申请的重点是阐明转氨酶2（TG 2）调节缺氧诱导因子（HIF）信号传导并减弱缺血诱导的细胞死亡的分子机制。响应于缺血，存在可促进细胞存活或细胞死亡的基因的上调。最终结果是细胞存活还是延迟细胞死亡取决于表达的缺血诱导基因的互补性，其可以根据变量如损伤的持续时间和严重程度以及调节蛋白的存在而不同。由氧敏感性HIF 1亚基和组成型表达的HIF 12组成的HIF是主要负责低氧应答基因上调的转录因子。我们已经证明，TG 2结合HIF 12，减弱HIF信号传导，减弱特异性HIF应答基因的表达，并保护免受缺血性损伤。此外，我们有令人信服的数据表明，在小鼠中风模型中，人TG2的神经元表达显著减少了梗死体积。本申请的中心假设是TG2是缺氧诱导的转录信号传导的调节剂，并且TG2在保护神经元免受缺血诱导的细胞死亡中起着重要作用。本提案的目标将通过检验假设的三个具体目标来实现：（1）TG 2介导的HIF信号传导的抑制依赖于TG 2在细胞核的定位，不依赖于转酰胺活性，并且需要与HIF 12的相互作用，（2）TG 2对缺血诱导的细胞死亡的保护作用不依赖于转酰胺活性，但是需要核定位，并且进一步地，TG 2与HIF 1 α的相互作用对于TG 2对抗缺血性损伤的保护作用是必需的，以及（3）TG 2减弱体内响应于中风而发生的延迟细胞死亡。总的来说，这是一个综合性、机制性和整体性的建议。在我们的研究中，我们将使用重组蛋白来定义TG 2和HIF 1？之间的相互作用域，使用细胞模型来理解TG 2如何调节HIF依赖的转录事件并减弱缺血诱导的细胞死亡，使用小鼠模型来描述神经元TG 2在减少中风损伤中的作用。我们相信，通过使用这种完整的补充方法，我们将能够更全面地了解TG2在改善缺血诱导的细胞死亡中的作用。这些都是令人兴奋和创新的研究，因为它们不仅为理解TG2的功能定义了一个新的框架，而且还为缺血诱导的细胞死亡过程的调控提供了新的见解。 公共卫生相关性：中风是一种常见的病症，其导致大脑中氧气可用性的紊乱，并且具有重要的医学意义，因为在美国每年约有500，000例新发缺血性中风。在缺血性脑中，细胞死亡在梗塞核心处非常迅速地发生，然而在周围的半影区中，细胞死亡较慢并且具有更可控的性质，因此这一直是介入治疗的重要靶点。我们最近发现，转氨酶2（TG2）减弱缺血诱导的细胞死亡的细胞和小鼠模型，因此我们的长期目标是充分了解TG2在改善中风诱导的细胞死亡的作用，使有效的治疗方法可以开发。

DESCRIPTION (provided by applicant): The focus of this application is on elucidating the molecular mechanisms by which transglutaminase 2 (TG2) regulates hypoxia inducible factor (HIF) signaling and attenuates ischemic-induced cell death. In response to ischemia there is an upregulation of genes that can facilitate either cell survival or cell death. Whether cell survival or delayed cell death is the final outcome is dependent on the complement of ischemia-induced genes expressed, which can differ depending on variables such as duration and severity of the insult and the presence of regulatory proteins. HIF, which is composed of the oxygen sensitive HIF1 subunit and the constitutively expressed HIF12, is the transcription factor that is primarily responsible for the upregulation of hypoxic responsive genes. We have demonstrated that TG2 binds HIF12, attenuates HIF signaling, attenuates the expression of specific HIF- responsive genes and protects against ischemic insult. Furthermore, we have compelling data demonstrating that neuronal expression of human TG2 significantly reduces infarct volume in a mouse stroke model. The central hypothesis of this application is that TG2 is a regulator of hypoxic-induced transcriptional signaling, and that TG2 plays a fundamental role in protecting neurons against ischemia-induced cell death. The objectives of this proposal will be met through three specific aims that test the hypotheses: (1) that the TG2- mediated suppression of HIF signaling is dependent on the localization of TG2 to the nucleus, independent of transamidating activity and requires interaction with HIF12, (2) that the protective effects of TG2 against ischemic-induced cell death are independent of transamidating activity, but require nuclear localization and further, that the interaction of TG2 with HIF1¿ is necessary for the protective role of TG2 against ischemic insult, and (3) that TG2 attenuates the delayed cell death that occurs in response to stroke in vivo. Overall, this is an integrated, mechanistic and holistic proposal. In our studies we will be using recombinant proteins to define the interacting domains between TG2 and HIF1¿, cell models to understand how TG2 modulates HIF dependent transcriptional events and attenuates ischemia-induced cell death, and mouse models to delineate the role of neuronal TG2 in decreasing stroke damage. We believe that by using this full complement of approaches we will be able to more completely understand the role of TG2 in ameliorating ischemia-induced cell death. These are exciting and innovative studies in that they not only define a new framework for understanding the function of TG2, but that they also are providing new insights into the regulation of ischemia-induced cell death processes. PUBLIC HEALTH RELEVANCE: Stroke is a common condition that results in a disturbance in the availability of oxygen in the brain and is of major medical importance as there are approximately 500,000 new ischemic strokes yearly in the USA. In the ischemic brain, cell death occurs very rapidly at the infarct core, however in the surrounding penumbra the cell death is slower and of a more controlled nature and therefore this has been a significant target for interventive therapies. We have recently found that Transglutaminase 2 (TG2) attenuates ischemic-induced cell death in both cell and mouse models and therefore our long range goal is to fully understand the role of TG2 in ameliorating stroke-induced cell death so that effective therapeutics can be developed.