Molecular and functional analysis of necroptosis initiation complex

描述(由申请人提供)： 广泛的证据表明，两种典型的细胞死亡途径，即细胞凋亡和坏死，并不涵盖各种生理和病理细胞死亡机制。我们和其他实验室已经证实存在一种常见的第三种途径，称为“程序性坏死”或“坏死性下垂”。坏死性下垂是一种受调节的细胞死亡途径，具有坏死的表型特征。它在细胞中被激活，这些细胞被诱导经历凋亡，但阻止了它的完成。我们最近开发了一种有效的和选择性的坏死性下垂的小分子抑制剂-Necrostatin-1，并使用该分子在体外和体内证明了坏死性下垂在各种病理性细胞死亡中的重要作用。坏死性下垂的发现为开发专门针对病理性细胞死亡的坏死成分的新疗法提供了独特的机会，以前没有人追求这一点，因为死亡是一种不受监管的死亡形式。然而，目前对坏死性下垂的激活和执行的具体机制知之甚少。丝氨酸/苏氨酸激酶RIP已成为坏死性下垂的关键上游激活剂。此外，我们最近确定RIP激酶活性是Necrostatin-1和其他几个结构上无关的有效Necrostatin的特异性细胞靶点，这突显了RIP激酶在坏死性下垂中的关键作用。在我们的初步研究中，我们开发了新的检测方法来专门测量RIP激酶的激活和坏死性下垂的诱导。我们对RIP激酶进行了初步的基于质谱学的表征，这导致了一些新的和特定的翻译后修饰(磷酸化)事件的识别，这些事件可能涉及RIP的坏死链活性的调节。我们还证明了利用质谱分析评估内源性RIP相互作用组组成动态变化的可行性。我们目前的建议集中在进一步研究RIP激酶诱导坏死性下垂的机制。该项目的具体目标包括：确认我们先前确定的RIP磷酸化变化在坏死性下垂的激活中的作用；使用磷酸化特异性RIP抗体和RIP激酶分析来表征体外和体内的RIP激酶激活过程；使用高分辨率质谱仪剖析RIP相互作用组，然后对RIP相互作用因子在坏死性下垂发生中的作用进行功能表征；以及建立死抑素抑制坏死性下垂作为针对急性病理性坏死的细胞保护治疗的新方向的可行性。总体而言，我们的研究将通过阐明诱导坏死性下垂的关键上游步骤-RIP激酶激活的分子基础，为调节坏死性上睑下垂提供重要的新见解，并将验证通过选择性靶向坏死性下垂特异性启动因子来治疗抑制病理性坏死的潜在新方向。坏死性下垂的发现不仅为基础生物学调控提供了新的视角，也为多种坏死性疾病的治疗干预提供了新的方向。我们的建议旨在进一步了解坏死性下垂的分子机制，这将使我们能够充分利用这一令人兴奋的发现来开发新的治疗方法。

DESCRIPTION (provided by applicant): Extensive evidence suggests that two "classic" cell death pathways, apoptosis and necrosis, do not encompass the full variety of physiological and pathological cell death mechanisms. Our and other laboratories have established the existence of a common third pathway, termed "programmed necrosis" or "necroptosis." Necroptosis is a regulated cell death pathway with phenotypic features of necrosis. It is activated in cells that are induced to undergo apoptosis, yet prevented from its completion. We have recently developed a potent and selective small molecule inhibitor of necroptosis, Necrostatin-1, and using this molecule have demonstrated the important role of necroptosis in various paradigms of pathologic cell death in vitro and in vivo. Discovery of necroptosis offers unique opportunity to develop novel therapies specifically targeting necrotic component of pathologic cell death, which was previously not pursued due to the notion that necrosis is an unregulated form of death. However, little is currently known regarding the specific mechanisms of activation and execution of necroptosis. Ser/Thr kinase RIP has emerged as the key upstream activator of necroptosis. Furthermore, we have recently established that RIP kinase activity is a specific cellular target of Necrostatin-1 and several other structurally unrelated potent necrostatins that were also developed in our laboratory, highlighting the critical role of RIP kinase in necroptosis. In our Preliminary studies, we developed new assays to specifically measure RIP kinase activation and necroptosis induction. We performed preliminary mass spectrometry-based characterization of RIP kinase that led to the identification of a number of novel and specific posttranslational modification (phosphorylation) events that are potentially involved in the regulation of necroptotic activity of RIP. We also demonstrated the feasibility of assessing dynamic changes in the composition of the endogenous RIP interactome using mass spectrometry analysis. Our current proposal focuses on further studies of the mechanism of necroptosis induction by RIP kinase. The specific aims of the project include confirming the role of RIP phosphorylation changes, previously identified by us, in the activation of necroptosis; characterization of RIP kinase activation process in vitro and in vivo using phospho-specific RIP antibodies and RIP kinase assay; dissection of RIP interactome using high resolution mass spectrometry followed by functional characterization of the role of RIP interacting factors in necroptosis initiation; and establishing the feasibility of the necroptosis inhibition by necrostatins as a new direction for cytoprotective therapies against acute pathologic necrosis. Overall, our studies will provide important new insights into the regulation of necroptosis through elucidating the molecular basis of the induction of the key upstream step in necroptosis, RIP kinase activation, and will validate a potential new direction for therapeutic inhibition of pathologic necrosis through selective targeting of necroptosis-specific initiation factors. Discovery of necroptosis not only provides novel insight into the basic biological regulation, but also offers new direction for therapeutic intervention against a variety of necrotizing disorders. Our proposal aims to provide further understanding of the molecular mechanism of necroptosis, which would allow us to take full advantage of this exciting discovery for development of new therapies.