Molecular and functional analysis of necroptosis initiation complex

坏死性凋亡起始复合物的分子和功能分析

• 批准号: 7527738
• 负责人: ALEXEI DEGTEREV
• 金额: $ 39.1万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7527738
• 关键词: Acute; Acute Brain Injuries; Address; Animal Model; Apoptosis; Apoptotic; Biological; Biological Assay; Brain; Caspase; Cell Death; Cell Death Process; Cells; Cessation of life; Characteristics; Collaborations; Complex; Condition; Cultured Cells; Data; Death Domain; Detection; Development; Disease; Dissection; Docking; Event; Fc Receptor; Goals; Heart; In Vitro; Injury; Ischemic Brain Injury; Knowledge; Laboratories; Ligands; Liver; Mass Spectrum Analysis; Measures; Mediator of activation protein; Methods; Molecular; Morphology; Nature; Necrosis; Numbers; Pathologic; Pathway interactions; Peptide Initiation Factors; Phosphorylation; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Process; Protein Binding; Protein Kinase; Regulation; Resolution; Role; Signal Transduction; Site; Stimulus; TNF gene; TNFSF10 gene; Therapeutic; Therapeutic Intervention; Tissues; Traumatic Brain Injury; Tumor Necrosis Factor Ligand Superfamily Member 6; base; cellular targeting; human RIPK1 protein; in vivo; inhibitor/antagonist; insight; novel; novel therapeutics; prevent; programs; protein activation; protein protein interaction; receptor; research study; retinal ischemia; small molecule; tool; tool development

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.

描述（由申请人提供）： 大量的证据表明，两个“经典”的细胞死亡途径，凋亡和坏死，不包括各种生理和病理细胞死亡机制。我们的实验室和其他实验室已经确定了一种常见的第三种途径的存在，称为“程序性坏死”或“坏死性凋亡”。“坏死性凋亡是一种受调节的细胞死亡途径，具有坏死的表型特征。它在被诱导进行凋亡的细胞中被激活，但阻止其完成。我们最近开发了一种有效的和选择性的小分子抑制剂坏死凋亡，Necrostatin-1，并使用这种分子已经证明了在体外和体内的病理性细胞死亡的各种范例的坏死凋亡的重要作用。坏死性凋亡的发现为开发特异性靶向病理性细胞死亡的坏死组分的新疗法提供了独特的机会，由于坏死是一种不受调节的死亡形式的概念，这在以前是不被追求的。然而，目前对坏死性凋亡的激活和执行的具体机制知之甚少。Ser/Thr激酶RIP已成为坏死性凋亡的关键上游激活因子。此外，我们最近确定RIP激酶活性是Necrostatin-1和我们实验室开发的其他几种结构无关的强效necrostatin的特异性细胞靶点，突出了RIP激酶在坏死性凋亡中的关键作用。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激酶激活和坏死性凋亡诱导。我们对RIP激酶进行了初步的基于质谱的表征，从而确定了一些新的和特异性的翻译后修饰（磷酸化）事件，这些事件可能参与RIP坏死性凋亡活性的调节。我们还证明了使用质谱分析评估内源性RIP相互作用组组成的动态变化的可行性。我们目前的建议集中在RIP激酶诱导坏死性凋亡的机制的进一步研究。该项目的具体目标包括：证实我们先前发现的RIP磷酸化变化在坏死性凋亡激活中的作用;使用磷酸化特异性RIP抗体和RIP激酶测定法在体外和体内表征RIP激酶激活过程;使用高分辨率质谱法解剖RIP相互作用组，随后功能表征RIP相互作用因子在坏死性凋亡起始中的作用;并建立了通过坏死抑制素抑制坏死性凋亡作为针对急性病理性坏死的细胞保护疗法的新方向的可行性。总体而言，我们的研究将提供重要的新的见解，通过阐明诱导的关键上游步骤，在坏死性凋亡，RIP激酶激活的分子基础，调节坏死性凋亡，并将验证一个潜在的新方向，通过选择性靶向坏死性凋亡特异性起始因子的治疗抑制病理性坏死。坏死性凋亡的发现不仅为研究其基本的生物学调控机制提供了新的视角，而且为各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凋亡的分子机制，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。