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: 8118275
• 负责人: ALEXEI DEGTEREV
• 金额: $ 36.8万
• 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/8118275
• 关键词: Acute; Address; Animal Model; Apoptosis; Apoptotic; Biological; Biological Assay; Brain Ischemia; Caspase; Cell Culture Techniques; Cell Death; Cell Death Process; Cells; Cessation of life; Characteristics; Collaborations; Complex; Data; Death Domain; Detection; Development; Disease; Dissection; Docking; Event; Fc Receptor; Goals; In Vitro; Injury; Ischemic Brain Injury; Knowledge; Laboratories; Ligands; Liver; Mass Spectrum Analysis; Measures; Mediator of activation protein; Methods; Molecular; Morphology; Myocardial Ischemia; Nature; Necrosis; 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）： 大量证据表明，两种“经典”细胞死亡途径，细胞凋亡和坏死，不包括各种生理和病理细胞死亡机制。我们和其他实验室已经建立了一种常见的第三个途径，称为“编程坏死”或“坏死”。坏死性是一种受调节的细胞死亡途径，具有坏死的表型特征。它在被诱导的细胞中被激活，但可阻止其完成。我们最近开发了一种有效的选择性小分子抑制坏死蛋白1，并且使用该分子证明了坏死性在体外和体内病理细胞死亡的各种范式中的重要作用。坏死性的发现提供了独特的机会来开发新的疗法，专门针对病理细胞死亡的坏死成分，由于死亡是一种不受管制的死亡形式，以前没有追求这种疗法。然而，目前知之甚少，关于坏死性激活和执行的特定机制知之甚少。 Ser/Thr激酶RIP已成为坏死凋亡的关键上游激活剂。此外，我们最近确定RIP激酶活性是坏死蛋白1的特定细胞靶标，以及其他几种在我们的实验室中也开发的有效的有效坏死蛋白，突出了RIP激酶在坏死性中的关键作用。在我们的初步研究中，我们开发了新的测定方法，以特别测量RIP激酶激活和坏死诱导。我们对RIP激酶进行了初步的质谱表征，这导致了许多新型和特定的翻译后修饰（磷酸化）事件，这些事件可能与RIP的坏死活性有关。我们还证明了使用质谱分析评估内源性RIP Interactome组成的动态变化的可行性。我们目前的建议集中于RIP激酶对坏死诱导的机理的进一步研究。该项目的具体目的包括确认我们先前确定的RIP磷酸化变化的作用，在激活坏死性中。使用磷酸特异性RIP抗体和RIP激酶测定法中RIP激酶激活过程的体外和体内表征；使用高分辨率的质谱法解剖RIP相互作用，然后是RIP相互作用因子在坏死性开始中的作用的功能表征。并确定坏死蛋白抑制坏死性抑制作用，是针对急性病理坏死的细胞保护疗法的新方向。总体而言，我们的研究将通过阐明诱导致命性上游促进，RIP激酶激活的关键上游步骤的分子基础的分子基础来提供对坏死性调节的重要新见解，并将通过选择性抑制致病性坏死的治疗性抑制，通过选择性靶向致命性促毒性剂量，并验证潜在的新方向。坏死性的发现不仅提供了对基本生物学调节的新见解，而且还为针对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的分子机制，这将使我们能够充分利用这一激动人心的新疗法发现。