Non-canonical roles of the apoptotic caspases in T-cell activation

凋亡 caspase 在 T 细胞激活中的非典型作用

• 批准号: 8889850
• 负责人: Dennis William Wolan
• 金额: $ 23.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8889850
• 关键词: Amino Acids; Apoptosis; Apoptotic; Aspartic Endopeptidases; Behavior; Biological; Biological Assay; Biological Phenomena; Biological Process; Caspase; Cell physiology; Cells; Cellular biology; Chemicals; Cleaved cell; Communities; Complex; Cysteine; DNA-Directed RNA Polymerase; Data; Disease; Ensure; Environment; Enzyme-Linked Immunosorbent Assay; Equipment and supply inventories; Erythropoiesis; Event; Family; Family member; Fluorescent Probes; Genetic Transcription; Goals; Health; Human; Immune; Individual; Induction of Apoptosis; Inflammation; Kinetics; Laboratories; Left; Libraries; Light; Location; Lymphoproliferative Disorders; Maps; Mass Spectrum Analysis; Measures; Memory; Methods; Neurons; Nuclear; Nuclear Proteins; Pathway interactions; Peptide Hydrolases; Peptides; Procedures; Process; Production; Protein Biosynthesis; Protein Isoforms; Proteins; Proteomics; RNA Interference; RNA Splicing; Resolution; Ribonucleoproteins; Role; Specific qualifier value; Substrate Specificity; Synaptic plasticity; T-Cell Activation; T-Cell Proliferation; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Translations; Validation; base; caspase-2; caspase-3; caspase-8; cell motility; cellular targeting; cytokine; design; helicase; inhibitor/antagonist; insight; leukemia/lymphoma; mRNA Expression; member; novel; public health relevance; stem cell differentiation; tool; transcription factor; transcriptome sequencing; transcriptomics

 DESCRIPTION (provided by applicant): The goal of this application is to determine the specific roles of individual caspase family members in T-cell activation and proliferation. We will use novel caspase-specific cell-permeable inhibitors and activity-based probes, which we recently designed and characterized, to probe caspase function. Caspases are a family of cysteine-aspartyl proteases that are essential for apoptosis, pyroptosis, and inflammation. Humans have 11 caspase isoforms where initiator caspases-2, -8, -9 and -10 and executioner caspases-3, -6, and -7 are responsible for the induction of apoptosis and cellular dismantling, respectively. Exciting new data suggest that several of these apoptosis-associated caspases have unexpected non-canonical functions in cellular differentiation and proliferation, neuronal pruning, and synaptic plasticity. However, very few details about the roles and contributions of distinct caspases to these biological phenomena have been forthcoming. The lack of mechanistic insights is primarily due to the close structural homology and overlapping substrate specificity among the caspase family members. Another limitation is the high promiscuity of the currently available library of caspase peptide-based inhibitors, substrates, and probes. Therefore, the current set of caspase-targeted molecules does not provide adequate resolution to precisely assign biological roles to individual caspases in a complex cellular environment. We recently developed a unique approach that incorporates unnatural amino acids into the canonical caspase-recognition peptides and optimized the reactivity of chemical leaving groups to ensure our peptide-based molecules are specifically targeted to individual caspases. We have amassed sufficient preliminary data to identify first-in-class caspase-3 and caspase-8 specific peptide probes, thereby targeting a critical executioner and an initiator caspase, respectively. Here, we will apply cell-permeable versions of our designed molecules to investigate the non-canonical functions of caspases-3 and -8 in human T-cell activation. During apoptosis, caspases cleave nuclear proteins involved in transcription and translation and we hypothesize that a subset of these substrates are selectively targeted by caspases-3 and/or -8 during T-cell expansion. Therefore, the proteolytic activity of caspases-3 and -8 will significantl impact the transcription and translation of many key cellular proteins that determine T-cell function. We propose to use mass spectrometry-based proteomics to elucidate the cellular substrates of caspases-3 and -8 and RNA-Seq transcriptomics to shed light on downstream translational and transcriptional consequences on T-cell reprogramming. Our efforts will generate a universally applicable set of tools that can be extended to probe the functions of individual caspases during other biological processes, including stem cell differentiation, erythropoiesis, and cell migration as well as produce a paradigm shift in how caspases control non-apoptotic biological processes in health and disease.

 描述（由应用程序提供）：本应用程序的目的是确定单个caspase家族成员在T细胞激活和增殖中的特定作用。我们将 使用我们最近设计和表征的新型caspase特异性细胞抑制剂和基于活性的问题，以探测caspase功能。胱天蛋白酶是半胱氨酸 - 天冬氨酸蛋白酶的家族，对于细胞凋亡，凋亡和注射至关重要。人类具有11个caspase同工型，其中启动器caspase -2，-8，-9和-10和executioner caspase -3，-6和-7分别负责诱导细胞凋亡和细胞拆卸。令人兴奋的新数据表明，这些与凋亡相关的胱天蛋白酶中的几个在细胞分化和增殖，神经元修剪和突触可塑性中具有意想不到的非规范功能。但是，关于这些生物学现象的不同胱天蛋白酶的作用和贡献的细节很少。缺乏机械洞察的主要原因是caspase家族成员之间的紧密结构同源性和重叠的底物特异性。另一个限制是当前可用的基于caspase的抑制剂，底物和问题的库的高希望。因此，当前的caspase靶向分子集不能为在复杂的细胞环境中精确地分配给单个caspase的生物学作用提供足够的分辨率。我们最近开发了一种独特的方法，该方法将不自然的氨基酸纳入规范的caspase-rake识别辣椒，并优化了化学静止组的反应性，以确保我们的基于肽的分子专门针对单个胱天蛋白酶。我们积累了足够的初步数据，以识别一流的caspase-3和caspase-8特定肽问题，从而分别针对关键的execution子手和启动器caspase。在这里，我们将应用我们设计的分子的细胞渗透版本来研究caspases-3和-8的非典型功能在人类T细胞激活中。在凋亡过程中，胱天蛋白酶清除参与转录和翻译的核蛋白，我们假设这些子材料的一部分在T细胞膨胀过程中由caspases-3和/或-8选择性靶向。因此，caspases-3和-8的蛋白水解活性将显着影响许多确定T细胞功能的许多关键细胞蛋白的转录和翻译。我们建议使用基于质谱的蛋白质组学来阐明caspases-3和-8和RNA-Seq转录组学的细胞底物，以阐明T细胞重编程的下游翻译和转录后果。我们的努力将产生一套普遍适用的工具，可以扩展以探测其他生物学过程中各个caspase的功能，包括干细胞分化，红细胞生成和细胞迁移，以及在健康和疾病中控制非膜质生物学过程的caspase如何在caspase中产生范式转移。