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（由申请人提供）：本申请的目的是确定单个胱天蛋白酶家族成员在T细胞活化和增殖中的特定作用。我们将 使用新的半胱天冬酶特异性细胞渗透性抑制剂和基于活性的探针，我们最近设计和表征，探测半胱天冬酶功能。半胱天冬酶是一个半胱氨酸-天冬酰蛋白酶家族，其对于细胞凋亡、焦亡和炎症是必需的。人类有11种半胱氨酸蛋白酶亚型，其中起始半胱氨酸蛋白酶-2、-8、-9和-10以及执行半胱氨酸蛋白酶-3、-6和-7分别负责诱导细胞凋亡和细胞解体。令人兴奋的新数据表明，这些阿尔茨海默病相关的半胱天冬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经典功能。然而，关于不同半胱天冬酶对这些生物现象的作用和贡献的细节却很少。缺乏机制的见解，主要是由于密切的结构同源性和重叠的底物特异性之间的胱天蛋白酶家族成员。另一个限制是目前可用的基于半胱天冬酶肽的抑制剂、底物和探针的文库的高度混杂性。因此，目前的一组半胱天冬酶靶向分子不能提供足够的分辨率，以精确地分配在复杂的细胞环境中的单个半胱天冬酶的生物学作用。我们最近开发了一种独特的方法，将非天然氨基酸掺入到典型的半胱天冬酶识别肽中，并优化了化学离去基团的反应性，以确保我们的肽基分子特异性靶向单个半胱天冬酶。我们已经积累了足够的初步数据，以确定一流的caspase-3和caspase-8特异性肽探针，从而分别针对关键的执行者和启动器caspase。在这里，我们将应用我们设计的分子的细胞可渗透版本来研究半胱天冬酶-3和-8在人类T细胞活化中的非经典功能。在细胞凋亡过程中，半胱天冬酶切割参与转录和翻译的核蛋白，我们假设这些底物的一个子集在T细胞扩增过程中被半胱天冬酶-3和/或-8选择性靶向。因此，半胱天冬酶-3和半胱天冬酶-8的蛋白水解活性将显著影响决定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半胱天冬酶-3和-8的细胞底物，以及RNA-Seq转录组学来阐明下游翻译和转录对T细胞重编程的影响。我们的努力将产生一套普遍适用的工具，可以扩展到探测单个半胱天冬酶在其他生物过程中的功能，包括干细胞分化，红细胞生成和细胞迁移，以及产生一个范式转变半胱天冬酶如何控制非凋亡的生物过程中的健康和疾病。