Apoptotic Death in T Lymphocytes

T 淋巴细胞凋亡

• 批准号: 6433143
• 负责人: Pierre A Henkart
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433143
• 关键词: T cell receptor; T lymphocyte; apoptosis; biological signal transduction; calpain; cytokine; cytotoxic T lymphocyte; endopeptidases; flow cytometry; helper T lymphocyte; human tissue; hybridomas; immunoregulation; leukocyte activation /transformation; oxidative stress; protease inhibitor

This project aims to assess the role of caspases in T cell death generally, and in particular to identify the cytoplasmic caspase substrates whose cleavage gives rise to cell death. We have found caspase-dependent increases in the rate endocytosis in apoptotic cells of the human Jurkat T cell line and in mouse thymocytes in short term culture. These increases were triggered by a variety of different apoptotic stimuli in each case, and were specifically blockable by peptide-FMK caspase inhibitors, leading us to conclude that increased endocytosis is a new and previously unknown aspect of apoptosis. A molecular hypothesis to explain this increase is that caspases may cleave a regulatory protein normally restraining membrane fusion. One such protein is the syntaxin-binding protein munc-18, whose sequence shows two potential recognition sites for caspase 6. In vitro translated recombinant munc-18 was shown to be digested by recombinant caspase 6 into two predicted fragments indicating cleavage occurred at both sites. This munc-18 cleavage was blocked by the specific caspase 6 inhibitor VEID-CHO. Using an anti-munc-18 Mab to detect this protein in cell extracts by Western blotting, we found that one of the munc-18 fragments produced by caspase-6 appeared in anti-Fas-treated Jurkat cells and dexamethasone-treated thymocytes. Since munc-18 is known to inhibit exocytosis, we examined the rate of exocytosis of 125I-labeled transferring in apoptotic Jurkat cells. We found that anti-Fas treatment induced a ~4x increase in the rate of exocytosis, as predicted by the munc-18 cleavage model. To definitively test whether munc-18 cleavage causes apoptotic increases in vesicle trafficking, we are currently assessing this in cells overexpressing munc-18 mutated at the cleavage sites.We have also studied caspase activation in intact apoptotic cells using a novel class of cell-permeable fluorogenic caspase substrates. These substituted peptides were synthesized to contain the optimal tetrapeptide recognition motifs for caspases 1,3/7,6,8 and 9. Examination of thymocytes by flow cytometry showed that for each substrate a discrete brightly fluorescent cell population appeared starting 2-3 hours after dexamethasone addition, and the proportion of these bright cells increased over the next 5 hours. The sequential order of increase of caspase activities was LEHDase, WEHDase, VEIDase, IETDase and DEVDase, largely corresponding to the caspase cascade predicted from studies on apoptotic extracts. When thymocytes were treated with anti-Fas antibody, the order of caspase activities was clearly different, with IETDase appearing first, and DEVDase preceding VEIDase, Thus fluorogenic intracellullar caspase substrates allow direct observations of the caspase cascade in apoptotic cells and complement traditional biochemical techniques.

本项目旨在评估半胱天冬酶在T细胞死亡中的作用，特别是鉴定其裂解导致细胞死亡的细胞质半胱天冬酶底物。我们发现caspase依赖性地增加了人Jurkat T细胞系和小鼠胸腺细胞短期培养中凋亡细胞的内吞率。在每种情况下，这些增加是由各种不同的凋亡刺激触发的，并且可以被肽- fmk caspase抑制剂特异性阻断，这使我们得出结论，内吞作用的增加是凋亡的一个新的和以前未知的方面。一种分子假说可以解释这种增加，即半胱天冬酶可以切割一种通常抑制膜融合的调节蛋白。其中一种蛋白质是syntaxin结合蛋白munc-18，其序列显示了caspase 6的两个潜在识别位点。体外翻译的重组munc-18被重组caspase 6消化成两个预测片段，表明两个位点都发生了裂解。这种munc-18的裂解被特异性caspase 6抑制剂VEID-CHO阻断。使用抗munc-18单抗，通过Western blotting检测细胞提取物中的该蛋白，我们发现caspase-6产生的munc-18片段之一出现在抗fas处理的Jurkat细胞和地塞米松处理的胸腺细胞中。由于已知munc-18抑制胞吐，我们检测了凋亡Jurkat细胞中125i标记转移的胞吐率。我们发现抗fas处理诱导胞吐率增加了4倍，正如munc-18切割模型所预测的那样。为了明确测试munc-18切割是否会导致囊泡运输中凋亡的增加，我们目前正在对在切割位点突变的过表达munc-18的细胞进行评估。我们还研究了caspase在完整凋亡细胞中的激活，使用了一类新的细胞渗透性荧光caspase底物。这些取代肽被合成为含有半胱天冬酶1、3/7、6、8和9的最佳四肽识别基序。流式细胞术检测胸腺细胞显示，在加入地塞米松后2-3小时，每种底物都出现离散的明亮荧光细胞群，在接下来的5小时内，这些明亮细胞的比例增加。caspase活性升高的顺序为lehase、wehase、VEIDase、ietase和DEVDase，与凋亡提取物研究预测的caspase级联反应基本一致。当胸腺细胞被抗fas抗体处理时，caspase的活性顺序明显不同，首先出现的是ietase，而DEVDase在VEIDase之前，因此荧光性的细胞内caspase底物可以直接观察凋亡细胞中caspase级联反应，补充了传统的生化技术。