Apoptotic Death in T Lymphocytes

本项目旨在评估caspase在T细胞死亡中的作用，特别是确定其裂解导致细胞死亡的细胞质caspase底物。我们发现，在短期培养中，人Jurkat T细胞系和小鼠胸腺细胞的凋亡细胞中caspase依赖的内吞速率增加。在每种情况下，这些增加都是由各种不同的凋亡刺激触发的，并且可以被多肽-FMK caspase抑制剂特异性地阻断，这使得我们得出结论，内吞增加是细胞凋亡的一个新的和以前未知的方面。解释这种增加的一个分子假说是，caspase可能会切割通常抑制膜融合的调节蛋白。其中一个蛋白质是合成素结合蛋白MUNC-18，它的序列显示了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，表明这两个位点都发生了切割。这种MUNC-18裂解被特异性的caspase 6抑制剂VEID-CHO阻断。用抗MUNC-18的单抗通过Western blotting检测细胞提取物中的MUNC-18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可以抑制胞吐作用，我们检测了125I标记转移在凋亡的Jurkat细胞中的胞吐速率。我们发现，正如MUNC-18切割模型所预测的那样，抗Fas处理导致胞吐速度增加约4倍。为了确定MUNC-18裂解是否导致囊泡运输中的凋亡增加，我们目前正在评估在裂解位点突变的高表达MUNC-18的细胞中的这一点。我们还使用一种新型的细胞渗透性荧光半胱氨酸天冬氨酸酶底物研究了完整凋亡细胞中的半胱氨酸天冬氨酸酶的激活。用流式细胞仪检测胸腺细胞发现，每种底物在加入地塞米松后2-3h开始出现一个离散的明亮的荧光细胞群，并在接下来的5h内这些明亮细胞的比例增加。Caspase活性升高的顺序为：LEHDase>WEHDase>VEIDase>IETDase>DEVDase，这在很大程度上与通过对细胞凋亡提取物的研究预测的caspase级联反应相对应。当胸腺细胞用抗Fas抗体处理时，caspase活性的顺序明显不同，首先是IETDase，然后是DEVDase，然后是VEIDase，因此荧光细胞内caspase底物可以直接观察到凋亡细胞中caspase的级联，补充了传统的生化技术。

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.