Apoptotic Death in T Lymphocytes

该项目旨在评估caspase在T细胞死亡中的作用，特别是确定其裂解引起细胞死亡的细胞质caspase底物。 我们已经发现，在短期培养的人Jurkat T细胞系的凋亡细胞和小鼠胸腺细胞中，内吞率的半胱天冬酶依赖性增加。 在每种情况下，这些增加是由各种不同的细胞凋亡刺激触发的，并且可以被肽-FMK半胱天冬酶抑制剂特异性阻断，这使我们得出结论，增加的内吞作用是细胞凋亡的一个新的和以前未知的方面。 解释这种增加的分子假说是半胱天冬酶可以切割通常抑制膜融合的调节蛋白。 一种这样的蛋白质是突触融合蛋白结合蛋白munc-18，其序列显示了半胱天冬酶6的两个潜在识别位点。 体外翻译的重组munc-18被重组半胱天冬酶6消化成两个预测的片段，表明在两个位点都发生了切割。 这种munc-18切割被特异性半胱天冬酶6抑制剂VEID-CHO阻断。 使用抗munc-18单克隆抗体检测这种蛋白质在细胞提取物中的Western印迹，我们发现，一个munc-18片段产生的caspase-6出现在抗Fas处理的Jurkat细胞和地塞米松处理的胸腺细胞。 由于已知munc-18抑制胞吐作用，我们检测了凋亡Jurkat细胞中125 I标记转移的胞吐率。 我们发现抗Fas处理诱导胞吐速率增加约4倍，如通过munc-18切割模型所预测的。 为了明确测试是否munc-18切割导致细胞凋亡增加的囊泡运输，我们目前正在评估这在细胞中过表达munc-18突变的切割sites.We还研究了半胱天冬酶激活在完整的凋亡细胞中使用一类新的细胞可渗透的荧光半胱天冬酶底物。 这些取代的肽被合成为含有对半胱天冬酶1、3/7、6、8和9的最佳四肽识别基序。 通过流式细胞术检查胸腺细胞显示，对于每种底物，在地塞米松添加后2-3小时开始出现离散的明亮荧光细胞群，并且这些明亮细胞的比例在接下来的5小时内增加。 半胱天冬酶活性增加的顺序是LEHDase、WEHDase、VEIDase、IETDase和DEVDase，这在很大程度上对应于从凋亡提取物研究中预测的半胱天冬酶级联反应。 当用抗Fas抗体处理胸腺细胞时，caspase活性的顺序明显不同，IETDase首先出现，DEVDase在VEIDase之前出现。因此，荧光胞内caspase底物允许直接观察凋亡细胞中的caspase级联反应，并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.