Cleavage of Bcl-xL by Caspases contributes to cell death in vivo

 描述(由申请人提供)：本项目的目标是确定半胱氨酸天冬氨酸氨基转移酶对细胞死亡的作用。细胞底物的半胱氨酸天冬氨酸氨基转移酶裂解是介导细胞凋亡的关键事件。Bcl2家族成员Bclxl是细胞凋亡的抑制因子，也是半胱氨酸天冬氨酸酶的底物。切割Bclxl不仅可以消除其抗死亡活性，而且可以将Bclxl从一个有效的抗凋亡蛋白转变为有效的促死亡分子。N-末端截短的BclxL裂解片段(ΔN-Bclxl)可加速细胞死亡。为了探讨Bclxl的体内切割效应，通过突变Bclxl内的两个caspase裂解位点，获得了caspase不能切割的Bclxl敲入小鼠。有趣的是，抗caspase基因的Bclxl小鼠在老年时仍能保持健康的胸腺，这表明Bclxl的裂解会导致细胞死亡，从而导致胸腺萎缩。我的初步数据表明，这些小鼠可能有缺陷的阳性选择(产生一个可行的T细胞受体)。因此，我们将探讨是否有可能 半胱氨酸天冬氨酸氨基转移酶裂解bc l-xl可杀死未通过阳性选择检查点的发育中的胸腺细胞。通过这样做，我们将为所谓的“疏忽致死”提供机械论的见解，即胸腺细胞无法接收生存线索。由于这些小鼠一生中保持着形态正常的胸腺，我将确定它们是否也保持对流感病毒的正常免疫反应。流感对老年人的影响尤其严重，他们对病毒的T细胞反应很差。这被认为是由于免疫衰老，部分归因于胸腺萎缩。一个长期的假设是，胸腺萎缩导致免疫反应减弱，这是由于T细胞产生减少，但一直缺乏明确测试这一假设的小鼠模型。因此，我们处于一个独特的位置来确定保护胸腺功能是否也能保护老年动物免受流感感染。我们将严格评估胸腺萎缩是否影响控制流感所需的免疫反应。这些研究还将探讨胸腺选择和T细胞在流感病毒免疫中的作用。

 DESCRIPTION (provided by applicant): The goal for this project is to determine how cleavage of Bcl-xL by caspases contributes to cell death in vivo. Caspase cleavage of cellular substrates is a key event in mediating apoptosis. The Bcl-2 family member Bcl-xL is an inhibitor of apoptotic cell death, and can also be a substrate of caspases. Cleavage of Bcl-xL [not only abolishes its anti-death activity] but also convert Bcl-xL from a potent anti-apoptotic protein to potent pro-death molecule. Cells transfected with the N-terminally truncated Bcl-xL cleavage fragment (ΔN-Bcl-xL) display accelerated cell death. To probe the in vivo effects of Bcl-xL cleavage, a caspase-uncleavable Bcl-xL knock-in mouse was generated by mutating the two caspase cleavage sites within Bcl-xL. Interestingly, caspase-resistant Bcl-xL mice retain a healthy thymus in old age suggesting that cleavage of Bcl-xL leads to cell death that results in thymic atrophy. My preliminary data suggest that these mice may have defective positive selection (generation of a viable T-cell receptor). Therefore, we will explore the possibility that caspase-cleavage of Bcl-xL kills developing thymocytes that fail the positive selection checkpoint. By doing so, we will provide mechanistic insights into what is termed "death by neglect", the failure of thymocytes to receive survival cues. Because these mice maintain a morphologically normal thymus throughout life, I will determine if they also maintain normal immune responses to influenza virus. Influenza particularly affects the elderly, who display poor T-cell responses to the virus. This is presumed to be because of immunosenescence, in part attributed to thymic atrophy. A long-standing assumption is that thymic atrophy contributes to waning immune responses due to decreased T-cell production, but mouse models to definitively test this hypothesis have been lacking. Therefore, we are in a unique position to determine if preserved thymic function also confers protection from influenza infection in aged animals. We will critically evaluate whether thymic atrophy compromises the immune responses needed to control influenza. These studies will also address the role of thymic selection and T-cell contributions to influenza virus immunity.