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天冬酶如何切割Bcl-xL导致体内细胞死亡。细胞底物的半胱天冬酶切割是介导细胞凋亡的关键事件。Bcl-2家族成员Bcl-xL是凋亡性细胞死亡的抑制剂，也可以是半胱天冬酶的底物。Bcl-xL的切割[不仅消除了其抗死亡活性]，而且还将Bcl-xL从有效的抗凋亡蛋白转化为有效的促死亡分子。用N末端截短的Bcl-xL切割片段（ΔN-Bcl-xL）转染的细胞显示加速的细胞死亡。为了探测Bcl-xL切割的体内作用，通过突变Bcl-xL内的两个半胱天冬酶切割位点产生半胱天冬酶不可切割的Bcl-xL敲入小鼠。有趣的是，半胱天冬酶抗性Bcl-xL小鼠在老年时保留了健康的胸腺，这表明Bcl-xL的裂解导致细胞死亡，导致胸腺萎缩。我的初步数据表明，这些小鼠可能有缺陷的正选择（产生一个可行的T细胞受体）。因此，我们将探讨 Bcl-xL的半胱天冬酶裂解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，我们将提供机械的见解，什么是所谓的“忽视死亡”，失败的胸腺细胞接受生存的线索。因为这些小鼠在一生中保持着形态正常的胸腺，我将确定它们是否也保持着对流感病毒的正常免疫反应。流感特别影响老年人，他们对病毒的T细胞反应很差。这被认为是因为免疫衰老，部分归因于胸腺萎缩。一个长期存在的假设是，胸腺萎缩有助于减弱免疫反应，由于减少T细胞的生产，但小鼠模型，以明确测试这一假设一直缺乏。因此，我们处于一个独特的位置，以确定是否保留胸腺功能也赋予保护免受流感感染的老年动物。我们将严格评估胸腺萎缩是否会损害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细胞对流感病毒免疫的作用。

 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.