An inhibitory mono-ubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways

Apoptosis is an evolutionary conserved cell death mechanism, which requires activation of initiator and effector caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian Caspase-2 and Caspase-9, has an N-terminal CARD domain that recruits Dronc into the apoptosome for activation. In addition to its role in apoptosis, Dronc also has non-apoptotic functions such as compensatory proliferation. One mechanism to control the activation of Dronc is ubiquitylation. However, the mechanistic details of ubiquitylation of Dronc are less clear. For example, monomeric inactive Dronc is subject to non-degradative ubiquitylation in living cells, while ubiquitylation of active apoptosome-bound Dronc triggers its proteolytic degradation in apoptotic cells. Here, we examined the role of non-degradative ubiquitylation of Dronc in living cells in vivo, i.e. in the context of a multi-cellular organism. Our in vivo data suggest that in living cells Dronc is mono-ubiquitylated on Lys78 (K78) in its CARD domain. This ubiquitylation prevents activation of Dronc in the apoptosome and protects cells from apoptosis. Furthermore, K78 ubiquitylation plays an inhibitory role for non-apoptotic functions of Dronc. We provide evidence that not all of the non-apoptotic functions of Dronc require its catalytic activity. In conclusion, we demonstrate a mechanism whereby Dronc’s apoptotic and non-apoptotic activities can be kept silenced in a non-degradative manner through a single ubiquitylation event in living cells.

细胞凋亡（Apoptosis）是一种进化保守的细胞死亡机制，其激活依赖于起始胱天蛋白酶与效应胱天蛋白酶的活化。果蝇的起始胱天蛋白酶Dronc是哺乳动物胱天蛋白酶-2（Caspase-2）与胱天蛋白酶-9（Caspase-9）的同源物，其N端带有半胱天冬酶募集结构域（CARD domain），可将Dronc招募至凋亡小体（apoptosome）中完成激活。除介导细胞凋亡外，Dronc还具备非凋亡相关功能，例如代偿性增殖。泛素化（ubiquitylation）是调控Dronc激活的重要机制之一，但目前关于Dronc泛素化的具体分子机制仍不甚明晰。例如，活细胞内以单体形式存在的无活性Dronc会发生非降解型泛素化；而结合于凋亡小体的活性Dronc经泛素化修饰后，会触发其在凋亡细胞中的蛋白水解降解。本研究针对活细胞体内（in vivo，即多细胞生物体的环境中）中Dronc的非降解型泛素化作用展开了探究。我们的体内实验数据表明，在活细胞中，Dronc的半胱天冬酶募集结构域（CARD domain）上的赖氨酸78位（Lys78，简称K78）会发生单泛素化修饰。该修饰可阻止Dronc在凋亡小体中被激活，进而使细胞免于凋亡。此外，赖氨酸78位的泛素化修饰还对Dronc的非凋亡相关功能发挥抑制作用。我们的研究证据显示，并非Dronc的所有非凋亡相关功能都依赖其催化活性。综上，本研究证实了一种调控机制：在活细胞内，仅需一次单泛素化修饰事件，即可通过非降解途径同时沉默Dronc的凋亡相关与非凋亡相关活性。