Ditzel Mark, Broemer Meike, Tenev Tencho, Bolduc Clare, Lee Tom V, Rigbolt Kristoffer T G, Elliott Richard, Zvelebil Marketa, Blagoev Blagoy, Bergmann Andreas, Meier Pascal
The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, UK.
Mol Cell. 2008 Nov 21;32(4):540-53. doi: 10.1016/j.molcel.2008.09.025.
Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 "activation" and nondegradative polyubiquitylation.
泛素介导的半胱天冬酶失活长期以来一直被认为有助于细胞凋亡的调控。然而，半胱天冬酶泛素化的详细机制和功能后果尚未得到证实。在这里，我们表明果蝇凋亡抑制因子1（DIAP1）通过将效应半胱天冬酶靶向多聚泛素化和非蛋白酶体失活来阻断它们。我们证明泛素与drICE的结合抑制了其切割半胱天冬酶底物的催化潜力。我们的数据表明，泛素结合在空间上干扰底物进入并降低半胱天冬酶的蛋白水解速度。通过DIAP1的E3活性突变或drICE的泛素受体赖氨酸突变破坏drICE的泛素化，消除了DIAP1在体内中和drICE并抑制细胞凋亡的能力。我们还表明，DIAP1处于一种“无活性”构象，需要半胱天冬酶介导的切割才能随后对半胱天冬酶进行泛素化。综上所述，我们的研究结果表明，效应半胱天冬酶通过一种涉及DIAP1“激活”和非降解性多聚泛素化的负反馈机制来调节自身的抑制作用。
