The death inhibitory molecules CED-9 and CED-4L use a common mechanism to inhibit the CED-3 death protease.
The apoptotic machinery of Caenorhabditis elegans includes three core interacting components: CED-3, CED-4, and CED-9. CED-3 is a death protease composed of a prodomain and a protease domain. CED-4 is a P-loop-containing, nucleotide-binding molecule that complexes with the single polypeptide zymogen form of CED-3, promoting its activation by autoprocessing. CED-9 blocks death by complexing with CED-4 and suppressing its ability to promote CED-3 activation. A naturally occurring alternatively spliced form of CED-4 that contains an insertion within the nucleotide-binding region (CED-4L) functions as a dominant negative inhibitor of CED-3 processing and attenuates cell death. Domain mapping studies revealed that distinct regions within CED-4 bind to the CED-3 prodomain and protease domain. Importantly, the CED-4 P-loop was involved in prodomain binding. Disruption of P-loop geometry because of mutation of a critical lysine (K165R) or insertional inactivation (CED-4L) abolished prodomain binding. Regardless, K165R and CED-4L still retained CED-3 binding through the protease domain but were unable to initiate CED-3 processing. Therefore, the P-loop-prodomain interaction is critical for triggering CED-4-mediated CED-3 processing. Underscoring the importance of this interaction was the finding that CED-9 contacted the P-loop and selectively inhibited its interaction with the CED-3 prodomain. These results provide a simple mechanism for how CED-9 functions to block CED-4-mediated CED-3 processing and cell death.
Chaudhary
D
D
Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
O'Rourke
K
K
Chinnaiyan
A M
AM
Dixit
V M
VM
D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology*; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D015801:Helminth Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D017384:Sequence Deletion
D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; C089224:Ced-4 protein, C elegans; C087233:Ced-9 protein, C elegans; D015853:Cysteine Proteinase Inhibitors; D015801:Helminth Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D020169:Caspases; D003546:Cysteine Endopeptidases; C089223:ced-3 protein, C elegans