Modeling the role of cyclin C in connecting stress‐induced mitochondrial fission to apoptosis

For normal cell function, exogenous signals must be correctly interpreted, and the proper response executed. The mitochondria are key regulatory nodes of cellular fate. For example, mitochondria undergo fission and fusion cycles depending on the energetic needs of the cell. Additionally, regulated cell death pathways also function at the mitochondria. Cyclin C is a transcriptional regulator of stress response and growth control genes. Following stress, a portion of cyclin C translocates to the cytoplasm, where it interacts with both the mitochondrial fission and apoptotic machinery. Based on these findings, we hypothesize that cyclin C represents a key mediator linking transcription to mitochondrial fission and intrinsic regulated cell death. Members of the cyclin protein family typically contain two conserved cyclin box domains each composed of five alpha-helices. The N-terminal cyclin box domain, CB1, is responsible for binding cyclin-dependent kinases (Cdks), while the C-terminal cyclin box, CB2, typically facilitates correct protein folding. For cyclin C, CB1 and CB2 mediate protein-protein interactions with regards to transcriptional regulation (Cdk8) and mitochondrial fission (Drp1), respectively. Pull down assays show that pro-apoptotic protein Bax interacts in a direct manner with cyclin C and this interaction is required for Bax activation and ultimately efficient intrinsic regulated cell death. Using the docking simulator ClusPro, cyclin C and Bax yielded consistent protein-protein interaction sites within amino acids 160-170 of cyclin C. Important residues in order of decreasing frequency include: Q161, D165, E169, Y162, Q164, and D170. Six sets of docking simulations yielded identical results, using multiple cyclin C and Bax protein data bank file combinations as a means of accounting for potential differences in the starting .pdb files. Inspection of this region in cyclin C shows a high degree of homology to the BH2 domain found in members of the Bcl-2 protein family, such as Bax, Bak, and Bcl2. These data suggest that cyclin C possesses a BH2 domain directing Bax interaction and intrinsic regulated cell death. Interestingly, CB2 is also responsible for binding the fission protein Drp1. However preliminary modeling data suggest that this interaction is mediated by the latter half of CB2. Prior work also has demonstrated that while the BH2 domain is required for Bax binding, it is not required for interaction with Drp1. Co-immunoprecipitation assays show Drp1 is required for cyclin C-Bax interaction, suggesting a role for the interaction of all three proteins. Taken together, these results suggest a model that cyclin C physically bridges the fission and apoptotic machinery allowing the cell to coordinate mitochondrial dynamics with programmed cell death pathways.