Experimental and Computational Insights into Apc2 Independent Apc10-Apc11a Interaction and Oxidative Stress Response in the Anaphase Promoting Complex of Entamoeba histolytica.

PURPOSE: In eukaryotes, anaphase promoting complex (APC) functions as a multi-protein ubiquitin ligase that governs chromosome segregation. The dynamic interactions of its subunits are significant for APC’s functionality as these interactions are typically transient and changes with cell progression through different stages of the cell cycle. E. histolytica possesses seven homologs of six distinct APC subunits; however, Apc2 is notably absent. This raises intriguing questions about structural organization and functional mechanisms of E. histolytica APC. Given the essential role of the Apc2-Apc10-Apc11 interaction in APC functionality in eukaryote, we sought to investigate whether Apc10 and Apc11a can interact independently of Apc2 in E. histolytica. METHODS: Yeast two-hybrid (Y2H) assay, in vitro binding assay, molecular docking, simulation and mutation analysis were employed to study the interaction between EhApc10 and EhApc11a. Given the redox-sensitive nature of the RING domain of Apc11, we investigated how oxidative stress influences the gene expression of EhApc11a and EhApc10 through qRT-PCR. RESULTS: EhApc10 and EhApc11a were shown to interact independently of Apc2 in Y2H, in vitro binding assay and computational analysis. Under oxidative stress (H2O2), both EhApc11a and EhApc10 showed reduced expression, indicating downregulation during stress. After removal of oxidative stress, both genes were upregulated, indicating recovery. CONCLUSION: E. histolytica APC may employ a unique structural organization, where EhApc10 and EhApc11a interact without Apc2. The reduced expression of both genes under H2O2 suggests a cellular strategic shift to prioritize repair and survival over anaphase progression. Their post-stress upregulation suggests a compensatory mechanism that restores normal cell cycle dynamics.