Proteolytic processing mediated activation of orthocaspases accelerates stress induced regulated cell death in...

The relationship between orthocaspases and regulated cell death in cyanobacteria has been predicted, but their functional significance remains poorly defined. Here, we characterized two orthocaspases of Anabaena PCC 7120, AnaOC2 and AnaOC6, focusing on their activation and catalytic function. These orthocaspases were calcium-independent cysteine endopeptidase having substrate specificity for basic amino acid residues. AnaOC2 and AnaOC6 exhibited autoprocessing upon cleavage after Arg¹⁶⁵ and Arg¹⁶², respectively, within the linker between the catalytic p20 and p10 regions. This cleavage was critical for activation as their mutation ceased the proteolytic activity. Besides, AnaOC2 transactivated AnaOC6, resulting in 15-30-fold increment in catalytic efficacy (k_cat/K_m) and turnover (k_cat) for arginine-containing substrates without changing substrate affinity (K_m). This suggests conformational changes favouring a more efficient catalytic state, indicating possibilities of hierarchical activation, like caspases. Pull down assay revealed diverse interactome for AnaOC2 and AnaOC6, subsuming proteins from different metabolic pathways. Functional analysis of AnaOC2 and AnaOC6 using Synechococcus overexpression lines revealed their stress-induced activation. Under salt stress, orthocaspase attenuated antioxidative enzymes activity, disrupting redox homeostasis, causing oxidative DNA fragmentation and membrane damage, thereby accelerating cell death. Overall, this study elucidates the enzymatic properties of orthocaspases and highlights their physiological role in cyanobacterial stress response.