ProDy, an integrated application programming interface developed for modelling and analysing protein dynamics, has significantly evolved in recent years in response to the growing data and needs of the computational biology community. We present major developments that led to ProDy 2.0: (i) improved interfacing with databases and parsing new file formats, (ii) SignDy for signature dynamics of protein families, (iii) CryoDy for collective dynamics of supramolecular systems using cryo-EM density maps and (iv) essential site scanning analysis for identifying sites essential to modulating global dynamics. ProDy is open-source and freely available under MIT License from https://github.com/prody/ProDy. Supplementary data are available at Bioinformatics online.

Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox.

Significance: The NLRP3 inflammasome is a critical component of the innate immune response to infectious agents, but its overactivation is implicated in numerous pathologies, including atherosclerosis, Alzheimer’s Disease, gout, inflammatory bowel disease, and type 2 diabetes. Our study reveals a mechanism by which cells modulate NLRP3 inflammasome activation and identifies potential therapeutic targets to treat pathologies associated with NLRP3 inflammasome activation.

We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO^● suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NO^● interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NO^● suppresses ETE-PE oxidation. Our study reveals that O₂ and NO^● use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O₂ and NO^● to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NO^● donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NO^●, in further support of the ability of NO^● to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NO^●.