Karolina Mikulska-Ruminska

Ether (alkyl/alkenyl) phospholipids, particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC), are broadly represented in membranes, but their physiological functions are poorly characterized. The antioxidant role of plasmalogens realized via oxidation of sn -1 vinyl bond has been associated with anti-ferroptotic regulatory function. Alternatively, peroxidation of polyunsaturated fatty acid (PUFA) in sn -2-position of alkenyl-PEs can be pro-ferroptotic. Since 15-LOXs generate 15-HpETE-PEs as ferroptotic signals, we explored alkyl/alkenyl-ETE-PE as substrates of enzymatic peroxidation. Using redox lipidomics, biochemical, biophysical, genetic approaches, and molecular dynamics simulations, we established that both isoforms of 15-LOX (15-LOX-1 and 15-LOX-2) selectively oxidize alkyl/alkenyl-ETE-PE (but not alkyl/alkenyl-ETE-PC), forming 15-HpETE-PEs, triggering ferroptotic death, independently of the vinyl bond. We showed that LOX-catalyzed peroxidation rate of sn -1 vinyl bond is ~500-fold lower than sn -2-ETE-PE, thus excluding the antioxidant role of plasmalogens in ferroptosis. We showed 15-LOX-driven production of sn -1-alkenyl- sn -2-15-HpETE-PE acts as pathogenic factor in acute/chronic diseases: asthma, cancer, brain trauma, skin UVB-injury. Thus, 15-LOX-catalyzed bias towards oxidation of alkenyl-ETE-PE may represent a new therapeutic target.

The transmembrane alkane monooxygenase AlkB and rubredoxin AlkG form an electron transfer complex that hydroxylates terminal alkanes to produce alcohols. The recent cryoEM study of Fontimonas thermophila AlkB-AlkG complex (FtAlkBG) revealed its architecture, including a dodecane (D12) substrate in the diiron active site. However, the molecular mechanism of action of FtAlkBG remains unknown. Here, we examined the FtAlkBG dynamics and interactions by multiscale computations, including molecular dynamics (MD) simulations, elastic network model (ENM), and QM/MM study of the oxidative mechanism at the catalytic site of AlkB. D12 remained stably bound to the catalytic site during MD runs, coordinated by hydrophobic residues I267, L263, L264, and I133, critical to substrate stabilization. MD simulations further showed that D12 could exit the catalytic site via a well-defined translocation pathway gated by I54, S49, and F46, through two intermediate states before nearly leaving the protein, and diffuse back to the active site. Several salt bridges and conserved AlkB-AlkG contacts were enhanced upon substrate binding. Substrate binding also mediated the association/dissociation events required for efficient electron transfer. It promoted tighter coupling between the diiron center in AlkB and the iron in AlkG. The allosteric effects relevant to enzymatic activity regulated by the substrate binding and channeling were further delineated by ENM analysis which confirmed a strong coupling spanning the site of entry from the membrane, the catalytic site and the AlkB-AlkG interface. Our study provides new insights into key sites that could be targeted for developing AlkB-variants with desirable alkane conversion functions.

We introduce WatFinder, a tool designed to identify and visualize protein–water interactions (water bridges, water-mediated associations, or water channels, fluxes, and clusters) relevant to protein stability, dynamics, and function. WatFinder is integrated into ProDy, a Python API broadly used for structure-based prediction of protein dynamics. WatFinder provides a suite of functions for generating raw data as well as outputs from statistical analyses. The ProDy framework facilitates comprehensive automation and efficient analysis of the ensembles of structures resolved for a given protein or the time-evolved conformations from simulations in explicit water, as illustrated in five case studies presented in the Supplementary Material . ProDy is open-source and freely available under MIT License from https://github.com/ProDy/ProDy.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non‐oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA‐PLs, particularly sn2‐arachidonoyl(AA)‐ and sn2‐adrenoyl(AdA)‐containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1–2 mol %) of doubly PUFA‐acylated phospholipids (di‐PUFA‐PLs) whose role in ferroptosis remains enigmatic. Here we report that 15‐lipoxygenase (15LOX) exhibits unexpectedly high pro‐ferroptotic peroxidation activity towards di‐PUFA‐PEs. We revealed that peroxidation of several molecular species of di‐PUFA‐PEs occurred early in ferroptosis. Ferrostatin‐1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di‐PUFA‐PEs. Furthermore, co‐incubation of cells with di‐AA‐PE and 15LOX produced PUFA‐PE peroxidation and induced ferroptotic death. The decreased contents of di‐PUFA‐PEs in ACSL4 KO A375 cells was associated with lower levels of di‐PUFA‐PE peroxidation and enhanced resistance to ferroptosis. Thus, di‐PUFA‐PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non‐oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA‐PLs, particularly sn2‐arachidonoyl(AA)‐ and sn2‐adrenoyl(AdA)‐containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1–2 mol %) of doubly PUFA‐acylated phospholipids (di‐PUFA‐PLs) whose role in ferroptosis remains enigmatic. Here we report that 15‐lipoxygenase (15LOX) exhibits unexpectedly high pro‐ferroptotic peroxidation activity towards di‐PUFA‐PEs. We revealed that peroxidation of several molecular species of di‐PUFA‐PEs occurred early in ferroptosis. Ferrostatin‐1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di‐PUFA‐PEs. Furthermore, co‐incubation of cells with di‐AA‐PE and 15LOX produced PUFA‐PE peroxidation and induced ferroptotic death. The decreased contents of di‐PUFA‐PEs in ACSL4 KO A375 cells was associated with lower levels of di‐PUFA‐PE peroxidation and enhanced resistance to ferroptosis. Thus, di‐PUFA‐PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

Barth syndrome (BTHS) is a life-threatening genetic disorder with unknown pathogenicity caused by mutations in TAFAZZIN (TAZ) that affect remodeling of mitochondrial cardiolipin (CL). TAZ deficiency leads to accumulation of mono-lyso-CL (MLCL), which forms a peroxidase complex with cytochrome c (cyt c) capable of oxidizing polyunsaturated fatty acid-containing lipids. We hypothesized that accumulation of MLCL facilitates formation of anomalous MLCL–cyt c peroxidase complexes and peroxidation of polyunsaturated fatty acid phospholipids as the primary BTHS pathogenic mechanism. Using genetic, biochemical/biophysical, redox lipidomic and computational approaches, we reveal mechanisms of peroxidase-competent MLCL–cyt c complexation and increased phospholipid peroxidation in different TAZ-deficient cells and animal models and in pre-transplant biopsies from hearts of patients with BTHS. A specific mitochondria-targeted anti-peroxidase agent inhibited MLCL–cyt c peroxidase activity, prevented phospholipid peroxidation, improved mitochondrial respiration of TAZ-deficient C2C12 myoblasts and restored exercise endurance in a BTHS Drosophila model. Targeting MLCL–cyt c peroxidase offers therapeutic approaches to BTHS treatment.