Formation of protein adducts with Hydroperoxy-PE electrophilic cleavage products during ferroptosis
A.A. Amoscato,
T. Anthonymuthu,
O. Kapralov,
L.J. Sparvero,
I.H. Shrivastava,
K. Mikulska-Ruminska,
V.A. Tyurin,
A.A. Shvedova,
Y.Y. Tyurina,
I. Bahar,
S. Wenzel,
H. Bayir,
V.E. Kagan
Affiliations
A.A. Amoscato
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA; Corresponding author.
T. Anthonymuthu
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA; Adeptrix Corp, 100 Cummings Center, Suite 339c, Beverly, MA, 01915, USA
O. Kapralov
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
L.J. Sparvero
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
I.H. Shrivastava
NIOSH/HELD/EAB, 1095 Willowdale Road, Morgantown, WV, 26505, USA
K. Mikulska-Ruminska
Institute of Physics, Faculty of Physics Astronomy and Informatics, Nicolaus Copernicus University in Toruń, PL87100, Toruń, Poland
V.A. Tyurin
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
A.A. Shvedova
NIOSH/HELD/EAB, 1095 Willowdale Road, Morgantown, WV, 26505, USA; Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, WV, USA
Y.Y. Tyurina
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
I. Bahar
Department of Computational and Systems Biology, University of Pittsburgh, 800 Murdoch I Bldg., 3420 Forbes Avenue, Pittsburgh, PA, 15213, USA; Laufer Center for Physical and Quantitative Biology, Laufer Center, Z-5252, Stony Brook University, Stony Brook, NY, 11794, USA
S. Wenzel
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Asthma and Environmental Lung Health Institute at UPMC, University of Pittsburgh, Pittsburgh, PA, 15261, USA
H. Bayir
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA; Safar Center for Resuscitation Research, Department of Critical Care Medicine, University of Pittsburgh Medical Center, 4401 Penn Ave, Pittsburgh, PA, 15224, USA; Department of Pediatrics Critical Care, Columbia University, 3959 Broadway, CHN-10, New York, NY, 10032, USA
V.E. Kagan
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA; Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2 Trubetskaya Str, 11999, Moscow, Russia; Corresponding author. Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA.
Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.
