Redox Epiphospholipidome in Programmed Cell Death Signaling: Catalytic Mechanisms and Regulation

Abstract

A huge diversification of phospholipids, forming the aqueous interfaces of all biomembranes, cannot be accommodated within a simple concept of their role as membrane building blocks. Indeed, a number of signaling functions of (phospho)lipid molecules has been discovered. Among these signaling lipids, a particular group of oxygenated polyunsaturated fatty acids (PUFA), so called lipid mediators, has been thoroughly investigated over several decades. This group includes oxygenated octadecanoids, eicosanoids, and docosanoids and includes several hundreds of individual species. Oxygenation of PUFA can occur when they are esterified into major classes of phospholipids. Initially, these events have been associated with non-specific oxidative injury of biomembranes. An alternative concept is that these post-synthetically oxidatively modified phospholipids and their adducts with proteins are a part of a redox epiphospholipidome that represents a rich and versatile language for intra- and inter-cellular communications. The redox epiphospholipidome may include hundreds of thousands of individual molecular species acting as meaningful biological signals. This review describes the signaling role of oxygenated phospholipids in programs of regulated cell death. Although phospholipid peroxidation has been associated with almost all known cell death programs, we chose to discuss enzymatic pathways activated during apoptosis and ferroptosis and leading to peroxidation of two phospholipid classes, cardiolipins (CLs) and phosphatidylethanolamines (PEs). This is based on the available LC-MS identification and quantitative information on the respective peroxidation products of CLs and PEs. We focused on molecular mechanisms through which two proteins, a mitochondrial hemoprotein cytochrome c (cyt c), and non-heme Fe lipoxygenase (LOX), change their catalytic properties to fulfill new functions of generating oxygenated CL and PE species. Given the high selectivity and specificity of CL and PE peroxidation we argue that enzymatic reactions catalyzed by cyt c/CL complexes and 15-lipoxygenase/phosphatidylethanolamine binding protein 1 (15LOX/PEBP1) complexes dominate, at least during the initiation stage of peroxidation, in apoptosis and ferroptosis. We contrast cell-autonomous nature of CLox signaling in apoptosis correlating with its anti-inflammatory functions vs. non-cell-autonomous ferroptotic signaling facilitating pro-inflammatory (necro-inflammatory) responses. Finally, we propose that small molecule mechanism-based regulators of enzymatic phospholipid peroxidation may lead to highly specific anti-apoptotic and anti-ferroptotic therapeutic modalities.

Keywords: apoptosis; cardiolipin; cytochrome c; ferroptosis; lipoxygenase; phospholipid peroxidation; redox lipidomics; regulated cell death.

Figure 1

Preferential peroxidation of C18:2 residue in hetero-acylated cardiolipin (CL) molecule in apoptosis.

Figure 2

Structural models of phospholipid peroxidizing Fe-proteins. (A) Structure of cytochrome c (pdb ID 1hrc) (61) shown in ribbon diagram, with the heme molecule (in cyan) and the Cyt C residues (H18 and M80) highlighted in space filling representation. The inset shows the coordination of the Heme molecule by the Cyt C residues, including H18 and M80. (B) Structure of 15LOX Ipdb ID 4nre) (62), also shown in ribbon diagram, with the arachidonic mimic (AA) shown in magenta, stick representation. The catalytic site region is shown in detain in the inset. The b-barrel, which interfaces with the membrane, is labelled. (C) Structure of cyt c-cardiolipin complex. The residue M80 which coordinates the heme has moved away from the Heme molecule, leading to an unfolded cyt c conformation. This unfolded conformation, which was obtained from an earlier study (16) was used to dock a cardiolipin molecule (shown in pink). (D) Structure of 15LOX/PEBP1-HpETE-PE complex. The model of the complex, proposed by us (36) is shown in ribbon diagram. The PEBP1 (shown in cyan) is docked onto the 15LOX, and this complex model, was used to dock HpETE-PE molecule (shown in pink). The ligand docking for both cyt c and 15LOX/PEBP1 complex was performed by SMINA (63). The insets in (C, D) depict the interfacing of peroxidase complex and membrane bilayer. The models for the protein-membrane complexes were built using the Orientation of Proteins in Membrane webserver (https://opm.phar.umich.edu/ppm_server) which calculates translational and rotational position of membranes and proteins from their three-dimensional structures.

Figure 3

Metabolic redox pathways of iron in cells. (A) Tight control of redox-active iron in cells prevents its participation in peroxidation reactions; (B) redox activity of low molecular iron complexes (labile iron); (C) oxidation of CL in cyt c/CL complexes; (D) formation of lipid radicals by the catalytic site of 15LOX/PEBP1 complex.

Figure 4

Allosteric modification of lipid binding in the 15LOX/PEBP1 complex. (A) Surface representation of 15LOX, viewed from top, showing the entrance to the catalytic site, the residues of which are highlighted in blue (top panel). The opening of the entrance site is reduced in the 15LOX/PEBP1 complex (bottom panel, right), compared to that in 15LOX alone (bottom panel, left). (B) The binding of sn1-18:0/sn2-20:4-PE onto 15LOX alone (red) and 15LOX/PEBP1 complex (green), showing the position of the nearest carbon at the catalytic iron. In 15LOX alone, this carbon is C10, leading to peroxidation at C13, where is in the complex it is C13, which leads to peroxidation at C15. These figures were adapted from (98).

Figure 5

Oxidatively truncated electrophilic products formed from phosphatidylethanolamine (PE) and their conjugates with target proteins.

Figure 6

Enzymatic peroxidation of polyunsaturated CL by cyt c yields a variety of products (12, 110), including oxidatively truncated molecular species. Shown are LC-MS results identifying the production CL molecular species containing 9-oxo-nanoic acid. (A) Profiles of tetralinoleyl cardiolipin (LA₄-CL, upper panel) with m/z 1,447.9656 and 9-oxo-nanoyl (ONA)/LA₃-CL with m/z 1,339.8342 (lower panel); (B) MS² fragmentation pattern and structural formulae (inset) of molecular ion with m/z 1,339.8342. MS² analysis reveals the fragments with m/z 1,185.79, m/z 695.47, and 587.33 produced due to the loss of oxidatively truncated residue as well as to di-linoleoyl-glycerol phosphatidate and diacylglycerol phosphatidate containing linoleic acid (LA) and its oxidatively truncated residue. Further MS² fragmentation of ion with m/z 587 yield ions with m/z 307 and m/z 415. The fragments corresponding to ONA (m/z 171) and LA (m/z 279) were detected as well. ONA, 9-oxo-nonanoic acid; LA, linoleic acid.

Figure 7

Peroxidized CLs, including oxidatively truncated species, are produced in the small intestine (ileum) of mice in vivo after total body irradiation (9.5 Gy) (110, 116). Two truncated CL species, ONA/LA₃-CL (A) and OA/LA₂/ONA-CL (B), have been identified by MS²/MS³ fragmentation analysis (ONA, 9-oxo-nonanoic acid; LA, linoleic acid; OA, oleic acid). As previously described (110, 112, 113, 116), the levels of peroxidized CL (including oxidatively truncated CL species containing ONA) are elevated after irradiation. Insets: structural formulas of ONA/LA₃-CL (A) and OA/LA/ONA-CL (B).

Figure 8

Radiation protection and mitigation by TPP-IOA and TPP-ISA. C57BL/6NTac female mice were exposed to total body irradiation to a dose of 9.25 Gy using a cesium source (n = 31–35 mice per group). The mice were irradiated and injected i.p. with TPP-IOA or TPP-ISA (5 mg per kg body weight in 100 μl of water containing 25% ethanol) 10 min after irradiation. P < 0.0001 (a two-sided log-rank test)—TPP-IOA or TPP-ISA injected and exposed to total body irradiation mice vs. mice exposed to total body irradiation only. These figure was adapted from (116).