Phospholipase iPLA2β averts ferroptosis by eliminating a redox lipid death signal

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca^{2+} -independent phospholipase A2β (iPLA2β, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA_{2}β averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson’s disease (PD)-associated mutation (fPD^{R747W}) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9^{R748W/R748W} mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant Snca^{A53T} mice, with decreased iPLA2β expression and a PD-relevant phenotype. Thus, iPLA_{2}β is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis

Structural requirements for optimized delivery, inhibition of oxidative stress, and antiapoptotic activity of targeted nitroxides

Suppression of mitochondrial production of reactive oxygen species is a promising strategy against intrinsic apoptosis typical of degenerative diseases. Stable nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and its analogs combine several important features, including recycleability, electron acceptance from respiratory complexes, superoxide dismutase mimicry, and radical scavenging. Although successful in antioxidant protection, their effective concentrations are too high for successful in vivo applications. Recently (J Am Chem Soc 127:12460, 2005), we reported that 4-amino 2,2,6,6-tetramethyl-1-piperidinyloxy, covalently conjugated to a five-residue segment of gramicidin S (GS), was integrated into mitochondria and blocked actinomycin D (ActD)-induced superoxide generation and apoptosis. Using a model of ActD-induced apoptosis in mouse embryonic cells, we screened a library of nitroxides to explore structure-activity relationships between their antioxidant/antiapoptotic properties and chemical composition and three-dimensional (3D) structure. High hydrophobicity and effective mitochondrial integration are necessary but not sufficient for high antiapoptotic/antioxidant activity of a nitroxide conjugate. By designing conformationally preorganized peptidyl nitroxide conjugates and characterizing their 3D structure experimentally (circular dichroism and NMR) and theoretically (molecular dynamics), we established that the presence of the β-turn/β-sheet secondary structure is essential for the desired activity. Monte Carlo simulations in model lipid membranes confirmed that the conservation of the D-Phe-Pro reverse turn in hemi-GS analogs ensures the specific positioning of the nitroxide moiety at the mitochondrial membrane interface and maximizes their protective effects. These new insights into the structure-activity relationships of nitroxide-peptide and -peptide isostere conjugates are instrumental for development of new mechanism-based therapeutically effective agents. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics

Redox (phospho)lipidomics of signaling in inflammation and programmed cell death

In addition to the known prominent role of polyunsaturated (phospho)lipids as structural blocks of biomembranes, there is an emerging understanding of another important function of these molecules as a highly diversified signaling language utilized for intra- and extracellular communications. Technological developments in high-resolution mass spectrometry facilitated the development of a new branch of metabolomics, redox lipidomics. Analysis of lipid peroxidation reactions has already identified specific enzymatic mechanisms responsible for the biosynthesis of several unique signals in response to inflammation and regulated cell death programs. Obtaining comprehensive information about millions of signals encoded by oxidized phospholipids, represented by thousands of interactive reactions and pleiotropic (patho)physiological effects, is a daunting task. However, there is still reasonable hope that significant discoveries, of at least some of the important contributors to the overall overwhelmingly complex network of interactions triggered by inflammation, will lead to the discovery of new small molecule regulators and therapeutic modalities. For example, suppression of the production of AA-derived pro-inflammatory mediators, HXA(3) and LTB4, by an iPLA(2)gamma inhibitor, R-BEL, mitigated injury associated with the activation of pro-inflammatory processes in animals exposed to whole-body irradiation. Further, technological developments promise to make redox lipidomics a powerful approach in the arsenal of diagnostic and therapeutic instruments for personalized medicine of inflammatory diseases and conditions

Differing processing requirements of four recombinant antigens containing a single defined T-cell epitope for presentation by major histocompatibility complex class II

A set of predictive rules governing the likelihood of generating a particular peptide–major histocompatibility complex (MHC) class II complex from an intact antigen has not been fully elucidated. We investigated the influence of positional and structural constraints in the region of the epitope by designing a set of recombinant antigens that each contained the well-characterized T-cell epitope moth cytochrome c (MCC) (88–103), which is specifically recognized by the monoclonal antibody (mAb) D4 when complexed with H-2E(k). Our model antigens contained MCC(88–103) either peripherally, at or towards the C-terminus, or internally. Their abilities to bind directly to soluble H-2E(k), and the extent of D4 epitope formation from them by antigen processing-competent and -incompetent cell lines, were determined. Here we report that three of these four antigens yielded MCC(88–103)/H-2E(k) complexes independently of the conventional MHC class II antigen-processing and presentation pathway, and in each case the epitope was carried peripherally; two bound directly as intact proteins, probably as a result of spatial separation of the epitope from the major globular domain, and one was processed to peptide by a cell-surface protease. One protein, which carried the epitope inserted into an internal loop, acted as a conventional processing-dependent MCC(88–103) delivery vehicle. Thus, this epitope has different presentation requirements depending on its context. These antigens constitute a panel whose framework could be modified to further define predictive rules for antigen processing for presentation through the different MHC class II complex-generating pathways

Aminopeptidase N (APN)/CD13 inhibitor, Ubenimex, enhances radiation sensitivity in human cervical cancer

<p>Abstract</p> <p>Background</p> <p>Radiotherapy can be used to treat all stages of cervical cancer. For improving local control via radiotherapy, it is important to use additional antitumor agents. Aminopeptidase N (APN)/CD13, a 150-kDa metalloproteinase, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression in several human malignancies.</p> <p>Methods</p> <p>We investigated whether the suppression of APN/CD13 using Ubenimex, an inhibitor of APN/CD13 activity, may affect tumor radiosensitivity in cervical cancer cells both <it>in vitro </it>and <it>in vivo</it>. Cell surface APN/CD13 activity in HeLa cells was calculated using alanine-p-nitroanilido as a substrate. For colony formation assays, single-dose radiation and/or Ubenimex were administered to each dish of HeLa cells, and these dishes were cultured for 14 days. Molecular changes of apoptosis were determined by Western blot. Apoptosis was evaluated by Annexin-V PI staining (flow cytometry analysis) and the Tunel method. Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice.</p> <p>Results</p> <p>We demonstrated that Ubenimex enhanced the effectiveness of radiotherapy, acting as a radiosensitizer both <it>in vitro </it>and <it>in vivo</it>. In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells. Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups. We also showed that ubenimex enhanced radiation-induced apoptosis <it>in vitro </it>and <it>in vivo</it>.</p> <p>Conclusion</p> <p>Although further studies are needed, this report suggests that Ubeniemx acts as a radiosensitizer in cervical cancer treatment, and that the inhibition of APN/CD13 activity may represent a new approach for improving the therapeutic efficacy of radiotherapy for uterine cervical cancer.</p