Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis.

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.

Secretory nucleoside diphosphate kinases from both intra- and extracellular pathogenic bacteria are functionally indistinguishable.

Nucleoside diphosphate kinase (NDK), responsible for the maintenance of NTP pools, is an ATP-utilizing enzyme secreted by different pathogens. We found that NDK from Salmonella enterica serovar Typhimurium (S. Typhimurium) is also secretory in nature. Secretory NDK is known to play a crucial role in the survival of pathogenic microbes within host cells through their interaction with extracellular ATP. To elucidate this aspect, we assessed the contribution of secretory products containing NDK from intracellular (Mycobacterium tuberculosis and S. Typhimurium) and extracellular (Vibrio cholerae) pathogens to the process of ATP-induced J774 mouse macrophage cell lysis by monitoring lactate dehydrogenase (LDH) release in the culture medium. Compared with an untreated control, our results demonstrate that S. Typhimurium secretory products caused a greater than twofold decrease in LDH release from J774 macrophage cells treated with ATP. Furthermore, the secretory products from an ndk-deleted strain of S. Typhimurium did not display such behaviour. Contrary to this observation, the secretory products containing NDK of V. cholerae were found to be cytotoxic to J774 cells. At the amino acid level, the sequences of both the NDKs (S. Typhimurium and V. cholerae) exhibited 65 % identity, and their biochemical characteristics (autophosphorylation and phosphotransfer activities) were indistinguishable. However, to our surprise, the secretory product of an ndk-deleted strain of S. Typhimurium, when complemented with V. cholerae ndk, was able to prevent ATP-induced cytolysis. Taken together, our results unambiguously imply that the intrinsic properties of secretory NDKs are identical in intra- and extracellular pathogens, irrespective of their mode of manifestation.

Intermolecular phosphotransfer is crucial for efficient catalytic activity of nucleoside diphosphate kinase.

NDK (nucleoside diphosphate kinase) is primarily involved in maintaining cellular nucleotide pools in both prokaryotes and eukaryotes. We cloned ndk from Salmonella typhimurium and expressed it in Escherichia coli as a histidine-tagged protein. The Ni-NTA (Ni(2+)-nitrilotriacetate)-purified protein (sNDK) was found to be tetrameric with a monomeric unit molecular mass of approximately 18 kDa. The sNDK exhibited bivalent-cation-dependent autophosphorylation at a wide range of pH values and the phosphorylation withstands acid or alkali treatment. Surprisingly, nucleoside diphosphates did not behave as 'true inhibitors' of autophosphorylation activity. The sNDK displayed phosphotransfer activity from nucleoside triphosphates to nucleoside diphosphates; however, it was Mg(2+)/Mn(2+)-dependent. Mutational analysis established His(117) as the predominantly phosphorylating residue in sNDK. Although it is a histidine kinase, we found that substitution of Ser(119) with alanine/glutamate significantly affected the autophosphorylation, as well as the NTP-synthesizing ability of sNDK. Interestingly, the mixture of inactive (H117A) and partially active (S119A) proteins was found to be catalytically more efficient than the presence of corresponding amounts of active population, advocating transfer of phosphate from phospho-His(117) to Ser(119). Consistent with this observation, the Ni-NTA-purified H117A protein, obtained following co-expression of both of the mutant constructs [His-tagged H117A and GST (glutathione transferase)-tagged S119A] in E. coli, exhibited autophosphorylation, thereby alluding to intermolecular phosphotransfer between His(117) and Ser(119). Although this housekeeping enzyme has long been discovered and characterized from different sources, the results of the present study portray how Ser(119) in sNDK is phosphorylated. Furthermore, our findings illustrate for the first time that the intermolecular phosphotransfer is mandatory for the efficient NTP synthesis in any NDK.

Dichotomous roles for externalized cardiolipin in extracellular signaling: Promotion of phagocytosis and attenuation of innate immunity.

Among the distinct molecular signatures present in the mitochondrion is the tetra-acylated anionic phospholipid cardiolipin, a lipid also present in primordial, single-cell bacterial ancestors of mitochondria and multiple bacterial species today. Cardiolipin is normally localized to the inner mitochondrial membrane; however, when cardiolipin becomes externalized to the surface of dysregulated mitochondria, it promotes inflammasome activation and stimulates the elimination of damaged or nonfunctional mitochondria by mitophagy. Given the immunogenicity of mitochondrial and bacterial membranes that are released during sterile and pathogen-induced trauma, we hypothesized that cardiolipins might function as "eat me" signals for professional phagocytes. In experiments with macrophage cell lines and primary macrophages, we found that membranes with mitochondrial or bacterial cardiolipins on their surface were engulfed through phagocytosis, which depended on the scavenger receptor CD36. Distinct from this process, the copresentation of cardiolipin with the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide dampened TLR4-stimulated production of cytokines. These data suggest that externalized, extracellular cardiolipins play a dual role in host-host and host-pathogen interactions by promoting phagocytosis and attenuating inflammatory immune responses.

Three consecutive arginines are important for the mycobacterial peptide deformylase enzyme activity.

Genes encoding the peptide deformylase enzyme (def) are present in all eubacteria and are involved in the deformylation of the N-formyl group of newly synthesized polypeptides during protein synthesis. We compared the amino acid sequences of this enzyme in different mycobacterial species and found that they are highly conserved (76% homology with 62% identity); however, when this comparison was extended to other eubacterial homologs, it emerged that the mycobacterial proteins have an insertion region containing three consecutive arginine residues (residues 77-79 in Mycobacterium tuberculosis peptide deformylase (mPDF)). Here, we demonstrate that these three arginines are important for the activity of mPDF. Circular dichroism studies of wild-type mPDF and of mPDF containing individual conservative substitutions (R77K, R78K, or R79K) or combined substitutions incorporated into a triple mutant (R77K/R78K/R79K) indicate that such mutations cause mPDF to undergo structural alterations. Molecular modeling of mPDF suggests that the three arginines are distal to the active site. Molecular dynamics simulations of wild-type and mutant mPDF structures indicate that the arginines may be involved in the stabilization of substrate binding pocket residues for their proper interaction with peptide(s). Treatment with 5'-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against different regions of def from M. tuberculosis inhibits growth of Mycobacterium smegmatis in culture. Taken together, these results hold out the possibility of future design of novel mycobacteria-specific PDF inhibitors.