PRDX6 contributes to selenocysteine metabolism and ferroptosis resistance.

Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and has traditionally been thought to begin with the uptake of the Sec carrier selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP undergoes metabolisation via selenocysteine lyase (SCLY), producing selenide, a substrate used by selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor - selenophosphate - for the biosynthesis of the selenocysteine tRNA. Here, we report the discovery of an alternative pathway mediating Sec metabolisation that is independent of SCLY and mediated by peroxiredoxin 6 (PRDX6). Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the presence and functional significance of this alternative route in cancer cells where we reveal a notable association between elevated expression of PRDX6 with a highly aggressive neuroblastoma subtype. Altogether, our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering new avenues for therapeutic exploitation.

Catalytic role of histidine-114 in the hydrolytic dehalogenation of chlorothalonil by Pseudomonas sp. CTN-3.

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, ChdH114A exhibited catalytic activity with a kcat value of 1.07 s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on ChdH114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.

7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Inflammation and the pathological progression of Alzheimer's disease are associated with low circulating choline levels.

Deficiency of dietary choline, an essential nutrient, is observed worldwide, with ~ 90% of Americans being deficient. Previous work highlights a relationship between decreased choline intake and an increased risk for cognitive decline and Alzheimer's disease (AD). The associations between blood circulating choline and the pathological progression in both mild cognitive impairment (MCI) and AD remain unknown. Here, we examined these associations in a cohort of patients with MCI with presence of either sparse or high neuritic plaque density and Braak stage and a second cohort with either moderate AD (moderate to frequent neuritic plaques, Braak stage = IV) or severe AD (frequent neuritic plaques, Braak stage = VI), compared to age-matched controls. Metabolomic analysis was performed on serum from the AD cohort. We then assessed the effects of dietary choline deficiency (Ch-) in 3xTg-AD mice and choline supplementation (Ch+) in APP/PS1 mice, two rodent models of AD. The levels of circulating choline were reduced while pro-inflammatory cytokine TNFα was elevated in serum of both MCI sparse and high pathology cases. Reduced choline and elevated TNFα correlated with higher neuritic plaque density and Braak stage. In AD patients, we found reductions in choline, its derivative acetylcholine (ACh), and elevated TNFα. Choline and ACh levels were negatively correlated with neuritic plaque load, Braak stage, and TNFα, but positively correlated with MMSE, and brain weight. Metabolites L-Valine, 4-Hydroxyphenylpyruvic, Methylmalonic, and Ferulic acids were significantly associated with circuiting choline levels. In 3xTg-AD mice, the Ch- diet increased amyloid-ß levels and tau phosphorylation in cortical tissue, and TNFα in both blood and cortical tissue, paralleling the severe human-AD profile. Conversely, the Ch+ diet increased choline and ACh while reducing amyloid-ß and TNFα levels in brains of APP/PS1 mice. Collectively, low circulating choline is associated with AD-neuropathological progression, illustrating the importance of adequate dietary choline intake to offset disease.

High-frequency imagery to capture coral tissue (Montipora capricornis) response to environmental stress, a pilot study.

Environment stress is a major threat to the existence of coral reefs and has generated a lot of interest in the coral research community. Under the environmental stress, corals can experience tissue loss and/or the breakdown of symbiosis between the cnidarian host and its symbiotic algae causing the coral tissue to appear white as the skeleton can be seen by transparency. Image analysis is a common method used to assess tissue response under the environmental stress. However, the traditional approach is limited by the dynamic nature of the coral-algae symbiosis. Here, we observed coral tissue response in the scleractinian coral, Montipora capricornis, using high frequency image analysis throughout the experiment, as opposed to the typical start/end point assessment method. Color analysis reveals that the process can be divided into five stages with two critical stages according to coral tissue morphology and color ratio. We further explore changes to the morphology of individual polyps by means of the Pearson correlation coefficient and recurrence plots, where the quasi-periodic and nonstationary dynamics can be identified. The recurrence quantification analysis also allows the comparison between the different polyps. Our research provides a detailed visual and mathematical analysis of coral tissue response to environmental stress, which potentially shows universal applicability. Moreover, our approach provides a robust quantitative advancement for improving our insight into a suite of biotic responses in the perspective of coral health evaluation and fate prediction.

Applying model approaches in non-model systems: A review and case study on coral cell culture.

Model systems approaches search for commonality in patterns underlying biological diversity and complexity led by common evolutionary paths. The success of the approach does not rest on the species chosen but on the scalability of the model and methods used to develop the model and engage research. Fine-tuning approaches to improve coral cell cultures will provide a robust platform for studying symbiosis breakdown, the calcification mechanism and its disruption, protein interactions, micronutrient transport/exchange, and the toxicity of nanoparticles, among other key biological aspects, with the added advantage of minimizing the ethical conundrum of repeated testing on ecologically threatened organisms. The work presented here aimed to lay the foundation towards development of effective methods to sort and culture reef-building coral cells with the ultimate goal of obtaining immortal cell lines for the study of bleaching, disease and toxicity at the cellular and polyp levels. To achieve this objective, the team conducted a thorough review and tested the available methods (i.e. cell dissociation, isolation, sorting, attachment and proliferation). The most effective and reproducible techniques were combined to consolidate culture methods and generate uncontaminated coral cell cultures for ~7 days (10 days maximum). The tests were conducted on scleractinian corals Pocillopora acuta of the same genotype to harmonize results and reduce variation linked to genetic diversity. The development of cell separation and identification methods in conjunction with further investigations into coral cell-type specific metabolic requirements will allow us to tailor growth media for optimized monocultures as a tool for studying essential reef-building coral traits such as symbiosis, wound healing and calcification at multiple scales.

Lemon Extract Reduces Angiotensin Converting Enzyme (ACE) Expression and Activity and Increases Insulin Sensitivity and Lipolysis in Mouse Adipocytes.

Obesity is associated with insulin resistance and cardiovascular complications. In this paper, we examine the possible beneficial role of lemon juice in dieting. Lemon extract (LE) has been proposed to improve serum insulin levels and decrease angiotensin converting enzyme (ACE) activity in mouse models. ACE is also a biomarker for sustained weight loss and ACE inhibitors improve insulin sensitivity in humans. Here, we show that LE impacts adipose tissue metabolism directly. In 3T3-L1 differentiated adipocyte cells, LE improved insulin sensitivity as evidenced by a 3.74 ± 0.54-fold increase in both pAKT and GLUT4 levels. LE also induced lipolysis as demonstrated by a 16.6 ± 1.2 fold-change in pHSL protein expression levels. ACE gene expression increased 12.0 ± 0.1 fold during differentiation of 3T3-L1 cells in the absence of LE, and treatment with LE decreased ACE gene expression by 80.1 ± 0.5% and protein expression by 55 ± 0.37%. We conclude that LE's reduction of ACE expression causes increased insulin sensitivity and breakdown of lipids in adipocytes.

Insights into the Mechanism of Action of Highly Diluted Biologics.

The therapeutic use of Abs in cancer, autoimmunity, transplantation, and other fields is among the major biopharmaceutical advances of the 20th century. Broader use of Ab-based drugs is constrained because of their high production costs and frequent side effects. One promising approach to overcome these limitations is the use of highly diluted Abs, which are produced by gradual reduction of an Ab concentration to an extremely low level. This technology was used to create a group of drugs for the treatment of various diseases, depending on the specificity of the used Abs. Highly diluted Abs to IFN-γ (hd-anti-IFN-γ) have been demonstrated to be efficacious against influenza and other respiratory infections in a variety of preclinical and clinical studies. In the current study, we provide evidence for a possible mechanism of action of hd-anti-IFN-γ. Using high-resolution solution nuclear magnetic resonance spectroscopy, we show that the drug induced conformational changes in the IFN-γ molecule. Chemical shift changes occurred in the amino acids located primarily at the dimer interface and at the C-terminal region of IFN-γ. These molecular changes could be crucial for the function of the protein, as evidenced by an observed hd-anti-IFN-γ-induced increase in the specific binding of IFN-γ to its receptor in U937 cells, enhanced induced production of IFN-γ in human PBMC culture, and increased survival of influenza A-infected mice.

Lipid bodies containing oxidatively truncated lipids block antigen cross-presentation by dendritic cells in cancer.

Cross-presentation is a critical function of dendritic cells (DCs) required for induction of antitumor immune responses and success of cancer immunotherapy. It is established that tumor-associated DCs are defective in their ability to cross-present antigens. However, the mechanisms driving these defects are still unknown. We find that impaired cross-presentation in DCs is largely associated with defect in trafficking of peptide-MHC class I (pMHC) complexes to the cell surface. DCs in tumor-bearing hosts accumulate lipid bodies (LB) containing electrophilic oxidatively truncated (ox-tr) lipids. These ox-tr-LB, but not LB present in control DCs, covalently bind to chaperone heat shock protein 70. This interaction prevents the translocation of pMHC to cell surface by causing the accumulation of pMHC inside late endosomes/lysosomes. As a result, tumor-associated DCs are no longer able to stimulate adequate CD8 T cells responses. In conclusion, this study demonstrates a mechanism regulating cross-presentation in cancer and suggests potential therapeutic avenues.

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.

Structural re-arrangement and peroxidase activation of cytochrome c by anionic analogues of vitamin E, tocopherol succinate and tocopherol phosphate.

Cytochrome c is a multifunctional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cytochrome c into a peroxidase, we investigated two hydrophobic anionic analogues of vitamin E, α-tocopherol succinate (α-TOS) and α-tocopherol phosphate (α-TOP), as potential inducers of peroxidase activity of cytochrome c. NMR studies and computational modeling indicate that they interact with cytochrome c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met(80)) bond associated with unfolding of cytochrome c. We found that α-TOS and α-TOP stimulate peroxidase activity of cytochrome c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with α-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (mito-VES), was more efficient in inducing H2O2-dependent apoptosis in mouse embryonic cytochrome c(+/+) cells than in cytochrome c(-/-) cells. Essential for execution of the apoptotic program peroxidase activation of cytochrome c by α-TOS may contribute to its known anti-cancer pharmacological activity.

Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations.

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer-the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.

Oxidized lipids block antigen cross-presentation by dendritic cells in cancer.

Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide-MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation.

Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells.

Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased the delivery of mitochondria to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1 light chain 3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modelling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an 'eat-me' signal for the elimination of damaged mitochondria from neuronal cells.

The cytoplasmic rhodopsin-protein interface: potential for drug discovery.

The mammalian dim-light photoreceptor rhodopsin is a prototypic G protein coupled receptor (GPCR), interacting with the G protein, transducin, rhodopsin kinase, and arrestin. All of these proteins interact with rhodopsin at its cytoplasmic surface. Structural and modeling studies have provided in-depth descriptions of the respective interfaces. Overlap and thus competition for binding surfaces is a major regulatory mechanism for signal processing. Recently, it was found that the same surface is also targeted by small molecules. These ligands can directly interfere with the binding and activation of the proteins of the signal transduction cascade, but they can also allosterically modulate the retinal ligand binding pocket. Because the pocket that is targeted contains residues that are highly conserved across Class A GPCRs, these findings imply that it may be possible to target multiple GPCRs with the same ligand(s). This is desirable for example in complex diseases such as cancer where multiple GPCRs participate in the disease networks.

A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 h before through 24 h after the irradiation.

Virus interactions with human signal transduction pathways.

Viruses depend on their hosts at every stage of their life cycles and must therefore communicate with them via Protein-Protein Interactions (PPIs). To investigate the mechanisms of communication by different viruses, we overlay reported pairwise human-virus PPIs on human signalling pathways. Of 671 pathways obtained from NCI and Reactome databases, 355 are potentially targeted by at least one virus. The majority of pathways are linked to more than one virus. We find evidence supporting the hypothesis that viruses often interact with different proteins depending on the targeted pathway. Pathway analysis indicates overrepresentation of some pathways targeted by viruses. The merged network of the most statistically significant pathways shows several centrally located proteins, which are also hub proteins. Generally, hub proteins are targeted more frequently by viruses. Numerous proteins in virus-targeted pathways are known drug targets, suggesting that these might be exploited as potential new approaches to treatments against multiple viruses.

Ligand-independent antiapoptotic function of estrogen receptor-beta in lung cancer cells.

Recent studies have demonstrated the presence of estrogen receptor (ER)beta in the mitochondria in various cell types and tissues, but the exact function of this localization remains unclear. In this study, we have examined the function of mitochondrial ERbeta in non-small-cell lung cancer (NSCLC) cells. Down-regulation of ERbeta by short hairpin RNA constructs sensitized NSCLC cells to various apoptosis-inducing agents such as cisplatin, taxol, and etoposide. The increased growth inhibition and induction of apoptosis in ERbeta-knockdown cells was observed irrespective of estrogen treatment, suggesting a ligand-independent role of ERbeta in regulating the intrinsic apoptotic pathway. Further, ERbeta from the mitochondrial fraction physically interacted with the proapoptotic protein Bad, in a ligand-independent manner. Glutathione-S-transferase pull-down assays and molecular modeling studies revealed that the DNA-binding domain and hinge region of ERbeta, and the BH3 domain of Bad were involved in these interactions. Further investigations revealed that ERbeta inhibited Bad function by disrupting Bad-Bcl-X(L) and Bad-Bcl-2 interactions. Reintroduction of ERbeta in the mitochondria of ERbeta knockdown cells reversed their sensitivity to cisplatin. Overall, our results demonstrate a ligand-independent role of ERbeta in regulating apoptosis, revealing a novel function for ERbeta in the mitochondria.

Characterization of membrane protein non-native states. 2. The SDS-unfolded states of rhodopsin.

Little is known about the molecular nature of residual structure in unfolded states of membrane proteins. A screen of chemical denaturants to maximally unfold the mammalian membrane protein and prototypic G protein coupled receptor rhodopsin, without interference from aggregation, described in an accompanying paper (DOI 10.1021/bi100338e ), identified sodium dodecyl sulfate (SDS), alone or in combination with other chemicals, as the most suitable denaturant. Here, we initiate the biophysical characterization of SDS-denatured states of rhodopsin. Using absorption, steady-state and time-resolved fluorescence spectroscopy, dynamic light scattering, and cysteine accessibility studies, tertiary structure of denatured states was characterized. In agreement with the pattern of secondary structure changes detected by circular dichroism described in the accompanying paper (DOI 10.1021/bi100338e ), tertiary structure changes are distinct over four SDS concentration ranges based on the expected predominant micellar structures. Dodecyl maltoside (DM)/SDS mixed micelle spheres (0.05-0.3% SDS) turn into SDS spheres (0.3-3% SDS) that gradually (3-15% SDS) become cylindrical (above 15% SDS). Denatured states in SDS spheres and cylinders show a relatively greater burial of cysteine and tryptophan residues and are more compact as compared to the states observed in mixed micellar structures. Protein structural changes at the membrane/water interface region are most prominent at very low SDS concentrations but reach transient stability in the compact conformations in SDS spheres. This is the first experimental evidence for the formation of a compact unfolding intermediate state with flexible surface elements in a membrane protein.