Analysis of 1-Deoxysphingoid Bases and Their N-Acyl Metabolites and Exploration of Their Occurrence in Some Food Materials.

Most common sphingolipids are comprised of "typical" sphingoid bases (sphinganine, sphingosine, and structurally related compounds) and are produced via the condensation of l-serine with a fatty acyl-CoA by serine palmitoyltransferase. Some organisms, including mammals, also produce "atypical" sphingoid bases that lack a 1-hydroxyl group as a result of the utilization of l-alanine or glycine instead of l-serine, resulting in the formation of 1-deoxy- or 1-desoxymethylsphingoid bases, respectively. Elevated production of "atypical" sphingolipids has been associated with human disease, but 1-deoxysphingoid bases have also been found to have potential as anticancer compounds, hence, the importance of knowing more about the occurrence of these compounds in food. Most of the "typical" and "atypical" sphingoid bases are found as the N-acyl metabolites (e.g., ceramides and 1-deoxyceramides) in mammals, but this has not been uniformly assessed in previous studies nor determined in consumed food. Therefore, we developed a method for the quantitative analysis of "typical" and "atypical" sphingoid bases and their N-acyl derivatives by reverse-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. On the basis of these analyses, there was considerable variability in the amounts and molecular subspecies of atypical sphingoid bases and their N-acyl metabolites found in different edible sources. These findings demonstrate that a broader assessment of the types of sphingolipids in foods is needed because some diets might contain sufficient amounts of atypical as well as typical sphingolipids that could have beneficial or possibly deleterious effects on human health.

Phosphatidylserine expressing platelet microparticle levels at hospital presentation are decreased in sepsis non-survivors and correlate with thrombocytopenia.

BACKGROUND: Sepsis induced platelet activation releases platelet microparticles (PMPs). PMPs express phosphatidylserine (PS) and can serve as a scaffold for the prothrombinase complex, thereby promoting coagulation. Studies of PMPs in intensive care unit sepsis patients demonstrate mixed results, while the earliest changes and potential effects of clinical interventions remain understudied. We hypothesized PMPs would be associated with patient outcome and dysfunctional coagulation shortly after emergency department presentation with sepsis. METHODS: Cohort study of patients from a single center enrolled in a previously published randomized control trial comparing two early resuscitation strategies. Adults presenting to the emergency department (ED) with suspected infection, ≥2 SIRS criteria, and either systolic blood pressure <90 mm Hg or lactate >4 mmol/L were eligible. Triple positive platelet microparticles (PMPs) expressing phosphatidylserine and integrin complexes alphaIIb (CD41) and beta3 (CD61) were quantitated using plasma from the time of enrollment. The primary outcome was in-hospital mortality. Secondary outcomes included platelet count, disseminated intravascular coagulation (DIC), and prothrombin time (PT). RESULTS: 193 patients were enrolled and 184 had samples available. In-hospital mortality was 21%. 10 (5%) patients developed DIC. Median platelet count was 197 (IQR 135, 280) and PT was 13.2 (IQR 11.9, 16.8). Median triple positive PMP counts were 932 per µL (IQR 381, 1872). PMPs were significantly lower in non-survivors (575 vs 1128, p = 0.02) and non-significantly lower in DIC (387 vs 942, p = 0.17). PMPs demonstrated a positive linear association with platelet count (p < 0.001, R2 = 0.21). After adjusting for platelet count, PMPs were no longer significant predictors of mortality (p = 0.28). We observed no association between PMPs and PT. CONCLUSION: Similar to patients enrolled later in the intensive care unit, PS-expressing PMPs are lower in emergency department sepsis non-survivors. These changes primarily reflect the degree of thrombocytopenia, and an independent prognostic role was not observed. Future studies should control for platelet count in assessment of PMP prognosis in sepsis.

Rodent vertical sleeve gastrectomy alters maternal immune health and fetoplacental development.

Bariatric surgery is increasingly employed to improve fertility and reduce obesity-related co-morbidities in obese women. Surgical weight loss not only improves the chance of conception but reduces the risk of pregnancy complications including pre-eclampsia, gestational diabetes, and macrosomia. However, bariatric procedures increase the incidence of intrauterine growth restriction (IUGR), fetal demise, thromboembolism, and other gestational disorders. Using our rodent model of vertical sleeve gastrectomy (VSG), we tested the hypothesis that VSG in diet-induced, obese dams would cause immune and placental structural abnormalities that may be responsible for fetal demise during pregnancy. VSG dams studied on gestational day (G) 19 had reduced circulating T-cell (CD3+ and CD8+) populations compared with lean or obese controls. Further, local interleukin (IL) 1ß and IL 1 receptor antagonist (il1rn) cmRNA were increased in placenta of VSG dams. Placental barrier function was also affected, with increased transplacental permeability to small molecules, increased matrix metalloproteinase 9 expression, and increased apoptosis in VSG. Furthermore, we identified increased placental mTOR signaling that may contribute to preserving the body weight of the fetuses during gestation. These changes occurred in the absence of a macronutrient deficit or gestational hypertension in the VSG dams. In summary, previous VSG in dams may contribute to fetal demise by affecting maternal immune system activity and compromise placental integrity.

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.

Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

Monothiol glutaredoxins can bind linear [Fe3S4]+ and [Fe4S4]2+ clusters in addition to [Fe2S2]2+ clusters: spectroscopic characterization and functional implications.

Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has been shown to function in biological [Fe2S2](2+) cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically, after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear [Fe3S4](+) cluster. The excited-state electronic properties and ground-state electronic and vibrational properties of the linear [Fe3S4](+) cluster have been characterized using UV-vis absorption/CD/MCD, EPR, Mössbauer, and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear [Fe3S4](+) cluster with properties similar to those reported for synthetic linear [Fe3S4](+) clusters and the linear [Fe3S4](+) clusters in purple aconitase. Moreover, the results indicate that the Fe-S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of [Fe2S2](2+) clusters, rather than linear [Fe3S4](+) clusters or mixtures of linear [Fe3S4](+) and [Fe2S2](2+) clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one [Fe4S4](2+) cluster that is competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear [Fe3S4](+) and [Fe4S4](2+) clusters in Grx5 has been assessed by spectroscopic, mutational, and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear [Fe3S4](+) clusters released during protein unfolding under oxidative stress conditions and in maturation of [Fe4S4](2+) cluster-containing proteins are discussed in light of these results.

Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons.

BACKGROUND: Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson's disease (PD) in part because their oxidative environment in the substantia nigra (SN) may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution of ceramide sphingolipid signaling in TNF-dependent toxicity. RESULTS: Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics mass spectrometry we confirmed that TNF treatment not only promotes generation of ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases (DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons. CONCLUSIONS: We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons. Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective strategies that can delay or attenuate the progressive loss of nigral DA neurons in patients with PD.

Acid ceramidase (ASAH1) represses steroidogenic factor 1-dependent gene transcription in H295R human adrenocortical cells by binding to the receptor.

Adrenocorticotropin (ACTH) signaling increases glucocorticoid production by promoting the interaction of transcription factors and coactivator proteins with the promoter of steroidogenic genes. The nuclear receptor steroidogenic factor 1 (SF-1) is essential for steroidogenic gene transcription. Sphingosine (SPH) is a ligand for SF-1. Moreover, suppression of expression of acid ceramidase (ASAH1), an enzyme that produces SPH, increases the transcription of multiple steroidogenic genes. Given that SF-1 is a nuclear protein, we sought to define the molecular mechanisms by which ASAH1 regulates SF-1 function. We show that ASAH1 is localized in the nuclei of H295R adrenocortical cells and that cyclic AMP (cAMP) signaling promotes nuclear sphingolipid metabolism in an ASAH1-dependent manner. ASAH1 suppresses SF-1 activity by directly interacting with the receptor. Chromatin immunoprecipitation (ChIP) assays revealed that ASAH1 is recruited to the promoter of various SF-1 target genes and that ASAH1 and SF-1 colocalize on the same promoter region of the CYP17A1 and steroidogenic acute regulatory protein (StAR) genes. Taken together, these results demonstrate that ASAH1 is a novel coregulatory protein that represses SF-1 function by directly binding to the receptor on SF-1 target gene promoters and identify a key role for nuclear lipid metabolism in regulating gene transcription.

Acid ceramidase (ASAH1) is a global regulator of steroidogenic capacity and adrenocortical gene expression.

In H295R human adrenocortical cells, ACTH rapidly activates ceramide (Cer) and sphingosine (SPH) turnover with a concomitant increase in SPH-1-phosphate secretion. These bioactive lipids modulate adrenocortical steroidogenesis, primarily by acting as second messengers in the protein kinase A/cAMP-dependent pathway. Acid ceramidase (ASAH1) directly regulates the intracellular balance of Cer, SPH, and SPH-1-phosphate by catalyzing the hydrolysis of Cer into SPH. ACTH/cAMP signaling stimulates ASAH1 transcription and activity, supporting a role for this enzyme in glucocorticoid production. Here, the role of ASAH1 in regulating steroidogenic capacity was examined using a tetracycline-inducible ASAH1 short hairpin RNA H295R human adrenocortical stable cell line. We show that ASAH1 suppression increases the transcription of multiple steroidogenic genes, including Cytochrome P450 monooxygenase (CYP)17A1, CYP11B1/2, CYP21A2, steroidogenic acute regulatory protein, hormone-sensitive lipase, 18-kDa translocator protein, and the melanocortin-2 receptor. Induced gene expression positively correlated with enhanced histone H3 acetylation at target promoters. Repression of ASAH1 expression also induced the expression of members of the nuclear receptor nuclear receptor subfamily 4 (NR4A) family while concomitantly suppressing the expression of dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1. ASAH1 knockdown altered the expression of genes involved in sphingolipid metabolism and changed the cellular amounts of distinct sphingolipid species. Finally, ASAH1 silencing increased basal and cAMP-dependent cortisol and dehydroepiandrosterone secretion, establishing ASAH1 as a pivotal regulator of steroidogenic capacity in the human adrenal cortex.

Ablation of ceramide synthase 2 strongly affects biophysical properties of membranes.

Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a ceramide synthase 2 (CerS2)-null mouse, which is unable to synthesize C22-C24 ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.

Lipidomics reveals a remarkable diversity of lipids in human plasma.

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Iron-sulfur cluster biosynthesis.

Iron-sulfur (Fe-S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe-S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe-S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe-S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe-S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe-S clusters.

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters.

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.

A proposed role for the Azotobacter vinelandii NfuA protein as an intermediate iron-sulfur cluster carrier.

Iron-sulfur clusters ([Fe-S] clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require [Fe-S] clusters for their functions. Previous studies have shown that Azotobacter vinelandii produces at least two [Fe-S] cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other [Fe-S] proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a [4Fe-4S] cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one [4Fe-4S] cluster can be assembled in vitro within a dimeric form of NfuA. The resultant [4Fe-4S] cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate [Fe-S] cluster carriers involved in [Fe-S] protein maturation.

Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1.

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron-sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron-sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron-sulfur biosynthesis is discussed with respect to their intracellular localization.