Characterizing Human Mesenchymal Stromal Cells Immune Modulatory Potency Using Targeted Lipidomic Profiling of Sphingolipids

Cell therapies are expected to increase over the next decade due to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized bone marrow (BM), adipose derived and umbilical cord tissue MSCs sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography tandem mass spectrometry. We found ceramide levels differed based upon donors sex in BM MSCs. We detected fatty acyl chain variants in MSCs from all 3 sources. Principal component analysis showed IFNg; primed and untreated MSCs separated according to their SL signature. We detected higher ceramide levels in low IDO MSCs, indicating sphingomeylinase or ceramidase enzymatic activity may be involved in their immune potency. Lastly, linear discriminant analysis revealed that MSCs separated based on tissue source.

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The Role of Ceramide Metabolism and Signaling in the Regulation of Mitophagy and Cancer Therapy

Cancers ◽

10.3390/cancers13102475 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2475

Author(s):

Megan Sheridan ◽

Besim Ogretmen

Keyword(s):

Cancer Therapy ◽

Acyl Chain ◽

Fatty Acyl ◽

Fatty Acyl Chain ◽

Cancer Cell Proliferation ◽

Bioactive Lipids ◽

Cellular Functions ◽

Proliferation Response ◽

Ceramide Synthases

Sphingolipids are bioactive lipids responsible for regulating diverse cellular functions such as proliferation, migration, senescence, and death. These lipids are characterized by a long-chain sphingosine backbone amide-linked to a fatty acyl chain with variable length. The length of the fatty acyl chain is determined by specific ceramide synthases, and this fatty acyl length also determines the sphingolipid’s specialized functions within the cell. One function in particular, the regulation of the selective autophagy of mitochondria, or mitophagy, is closely regulated by ceramide, a key regulatory sphingolipid. Mitophagy alterations have important implications for cancer cell proliferation, response to chemotherapeutics, and mitophagy-mediated cell death. This review will focus on the alterations of ceramide synthases in cancer and sphingolipid regulation of lethal mitophagy, concerning cancer therapy.

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Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

Journal of Biological Chemistry ◽

10.1074/jbc.m607667200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4437-4446 ◽

Cited By ~ 21

Author(s):

Markus Liebscher ◽

Günther Jahreis ◽

Christian Lücke ◽

Susanne Grabley ◽

Satish Raina ◽

...

Keyword(s):

Protein Folding ◽

Minimal Inhibitory Concentration ◽

Inhibitory Concentration ◽

Acyl Chain ◽

Fatty Acyl ◽

Secondary Amide ◽

Fatty Acyl Chain ◽

Isomerase Activity

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. Nα-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 ± 20 μg/ml. The compounds were shown to compete with fluorophore-labeled σ32-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.

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Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement

Molecular Biology of the Cell ◽

10.1091/mbc.e16-04-0230 ◽

2016 ◽

Vol 27 (16) ◽

pp. 2590-2597 ◽

Cited By ~ 16

Author(s):

Michael Zick ◽

William Wickner

Keyword(s):

Acyl Chain ◽

Fatty Acyl ◽

Fatty Acyl Chain ◽

Rab Gtpase ◽

Lower Phase ◽

Yeast Vacuole ◽

Vacuole Fusion ◽

In Vitro Reconstitution

In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1.

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Effect of metformin on bioactive lipid metabolism in insulin-resistant muscle

Journal of Endocrinology ◽

10.1530/joe-16-0381 ◽

2017 ◽

Vol 233 (3) ◽

pp. 329-340 ◽

Cited By ~ 19

Author(s):

Piotr Zabielski ◽

Marta Chacinska ◽

Karol Charkiewicz ◽

Marcin Baranowski ◽

Jan Gorski ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Acyl Chain ◽

Principal Component ◽

Synthesis Rate ◽

Bioactive Lipids ◽

Insulin Resistant ◽

Isotope Tracer ◽

Lipid Species

Intramuscular accumulation of bioactive lipids leads to insulin resistance and type 2 diabetes (T2D). There is lack of consensus concerning which of the lipid mediators has the greatest impact on muscle insulin action in vivo. Our aim was to elucidate the effects of high-fat diet (HFD) and metformin (Met) on skeletal muscle bioactive lipid accumulation and insulin resistance (IR) in rats. We employed a [U-13C]palmitate isotope tracer and mass spectrometry to measure the content and fractional synthesis rate (FSR) of intramuscular long-chain acyl-CoA (LCACoA), diacylglycerols (DAG) and ceramide (Cer). Eight weeks of HFD-induced intramuscular accumulation of LCACoA, DAG and Cer accompanied by both systemic and skeletal muscle IR. Metformin treatment improved insulin sensitivity at both systemic and muscular level by the augmentation of Akt/PKB and AS160 phosphorylation and decreased the content of DAG and Cer and their respective FSR. Principal component analysis (PCA) of lipid variables revealed that altered skeletal muscle IR was associated with lipid species containing 18-carbon acyl-chain, especially with C18:0-Cer, C18:1-Cer, 18:0/18:2-DAG and 18:2/18:2-DAG, but not palmitate-derived lipids. It is concluded that the insulin-sensitizing action of metformin in skeletal muscle is associated with decreased 18-carbon acyl-chain-derived bioactive lipids.

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Differential effects of the cystic fibrosis lung inflammatory environment on mesenchymal stromal cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00218.2020 ◽

2020 ◽

Vol 319 (6) ◽

pp. L908-L925

Author(s):

Soraia C. Abreu ◽

Thomas H. Hampton ◽

Evan Hoffman ◽

Jacob Dearborn ◽

Alix Ashare ◽

...

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Lavage Fluid ◽

Cell Therapies ◽

Acute Exacerbations ◽

Aspergillus Sp

Growing evidence demonstrates that human mesenchymal stromal cells (MSCs) modify their in vivo anti-inflammatory actions depending on the specific inflammatory environment encountered. Understanding this better is crucial to refine MSC-based cell therapies for lung and other diseases. Using acute exacerbations of cystic fibrosis (CF) lung disease as a model, the effects of ex vivo MSC exposure to clinical bronchoalveolar lavage fluid (BALF) samples, as a surrogate for the in vivo clinical lung environment, on MSC viability, gene expression, secreted cytokines, and mitochondrial function were compared with effects of BALF collected from healthy volunteers. CF BALF samples that cultured positive for Aspergillus sp. (Asp) induced rapid MSC death, usually within several hours of exposure. Further analyses suggested the fungal toxin gliotoxin as a potential mediator contributing to CF BALF-induced MSC death. RNA sequencing analyses of MSCs exposed to either Asp+ or Asp− CF BALF samples identified a number of differentially expressed transcripts, including those involved in interferon signaling, antimicrobial gene expression, and cell death. Toxicity did not correlate with bacterial lung infections. These results suggest that the potential use of MSC-based cell therapies for CF or other lung diseases may not be warranted in the presence of Aspergillus.

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Changes in lipid and proteome composition accompany growth ofBacillus subterraneusMITOT1 under supercritical CO2and may promote acclimation to associated stresses

10.1101/320424 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kyle C. Peet ◽

Kodihalli C. Ravindra ◽

John S. Wishnok ◽

Roger E. Summons ◽

Janelle R. Thompson

Keyword(s):

Amino Acid ◽

Membrane Lipids ◽

Acyl Chain ◽

Acid Stress ◽

Principal Component ◽

Fatty Acyl ◽

Fatty Acyl Chain ◽

Membrane Composition ◽

Glycine Cleavage ◽

Batch Bioreactors

AbstractRecent demonstration that multipleBacillusstrains grow in batch bioreactors containing supercritical (sc) CO2(i.e. >73 atm, >31°C) is surprising given the recognized roles of scCO2as a sterilant and solvent. Growth under scCO2is of interest for biotechnological applications and for microbially-enhanced geologic carbon sequestration. We hypothesize thatBacillusspp. may alter cell wall and membrane composition in response to scCO2-associated stresses. In this study, protein expression and membrane lipids ofB. subterraneusMITOT1 were profiled in cultures grown under headspaces of 1 and 100 atm of CO2or N2. Growth under 100 atm CO2revealed significantly decreased fatty acid branching and increased fatty acyl chain lengths relative to 1 atm cultures. Proteomes of MITOT1 grown under 1 and 100 atm pressures of CO2and N2were similar (Spearman R>0.65), and principal component analysis revealed variation by treatment with the first two principal components corresponding to headspace gas (CO2or N2) and pressure (1 atm and 100 atm), respectively. Amino acid metabolic proteins were enriched under CO2, including the glycine cleavage system, previously shown to be upregulated in acid stress response. These results provide insights into the stationary phase physiology of strains grown under scCO2, suggesting modifications of cell membranes and amino acid metabolism may be involved in response to acidic, high CO2conditions under scCO2.

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Profile of Phosphatidylserine Modifications under Nitroxidative Stress Conditions Using a Liquid Chromatography-Mass Spectrometry Based Approach

Molecules ◽

10.3390/molecules24010107 ◽

2018 ◽

Vol 24 (1) ◽

pp. 107 ◽

Cited By ~ 4

Author(s):

Bruna Neves ◽

Pedro Domingues ◽

Maria Oliveira ◽

Maria Domingues ◽

Tânia Melo

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Acyl Chain ◽

Fatty Acyl ◽

Head Group ◽

Fatty Acyl Chain ◽

Polar Head Group ◽

Derivatives Of

Nitrated lipids have been detected in vitro and in vivo, usually associated with a protective effect. While nitrated fatty acids have been widely studied, few studies reported the nitration and nitroxidation of the phospholipid classes phosphatidylcholine, and phosphatidylethanolamine. However, no information regarding nitrated and nitroxidized phosphatidylserine can be found in the literature. This work aims to identify and characterize the nitrated and nitroxidized derivatives of 1-palmitoyl-2-oleoyl-sn-3-glycero-phosphoserine (POPS), obtained after incubation with nitronium tetrafluoroborate, by liquid chromatography (LC) coupled to mass spectrometry (MS) and tandem MS (MS/MS). Several nitrated and nitroxidized products were identified, namely, nitro, nitroso, nitronitroso, and dinitro derivatives, as well as some nitroxidized species such as nitrosohydroxy, nitrohydroxy, and nitrohydroperoxy. The fragmentation pathways identified were structure-dependent and included the loss of HNO and HNO2 for nitroso and nitro derivatives, respectively. Combined losses of PS polar head group plus HNO or HNO2 and carboxylate anions of modified fatty acyl chain were also observed. The nitrated POPS also showed antiradical potential, demonstrated by the ability to scavenge the ABTS●+ and DPPH● radicals. Overall, this in vitro model of nitration based on LC-MS/MS provided additional insights into the nitrated and nitroxidized derivatives of PS and their fragmentation fingerprinting. This information is a valuable tool for targeted analysis of these modified PS in complex biological samples, to further explore the new clues on the antioxidant potential of nitrated POPS.

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