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Scientific Reports Aug 2023The occurrence of methyl carbamates of phosphatidylethanolamines and phosphatidylserines in the lipid extract of mitochondria obtained from mouse embryonic fibroblasts...
The occurrence of methyl carbamates of phosphatidylethanolamines and phosphatidylserines in the lipid extract of mitochondria obtained from mouse embryonic fibroblasts was ascertained by hydrophilic interaction liquid chromatography with electrospray ionization single and multi-stage mass spectrometry, performed using sinergically a high resolution (quadrupole-Orbitrap) and a low resolution (linear ion trap) spectrometer. Two possible routes to the synthesis of methyl carbamates of phospholipids were postulated and evaluated: (i) a chemical transformation involving phosgene, occurring as a photooxidation by-product in the chloroform used for lipid extraction, and methanol, also used for the latter; (ii) an enzymatic methoxycarbonylation reaction due to an accidental bacterial contamination, that was unveiled subsequently on the murine mitochondrial sample. A specific lipid extraction performed on a couple of standard phosphatidyl-ethanolamines/-serines, based on purposely photo-oxidized chloroform and deuterated methanol, indicated route (i) as negligible in the specific case, thus highlighting the enzymatic route related to bacterial contamination as the most likely source of methyl carbamates. The unambiguous recognition of the latter might represent the starting point toward a better understanding of their generation in biological systems and a minimization of their occurrence when an artefactual formation is ascertained.
Topics: Animals; Mice; Phosphatidylethanolamines; Chloroform; Fibroblasts; Methanol; Phosphatidylserines; Carbamates; Mitochondria
PubMed: 37633960
DOI: 10.1038/s41598-023-40357-5 -
Proceedings of the National Academy of... Aug 2023The membrane contact site ER/PM junctions are hubs for signaling pathways, including Ca signaling. Phosphatidylserine (PtdSer) mediates various physiological functions;...
The membrane contact site ER/PM junctions are hubs for signaling pathways, including Ca signaling. Phosphatidylserine (PtdSer) mediates various physiological functions; however, junctional PtdSer composition and the role of PtdSer in Ca signaling and Ca-dependent gene regulation are not understood. Here, we show that STIM1-formed junctions are required for PI(4)P/PtdSer exchange by ORP5 and ORP8, which have reciprocal lipid exchange modes and function as a rheostat that sets the junctional PtdSer/PI(4)P ratio. Targeting the ORP5 and ORP8 and their lipid transfer ORD domains to PM subdomains revealed that ORP5 sets low and ORP8 high junctional PI(4)P/PtdSer ratio that controls STIM1-STIM1 and STIM1-Orai1 interaction and the activity of the SERCA pump to determine the pattern of receptor-evoked Ca oscillations, and consequently translocation of NFAT to the nucleus. Significantly, targeting the ORP5 and ORP8 ORDs to the STIM1 ER subdomain reversed their function. Notably, changing PI(4)P/PtdSer ratio by hydrolysis of PM or ER PtdSer with targeted PtdSer-specific PLA1a1 reproduced the ORPs function. The function of the ORPs is determined both by their differential lipid exchange modes and by privileged localization at the ER/PM subdomains. These findings reveal a role of PtdSer as a signaling lipid that controls the available PM PI(4)P, the unappreciated role of ER PtdSer in cell function, and the diversity of the ER/PM junctions. The effect of PtdSer on the junctional PI(4)P level should have multiple implications in cellular signaling and functions.
Topics: Phosphatidylserines; Signal Transduction; Cell Nucleus; Hydrolysis; Mitochondrial Membranes
PubMed: 37607230
DOI: 10.1073/pnas.2301410120 -
Journal For Immunotherapy of Cancer Dec 2023CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various...
BACKGROUND
CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach.
METHODS
To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography.
RESULTS
Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling.
CONCLUSION
Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.
Topics: Humans; Hepatitis A Virus Cellular Receptor 2; Single-Domain Antibodies; Phosphatidylserines; Annexin A5; T-Lymphocyte Subsets
PubMed: 38040419
DOI: 10.1136/jitc-2023-007631 -
Thrombosis and Haemostasis Dec 2023Although thrombosis events are the leading complication of uremia, their mechanism is largely unknown. The interaction between endothelial cells (ECs) and red blood...
BACKGROUND
Although thrombosis events are the leading complication of uremia, their mechanism is largely unknown. The interaction between endothelial cells (ECs) and red blood cells (RBCs) in uremic solutes and its prothrombotic role need to be investigated.
METHODS AND RESULTS
Here, we established an in vitro co-incubation model of uremic RBC and EC as well as a uremic rat model induced by adenine. Using flow cytometry, confocal microscopy, and electron microscopy, we found increased erythrophagocytosis by EC accompanied by increased reactive oxygen species, lipid peroxidation, and impairment of mitochondria, indicating that ECs undergo ferroptosis. Further investigations showed increased proteins' expression of heme oxygenase-1 and ferritin and labile iron pool accumulation in EC, which could be suppressed by deferoxamine (DFO). The ferroptosis-negative regulators glutathione peroxidase 4 and SLC7A11 were decreased in our erythrophagocytosis model and could be enhanced by ferrostatin-1 or DFO. In vivo, we observed that vascular EC phagocytosed RBC and underwent ferroptosis in the kidney of the uremic rat, which could be inhibited by blocking the phagocytic pathway or inhibiting ferroptosis. Next, we found that the high tendency of thrombus formation was accompanied by erythrophagocytosis-induced ferroptosis in vitro and in vivo. Importantly, we further revealed that upregulated TMEM16F expression mediated phosphatidylserine externalization on ferroptotic EC, which contributed to a uremia-associated hypercoagulable state.
CONCLUSION
Our results indicate that erythrophagocytosis-triggered ferroptosis followed by phosphatidylserine exposure of EC may play a key role in uremic thrombotic complications, which may be a promising target to prevent thrombogenesis of uremia.
Topics: Rats; Animals; Ferroptosis; Endothelial Cells; Phosphatidylserines; Erythrocytes; Thrombosis; Uremia
PubMed: 37364609
DOI: 10.1055/a-2117-7890 -
Scientific Reports Jan 2024SLMO2 is a lipid transporter that transports phosphatidylserine to the interior of mitochondria, also known as PRELID3B, which plays an important role in lipid...
SLMO2 is a lipid transporter that transports phosphatidylserine to the interior of mitochondria, also known as PRELID3B, which plays an important role in lipid metabolism. It has also been reported to be involved in the growth process of breast and lung tumors. However, its functions and underlying mechanisms in cancer progress remain elusive, and the potential as pan-cancer biomarker and therapeutic target remains unexplored. Using the TCGA project and GEO database, we performed pan-cancer analysis of SLMO2, which including the expression pattern, prognostic value, mutation landscape, methylation modification, protein-protein interaction network and the relationship between SLMO2 expression and immune infiltration. KEGG enrichment analysis was also performed to predict function and relevant cellular pathways of SLMO2. In addition, proliferation and migration assays were performed to detect the proliferation and metastasis capacity of breast cancer and lung cancer cells. In our study, we found that SLMO2 was overexpressed in pan-cancer and the elevated expression of SLMO2 was correlated with poorer prognosis. SLMO2 mutations were distributed in a variety of tumors and correlated with prognosis. Promoter methylation analysis showed that SLMO2 methylation levels were lower in most tumors compared with normal tissues, while a few tumors showed increased methylation levels of SLMO2. SLMO2 expression was also positively correlated with immune infiltration of MDSCs. Further pathway enrichment analysis indicated that SLMO2 was involved in regulating of cytoplasmic transport and other oncogenic processes. In vitro experiments have shown that SLMO2 promotes the proliferation and migration of breast cancer and lung cancer cells. In conclusion, our findings suggested that SLMO2 was a potential prognostic and immunological marker in pan-cancer. This study suggested a potential strategy for targeting SLMO2 to treat tumors, including manipulating tumor growth or the tumor microenvironment, especially the infiltration of MDSC.
Topics: Humans; Female; Prognosis; Breast Neoplasms; Lung Neoplasms; Biomarkers, Tumor; Biological Transport; Tumor Microenvironment
PubMed: 38212657
DOI: 10.1038/s41598-024-51720-5 -
Nature Microbiology Apr 2024Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles...
Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles (EVs) in semen and saliva inhibit Zika virus infection. However, the antiviral spectrum and underlying mechanism remained unclear. Here we applied lipidomics and flow cytometry to show that these EVs expose phosphatidylserine (PS). By blocking PS receptors, targeted by Zika virus in the process of apoptotic mimicry, they interfere with viral attachment and entry. Consequently, physiological concentrations of EVs applied in vitro efficiently inhibited infection by apoptotic mimicry dengue, West Nile, Chikungunya, Ebola and vesicular stomatitis viruses, but not severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus 1, hepatitis C virus and herpesviruses that use other entry receptors. Our results identify the role of PS-rich EVs in body fluids in innate defence against infection via viral apoptotic mimicries, explaining why these viruses are primarily transmitted via PS-EV-deficient blood or blood-ingesting arthropods rather than direct human-to-human contact.
Topics: Female; Humans; Phosphatidylserines; Viruses; Extracellular Vesicles; Virus Attachment; Zika Virus; Body Fluids; Zika Virus Infection
PubMed: 38528146
DOI: 10.1038/s41564-024-01637-6 -
Acta Biomaterialia Oct 2023At present, surgical debridement and systematic administration of antibiotics represent the mainstay of treatment for chronic osteomyelitis. However, it is now...
At present, surgical debridement and systematic administration of antibiotics represent the mainstay of treatment for chronic osteomyelitis. However, it is now understood that Staphylococcus aureus (S. aureus) can survive within excessively polarized M2 macrophages and evade antibiotics, accounting for the high recurrence of chronic osteomyelitis. Effective treatments for intracellular infection have rarely been reported. Herein, we designed an in situ sprayed liposomes hydrogels spray with macrophage-targeted effects and the ability to reverse polarization and eradicate intracellular bacteria to reduce the recurrence of osteomyelitis. Resiquimod (R848)-loaded and phosphatidylserine (PS)-coating nanoliposomes were introduced into fibrinogen and thrombin to form the PSL-R848@Fibrin spray. Characterization and phagocytosis experiments were performed to confirm the successful preparation of the PSL-R848@Fibrin spray. Meanwhile, in vitro cell experiments validated its ability to eliminate intracellular S. aureus by reprogramming macrophages from the M2 to the M1 phenotype. Additionally, we established a chronic osteomyelitis rat model to simulate the treatment and recurrence process. Histological analysis demonstrated a significant increase in M1 macrophages and the elimination of intracellular bacteria. Imaging revealed a significant decrease in osteomyelitis recurrence. Overall, the liposome hydrogels could target macrophages to promote antibacterial properties against intracellular infection and reduce the recurrence of chronic osteomyelitis, providing the foothold for improving the outcomes of this patient population. STATEMENT OF SIGNIFICANCE: Chronic osteomyelitis remains a high recurrence although undergoing traditional treatment of debridement and antibiotics. S. aureus can survive within the excessively polarized M2 macrophages to evade the effects of antibiotics. However, few studies have sought to investigate effective intracellular bacteria eradication. Herein, we designed a macrophage-targeted R848-containing liposomes fibrin hydrogels spray (PSL-R848@Fibrin) that can reprogram polarization of macrophages and eradicate intracellular bacteria for osteomyelitis treatment. With great properties of rapid gelation, strong adhesion, high flexibility and fit-to-shape capacity, the facile-operated immunotherapeutic in-situ-spray fibrin hydrogels exhibited huge promise of reversing polarization and fighting intracellular infections. Importantly, we revealed a hitherto undocumented treatment strategy for reducing the recurrence of chronic osteomyelitis and potentially improving the prognosis of chronic osteomyelitis patients.
Topics: Humans; Rats; Animals; Liposomes; Hydrogels; Staphylococcus aureus; Osteomyelitis; Anti-Bacterial Agents; Staphylococcal Infections; Fibrin
PubMed: 37516419
DOI: 10.1016/j.actbio.2023.07.039 -
Life (Basel, Switzerland) Dec 2023Eryptosis stimulated by anticancer drugs can lead to anemia in patients. β-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however,...
BACKGROUND
Eryptosis stimulated by anticancer drugs can lead to anemia in patients. β-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however, its effect on the structure and function of human red blood cells (RBCs) remains unexplored. The aim of this study was to investigate the hemolytic and eryptotic activities and underlying molecular mechanisms of CPO in human RBCs.
METHODS
Cells were treated with 10-100 μM of CPO for 24 h at 37 °C, and hemolysis, LDH, AST, and AChE activities were photometrically assayed. Flow cytometry was employed to determine changes in cell volume from FSC, phosphatidylserine (PS) externalization by annexin-V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (HDCFDA). Cells were also cotreated with CPO and specific signaling inhibitors and antihemolytic agents. Furthermore, whole blood was exposed to CPO to assess its toxicity to other peripheral blood cells.
RESULTS
CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca accumulation, oxidative stress, and reduced AChE activity. The toxicity of CPO was ameliorated by D4476, staurosporin, and necrosulfonamide. ATP and PEG 8000 protected the cells from hemolysis, while urea and isotonic sucrose had opposite effects.
CONCLUSIONS
CPO stimulates hemolysis and eryptosis through energy depletion, Ca buildup, oxidative stress, and the signaling mediators casein kinase 1α, protein kinase C, and mixed lineage kinase domain-like pseudokinase. Development of CPO as an anticancer therapeutic must be approached with prudence to mitigate adverse effects on RBCs using eryptosis inhibitors, Ca channel blockers, and antioxidants.
PubMed: 38137900
DOI: 10.3390/life13122299 -
Journal of Thrombosis and Haemostasis :... Sep 2023The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against...
BACKGROUND
The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against β2-glycoprotein I (β2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against β2GPI and prothrombin cause APC resistance is currently unclear.
OBJECTIVES
To investigate how anti-β2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance.
METHODS
The effects of anti-β2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies.
RESULTS
APC resistance was observed in LA-positive patients with anti-β2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-β2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-β2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-β2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306.
CONCLUSION
Anti-β2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.
Topics: Humans; Activated Protein C Resistance; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Factor V; Lupus Coagulation Inhibitor; Phosphatidylserines; Protein C; Prothrombin; Thrombosis
PubMed: 37290588
DOI: 10.1016/j.jtha.2023.05.024 -
Nature Communications Sep 2023The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is...
The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is activated by caspase-mediated cleavage and binding of the XRCC4 fragment. Here, we show that extracellular calcium is an additional factor needed to activate Xkr4. The constitutively active mutant of Xkr4 is found to induce phospholipid scrambling in an extracellular, but not intracellular, calcium-dependent manner. Importantly, other Xkr family members also require extracellular calcium for activation. Alanine scanning shows that D123 and D127 of TM1 and E310 of TM3 coordinate calcium binding. Moreover, lysine scanning demonstrates that the E310K mutation-mediated salt bridge between TM1 and TM3 bypasses the requirement of calcium. Cysteine scanning proves that disulfide bond formation between TM1 and TM3 also activates phospholipid scrambling without calcium. Collectively, this study shows that extracellular calcium functions as a molecular glue for TM1 and TM3 of Xkr proteins for activation, thus demonstrating a regulatory mechanism for multi-transmembrane region-containing proteins.
Topics: Calcium; Alanine; Biological Transport; Caspases; Phosphatidylserines
PubMed: 37696806
DOI: 10.1038/s41467-023-40934-2