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Biophysical Journal Jun 2024The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO, CO, Cl, Na, K, NH, and H, which are necessary for regulation of pH and ion...
The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO, CO, Cl, Na, K, NH, and H, which are necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl/HCO exchanger) and NBCe1 (Na-COcotransporter). Previous computational studies of the outward-facing state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed coarse-grained and atomistic molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1, and NDCBE (an Na-CO/Cl exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The recently resolved inward-facing state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, phosphatidylcholine, and sphingomyelin in the three studied proteins, and their potential roles in the SLC4 transport function, conformational transition, and protein dimerization are discussed.
Topics: Molecular Dynamics Simulation; Protein Binding; Sodium-Bicarbonate Symporters; Humans; Cholesterol
PubMed: 38760929
DOI: 10.1016/j.bpj.2024.05.013 -
Antonie Van Leeuwenhoek May 2024A nitrogen-fixing strain designated SG130 was isolated from paddy soil in Fujian Province, China. Strain SG130 was Gram-staining-negative, rod-shaped, and strictly...
A nitrogen-fixing strain designated SG130 was isolated from paddy soil in Fujian Province, China. Strain SG130 was Gram-staining-negative, rod-shaped, and strictly anaerobic. Strain SG130 showed the highest 16S rRNA gene sequence similarities with the type strains Dendrosporobacter quercicolus DSM 1736 (91.7%), Anaeroarcus burkinensis DSM 6283 (91.0%) and Anaerospora hongkongensis HKU 15 (90.9%). Furthermore, the phylogenetic and phylogenomic analysis also suggested strain SG130 clustered with members of the family Sporomusaceae and was distinguished from other genera within this family. Growth of strain SG130 was observed at 25-45 °C (optimum 30 °C), pH 6.0-9.5 (optimum 7.0) and 0-1% (w/v) NaCl (optimum 0.1%). The quinones were Q-8 and Q-9. The polar lipids were phosphatidylserine (PS), phosphatidylethanolamine (PE), glycolipid (GL), phospholipid (PL) and an unidentified lipid (UL). The major fatty acids (> 10%) were iso-C 3OH (26.6%), iso-C (15.6%) and iso-C F (11.4%). The genomic DNA G + C content was 50.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG130 and the most closely related type strain D. quercicolus DSM 1736 (ANI 68.0% and dDDH 20.3%) were both below the cut-off level for species delineation. The average amino acid identity (AAI) between strain SG130 and the most closely related type strain D. quercicolus DSM 1736 was 63.2%, which was below the cut-off value for bacterial genus delineation (65%). Strain SG130 possessed core genes (nifHDK) involved in nitrogen fixation, and nitrogenase activity (106.38 μmol CH g protein h) was examined using the acetylene reduction assay. Based on the above results, strain SG130 is confirmed to represent a novel genus of the family Sporomusaceae, for which the name Azotosporobacter soli gen. nov., sp. nov. is proposed. The type strain is SG130 (= GDMCC 1.3312 = JCM 35641).
Topics: Soil Microbiology; Phylogeny; RNA, Ribosomal, 16S; DNA, Bacterial; Base Composition; Fatty Acids; Bacterial Typing Techniques; China; Phospholipids; Nitrogen Fixation; Sequence Analysis, DNA; Nitrogen-Fixing Bacteria
PubMed: 38755437
DOI: 10.1007/s10482-024-01978-6 -
Microbes and Infection May 2024The changes in host membrane phospholipids are crucial in airway infection pathogenesis. Phospholipase A2 hydrolyzes host cell membranes, producing lyso-phospholipids...
CONTEXT
The changes in host membrane phospholipids are crucial in airway infection pathogenesis. Phospholipase A2 hydrolyzes host cell membranes, producing lyso-phospholipids and free fatty acids, including arachidonic acid (AA), which contributes significantly to lung inflammation.
AIM
Follow these changes and their evolution from day 1, day 3 to day 7 in airway aspirates of 89 patients with COVID-19-associated acute respiratory distress syndrome and examine whether they correlate with the severity of the disease. The patients were recruited in three French intensive care units. The analysis was conducted from admission to the intensive care unit until the end of the first week of mechanical ventilation.
RESULTS
In the airway aspirates, we found significant increases in the levels of host cell phospholipids, including phosphatidyl-serine and phosphatidyl-ethanolamine, and their corresponding lyso-phospholipids. This was accompanied by increased levels of AA and its inflammatory metabolite prostaglandin E2 (PGE2). Additionally, enhanced levels of ceramides, sphingomyelin, and free cholesterol were observed in these aspirates. These lipids are known to be involved in cell death and/or apoptosis, whereas free cholesterol plays a role in virus entry and replication in host cells. However, there were no significant changes in the levels of dipalmitoyl-phosphatidylcholine, the major surfactant phospholipid. A correlation analysis revealed an association between mortality risk and levels of AA and PGE2, as well as host cell phospholipids.
CONCLUSION
Our findings indicate a correlation between heightened cellular phospholipid modifications and variations in AA and PGE2 with the severity of the disease in patients. Nevertheless, there is no indication of surfactant alteration in the initial phases of the illness.
PubMed: 38754811
DOI: 10.1016/j.micinf.2024.105354 -
Reproduction, Fertility, and Development May 2024Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the...
Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.
Topics: Male; Animals; Receptor, Melatonin, MT1; Spermatozoa; Sperm Motility; Sheep; Genotype; Semen Analysis; Polymorphism, Genetic; Reactive Oxygen Species; DNA Fragmentation; Polymorphism, Single Nucleotide
PubMed: 38753960
DOI: 10.1071/RD23235 -
Nutrition and Cancer 2024Lipids participate in many important biological functions through energy storage, membrane structure stabilization, signal transduction, and molecular recognition....
Lipids participate in many important biological functions through energy storage, membrane structure stabilization, signal transduction, and molecular recognition. Previous studies have shown that patients with esophageal squamous cell carcinoma (ESCC) have abnormal lipid metabolism. However, studies characterizing lipid metabolism in ESCC patients through lipidomics are limited. Plasma lipid profiles of 65 ESCC patients and 42 healthy controls (HC) were characterized by lipidomics-based ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Single-factor and multi-factor statistical analysis were used to screen the differences in blood lipids between groups, and combined with component ratio analysis and receiver operating characteristic (ROC) curve diagnostic efficiency assessment, to reveal the potential mechanisms and biomarkers of ESCC. There were significant differences in lipid profiles between the ESCC and HC groups. Thirty-six differential lipids (11 up-regulated and 25 down-regulated) were selected based on the criteria of < .05 and fold change > 1.3 or < 0.77. Glycerophospholipids were the major differential lipids, suggesting that these lipid metabolic pathways exhibit a significant imbalance that may contribute to the development of esophageal squamous cell carcinoma. Among them, the seven candidate biomarkers for esophageal squamous cell carcinoma with the highest diagnostic value are three phosphatidylserine (PS), three fatty acids (FA) and one phosphatidylcholine (PC).
Topics: Humans; Esophageal Squamous Cell Carcinoma; Male; Esophageal Neoplasms; Lipidomics; Female; Middle Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Aged; Lipid Metabolism; Lipids; ROC Curve; Glycerophospholipids; Phosphatidylserines; Fatty Acids
PubMed: 38753560
DOI: 10.1080/01635581.2024.2350097 -
Biochemical and Biophysical Research... Aug 2024Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the...
Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, K at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3-5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.
Topics: Humans; Blood Platelets; Protein Binding; Factor IXa; Binding Sites; Blood Coagulation; Thrombin; Factor X; Flow Cytometry; Phosphatidylserines; Carrier Proteins; Peptides
PubMed: 38749192
DOI: 10.1016/j.bbrc.2024.150099 -
The Journal of Biological Chemistry Jun 2024
PubMed: 38749156
DOI: 10.1016/j.jbc.2024.107356 -
Frontiers in Oncology 2024Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this...
INTRODUCTION
Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated.
METHODS
The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The effects of MK591 were evaluated in pancreatic tumor xenograft model.
RESULTS
MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis.
DISCUSSION
These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.
PubMed: 38746674
DOI: 10.3389/fonc.2024.1387535 -
BioRxiv : the Preprint Server For... Apr 2024Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible...
Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible forms of the parasite, arise during the blood stage of the parasite and develop through 5 morphologically distinct stages. Immature gametocytes (stage I-IV) sequester and develop in the extravascular niche of the bone marrow and possibly spleen. Only mature stage V gametocytes re-enter peripheral circulation to be taken up by mosquitoes for successful onward transmission. We have recently shown that immature, but not mature gametocytes are targets of host immune responses and identified putative target surface antigens. We hypothesize that these antigens play a role in gametocyte sequestration and contribute to acquired transmission-reducing immunity. Here we demonstrate that surface antigen expression, serum reactivity by human IgG, and opsonic phagocytosis by macrophages all show similar dynamics during gametocyte maturation, i.e., on in immature and off in mature gametocytes. Moreover, the switch in surface reactivity coincides with reversal in phosphatidylserine (PS) surface exposure, a marker for red blood cell age and clearance. PS is exposed on the surface of immature gametocytes (as well as in late asexual stages) but is removed from the surface in later gametocyte stages (IV-V). Using parasite reverse genetics and drug perturbations, we confirm that parasite protein export into the host cell and phospholipid scramblase activity are required for the observed surface modifications in asexual and sexual stages. These findings suggest that the dynamic surface remodelling allows (i) immature gametocyte sequestration in bone marrow and (ii) mature gametocyte release into peripheral circulation and immune evasion, therefore contributing to mature gametocyte survival and onward transmission to mosquitoes. Importantly, blocking scramblase activity during gametocyte maturation results in efficient clearance of mature gametocytes, revealing a potential path for transmission blocking interventions. Our studies have important implications for our understanding of parasite biology and form a starting point for novel intervention strategies to simultaneously reduce parasite burden and transmission.
PubMed: 38746342
DOI: 10.1101/2024.04.30.591837 -
American Journal of Human Genetics Jun 2024Anoctamins are a family of Ca-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease...
Anoctamins are a family of Ca-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patch-clamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease.
Topics: Humans; Anoctamins; Mutation, Missense; Male; Female; Epilepsy; Child; Phospholipid Transfer Proteins; Genetic Association Studies; Pedigree; Calcium; Genes, Dominant; Child, Preschool; HEK293 Cells; Adolescent
PubMed: 38744284
DOI: 10.1016/j.ajhg.2024.04.014