scholarly journals The Antipsychotic Risperidone Alters Dihydroceramide and Ceramide Composition and Plasma Membrane Function in Leukocytes In Vitro and In Vivo

2021 ◽  
Vol 22 (8) ◽  
pp. 3919
Author(s):  
Alberto Canfrán-Duque ◽  
Óscar Pastor ◽  
David García-Seisdedos ◽  
Yessenia L. Molina ◽  
Bohdan Babiy ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Raft ◽  
Older Patients ◽  
In Vitro Assays ◽  
Fatty Acid Elongase ◽  
Membrane Function ◽  
Increased Risk ◽  
Raft Domains ◽  
Long Chain

Atypical or second-generation antipsychotics are used in the treatment of psychosis and behavioral problems in older persons with dementia. However, these pharmaceutical drugs are associated with an increased risk of stroke in such patients. In this study, we evaluated the effects of risperidone treatment on phospholipid and sphingolipid composition and lipid raft function in peripheral blood mononuclear cells (PBMCs) of older patients (mean age >88 years). The results showed that the levels of dihydroceramides, very-long-chain ceramides, and lysophosphatidylcholines decreased in PBMCs of the risperidone-treated group compared with untreated controls. These findings were confirmed by in vitro assays using human THP-1 monocytes. The reduction in the levels of very-long-chain ceramides and dihydroceramides could be due to the decrease in the expression of fatty acid elongase 3, as observed in THP-1 monocytes. Moreover, risperidone disrupted lipid raft domains in the plasma membrane of PBMCs. These results indicated that risperidone alters phospholipid and sphingolipid composition and lipid raft domains in PBMCs of older patients, potentially affecting multiple signaling pathways associated with these membrane domains.

scholarly journals A Specific Structural Requirement for Ergosterol in Long-chain Fatty Acid Synthesis Mutants Important for Maintaining Raft Domains in Yeast

2002 ◽  
Vol 13 (12) ◽  
pp. 4414-4428 ◽  
Author(s):  
Marlis Eisenkolb ◽  
Christoph Zenzmaier ◽  
Erich Leitner ◽  
Roger Schneiter
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Double Mutant ◽  
Chain Fatty Acid ◽  
Long Chain Fatty Acid ◽  
Structural Requirement ◽  
Fatty Acid Elongase ◽  
Synthetically Lethal ◽  
Raft Domains ◽  
Long Chain

Fungal sphingolipids contain ceramide with a very-long-chain fatty acid (C26). To investigate the physiological significance of the C26-substitution on this lipid, we performed a screen for mutants that are synthetically lethal with ELO3. Elo3p is a component of the ER-associated fatty acid elongase and is required for the final elongation cycle to produce C26 from C22/C24 fatty acids.elo3Δ mutant cells thus contain C22/C24- instead of the natural C26-substituted ceramide. We now report that under these conditions, an otherwise nonessential, but also fungal-specific, structural modification of the major sterol of yeast, ergosterol, becomes essential, because mutations in ELO3 are synthetically lethal with mutations in ERG6. Erg6p catalyzes the methylation of carbon atom 24 in the aliphatic side chain of sterol. The lethality of an elo3Δ erg6Δ double mutant is rescued by supplementation with ergosterol but not with cholesterol, indicating a vital structural requirement for the ergosterol-specific methyl group. To characterize this structural requirement in more detail, we generated a strain that is temperature sensitive for the function of Erg6p in an elo3Δ mutant background. Examination of raft association of the GPI-anchored Gas1p and plasma membrane ATPase, Pma1p, in the conditional elo3Δ erg6 ts double mutant, revealed a specific defect of the mutant to maintain raft association of preexisting Pma1p. Interestingly, in an elo3Δ mutant at 37°C, newly synthesized Pma1p failed to enter raft domains early in the biosynthetic pathway, and upon arrival at the plasma membrane was rerouted to the vacuole for degradation. These observations indicate that the C26 fatty acid substitution on lipids is important for establishing raft association of Pma1p and stabilizing the protein at the cell surface. Analysis of raft lipids in the conditional mutant strain revealed a selective enrichment of ergosterol in detergent-resistant membrane domains, indicating that specific structural determinants on both sterols and sphingolipids are required for their association into raft domains.

scholarly journals Studies of insulin resistance in patients with clinical and subclinical hypothyroidism

2009 ◽  
Vol 160 (5) ◽  
pp. 785-790 ◽  
Author(s):  
Eirini Maratou ◽  
Dimitrios J Hadjidakis ◽  
Anastasios Kollias ◽  
Katerina Tsegka ◽  
Melpomeni Peppa ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
Subclinical Hypothyroidism ◽  
Tolerance Test ◽  
Oral Glucose ◽  
Model Assessment ◽  
Increased Risk

ObjectiveAlthough clinical hypothyroidism (HO) is associated with insulin resistance, there is no information on insulin action in subclinical hypothyroidism (SHO).Design and methodsTo investigate this, we assessed the sensitivity of glucose metabolism to insulin both in vivo (by an oral glucose tolerance test) and in vitro (by measuring insulin-stimulated rates of glucose transport in isolated monocytes with flow cytometry) in 21 euthyroid subjects (EU), 12 patients with HO, and 13 patients with SHO.ResultsAll three groups had comparable plasma glucose levels, with the HO and SHO having higher plasma insulin than the EU (P<0.05). Homeostasis model assessment index was increased in HO (1.97±0.22) and SHO (1.99±0.13) versus EU (1.27±0.16, P<0.05), while Matsuda index was decreased in HO (3.89±0.36) and SHO (4.26±0.48) versus EU (7.76±0.87, P<0.001), suggesting insulin resistance in both fasting and post-glucose state. At 100 μU/ml insulin: i) GLUT4 levels on the monocyte plasma membrane were decreased in both HO (215±19 mean fluorescence intensity, MFI) and SHO (218±24 MFI) versus EU (270±25 MFI, P=0.03 and 0.04 respectively), and ii) glucose transport rates in monocytes from HO (481±30 MFI) and SHO (462±19 MFI) were decreased versus EU (571±15 MFI, P=0.04 and 0.004 respectively).ConclusionsIn patients with HO and SHO: i) insulin resistance was comparable; ii) insulin-stimulated rates of glucose transport in isolated monocytes were decreased due to impaired translocation of GLUT4 glucose transporters on the plasma membrane; iii) these findings could justify the increased risk for insulin resistance-associated disorders, such as cardiovascular disease, observed in patients with HO or SHO.

scholarly journals BAR Domain Proteins Rvs161 and Rvs167 Contribute to Candida albicans Endocytosis, Morphogenesis, and Virulence

2009 ◽  
Vol 77 (9) ◽  
pp. 4150-4160 ◽  
Author(s):  
Lois M. Douglas ◽  
Stephen W. Martin ◽  
James B. Konopka
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Antifungal Drugs ◽  
Host Immune System ◽  
Membrane Function ◽  
Histatin 5 ◽  
Essentially Normal ◽  
Altered Cell

ABSTRACT The Candida albicans plasma membrane plays critical roles in growth and virulence and as a target for antifungal drugs. Three C. albicans genes that encode Bin-Amphiphysin-Rvs homology domain proteins were mutated to define their roles in plasma membrane function. The deletion of RVS161 and RVS167, but not RVS162, caused strong defects. The rvs161Δ mutant was more defective in endocytosis and morphogenesis than rvs167Δ, but both were strongly defective in polarizing actin patches. Other plasma membrane constituents were still properly localized, including a filipin-stained domain at the hyphal tips. An analysis of growth under different in vitro conditions showed that the rvs161Δ and rvs167Δ mutants grew less invasively in agar and also suggested that they have defects in cell wall synthesis and Rim101 pathway signaling. These mutants were also more resistant to the antimicrobial peptide histatin 5 but showed essentially normal responses to the drugs caspofungin and amphotericin. Surprisingly, the rvs161Δ mutant was more sensitive to fluconazole, whereas the rvs167Δ mutant was more resistant, indicating that these mutations cause overlapping but distinct effects on cells. The rvs161Δ and rvs167Δ mutants both showed greatly reduced virulence in mice. However, the mutants were capable of growing to high levels in kidneys. Histological analyses of infected kidneys revealed that these rvsΔ mutants grew in a large fungal mass that was walled off by leukocytes, rather than forming disseminated microabscesses as seen for the wild type. The diminished virulence is likely due to a combination of the morphogenesis defects that reduce invasive growth and altered cell wall construction that exposes proinflammatory components to the host immune system.

scholarly journals TBC1D10C is a cytoskeletal functional linker that modulates cell spreading and phagocytosis in macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fabian R. Villagomez ◽  
Juan D. Diaz-Valencia ◽  
Erasmo Ovalle-García ◽  
Armando Antillón ◽  
Iván Ortega-Blake ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ex Vivo ◽  
Cell Spreading ◽  
Small Gtpases ◽  
Burkholderia Cenocepacia ◽  
In Vitro Assays ◽  
Inhibitory Protein ◽  
Intrinsically Disordered ◽  
Biological Potential

AbstractCell spreading and phagocytosis are notably regulated by small GTPases and GAP proteins. TBC1D10C is a dual inhibitory protein with GAP activity. In immune cells, TBC1D10C is one of the elements regulating lymphocyte activation. However, its specific role in macrophages remains unknown. Here, we show that TBC1D10C engages in functions dependent on the cytoskeleton and plasma membrane reorganization. Using ex vivo and in vitro assays, we found that elimination and overexpression of TBC1D10C modified the cytoskeletal architecture of macrophages by decreasing and increasing the spreading ability of these cells, respectively. In addition, TBC1D10C overexpression contributed to higher phagocytic activity against Burkholderia cenocepacia and to increased cell membrane tension. Furthermore, by performing in vitro and in silico analyses, we identified 27 TBC1D10C-interacting proteins, some of which were functionally classified as protein complexes involved in cytoskeletal dynamics. Interestingly, we identified one unreported TBC1D10C-intrinsically disordered region (IDR) with biological potential at the cytoskeleton level. Our results demonstrate that TBC1D10C shapes macrophage activity by inducing reorganization of the cytoskeleton-plasma membrane in cell spreading and phagocytosis. We anticipate our results will be the basis for further studies focused on TBC1D10C. For example, the specific molecular mechanism in Burkholderia cenocepacia phagocytosis and functional analysis of TBC1D10C-IDR are needed to further understand its role in health and disease.

scholarly journals AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion of Dictyostelium Contractile Vacuoles

2009 ◽  
Vol 20 (20) ◽  
pp. 4278-4288 ◽  
Author(s):  
Yujia Wen ◽  
Irene Stavrou ◽  
Kirill Bersuker ◽  
Rebecca J. Brady ◽  
Arturo De Lozanne ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Contractile Vacuole ◽  
Snare Proteins ◽  
In Vitro Assays ◽  
Accessory Proteins ◽  
Coated Pits ◽  
Contractile Vacuoles ◽  
Null Mutants

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane–coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.

scholarly journals In vitro and in vivo modulation of NADPH-oxidase activity and reactive oxygen species production in human neutrophils by alpha-1 antitrypsin

2021 ◽  
pp. 00234-2021
Author(s):  
Padraig Hawkins ◽  
Thomas McEnery ◽  
Claudie Gabillard-Lefort ◽  
David A Bergin ◽  
Bader Alfawaz ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Ex Vivo ◽  
Human Neutrophils ◽  
Driving Mechanism ◽  
Increased Risk ◽  
Alpha 1 Antitrypsin

Oxidative stress from innate immune cells is a driving mechanism that underlies COPD pathogenesis. Individuals with alpha-1 antitrypsin (AAT) deficiency (AATD) have a dramatically increased risk of developing COPD. To understand this further, the aim of this study was to investigate whether AATD presents with altered neutrophil NADPH-oxidase activation, due to the specific lack of plasma AAT. Experiments were performed using circulating neutrophils isolated from healthy controls and individuals with AATD. Superoxide anion (O2−) production was determined from the rate of reduction of cytochrome c. Quantification of membrane NADPH-oxidase subunits was performed by mass spectrometry and western blot analysis. The clinical significance of our in vitro findings were assessed in patients with AATD and severe COPD receiving intravenous AAT replacement therapy. In vitro, AAT significantly inhibited O2− production by stimulated neutrophils and suppressed receptor stimulation of cyclic adenosimonophosphate (cAMP) and extracellular-signal regulated kinase (ERK)1/2 phosphorylation. In addition, AAT reduced plasma membrane translocation of cytosolic phox components of the NADPH-oxidase. Ex vivo, AATD neutrophils demonstrated increased plasma membrane associated p67phox and p47phox and significantly increased O2− production. The described variance in phox protein membrane assembly was resolved post AAT augmentation therapy in vivo, the effects of which significantly reduced AATD neutrophil O2− production to that of healthy control cells. These results expand our knowledge on the mechanism of neutrophil driven airways disease associated with AATD. Therapeutic AAT augmentation modified neutrophil NADPH-oxidase assembly and ROS production, with implications for clinical use in conditions in which oxidative stress plays a pathogenic role.

pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation

2021 ◽  
Author(s):  
Heidi Pertl-Obermeyer ◽  
Ana Gimeno ◽  
Verena Kuchler ◽  
Evrim Servili ◽  
Shuai Huang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pollen Tube ◽  
Pollen Tube Growth ◽  
Pollen Grains ◽  
Tube Growth ◽  
In Vitro Assays ◽  
Sudden Increase ◽  
Ph Gradients ◽  
Threonine Residue

Abstract Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

scholarly journals Acyl-Coenzyme A Dehydrogenases Are Localized on GLUT4-Containing Vesicles via Association with Insulin-Regulated Aminopeptidase in a Manner Dependent on Its Dileucine Motif

2002 ◽  
Vol 16 (5) ◽  
pp. 1049-1059 ◽  
Author(s):  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Tetsuya Yamada ◽  
Toshifumi Aoyama ◽  
Yasushi Fukushima ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coenzyme A ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Medium Chain ◽  
Dileucine Motif ◽  
Chain Acyl ◽  
Acyl Coenzyme A ◽  
Long Chain

Abstract Insulin-regulated aminopeptidase (IRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of IRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55–82) is reportedly involved in retention of GLUT4-containing vesicles. The region of IRAP fused with glutathione-S-transferase [GST-IRAP(55–82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. The association was nearly abolished by mutation of the dileucine motif of IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.

Iron-mediated interaction of alpha synuclein with lipid raft model membranes

Nanoscale ◽  
2020 ◽  
Vol 12 (14) ◽  
pp. 7631-7640 ◽  
Author(s):  
Fabio Perissinotto ◽  
Chiaramaria Stani ◽  
Elena De Cecco ◽  
Lisa Vaccari ◽  
Valeria Rondelli ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Lipid Raft ◽  
Alpha Synuclein ◽  
Model Membranes ◽  
Membrane Systems ◽  
Pathological Conditions ◽  
Model Cell ◽  
Raft Domains ◽  
Model Cell Membrane

We demonstrated that pathological conditions as accumulation of iron cations promote fast formation of α-synuclein aggregation in vitro, which preferentially interact with lipid-raft domains in model cell membrane systems.

Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance

2020 ◽  
Vol 40 (6) ◽  
pp. 731-745
Author(s):  
Huilong Zhang ◽  
Chen Deng ◽  
Xia Wu ◽  
Jun Yao ◽  
Yanli Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Salt Tolerance ◽  
Transgenic Plants ◽  
Recombinant Protein ◽  
Atpase Activity ◽  
Populus Euphratica ◽  
Hydrolytic Activity ◽  
In Vitro Assays

Abstract Remorins (REMs) play an important role in the ability of plants to adapt to adverse environments. PeREM6.5, a protein of the REM family in Populus euphratica (salt-resistant poplar), was induced by NaCl stress in callus, roots and leaves. We cloned the full-length PeREM6.5 from P. euphratica and transformed it into Escherichia coli and Arabidopsis thaliana. PeREM6.5 recombinant protein significantly increased the H+-ATPase hydrolytic activity and H+ transport activity in P. euphratica plasma membrane (PM) vesicles. Yeast two-hybrid assay showed that P. euphratica REM6.5 interacted with RPM1-interacting protein 4 (PeRIN4). Notably, the PeREM6.5-induced increase in PM H+-ATPase activity was enhanced by PeRIN4 recombinant protein. Overexpression of PeREM6.5 in Arabidopsis significantly improved salt tolerance in transgenic plants in terms of survival rate, root growth, electrolyte leakage and malondialdehyde content. Arabidopsis plants overexpressing PeREM6.5 retained high PM H+-ATPase activity in both in vivo and in vitro assays. PeREM6.5-transgenic plants had reduced accumulation of Na+ due to the Na+ extrusion promoted by the H+-ATPases. Moreover, the H+ pumps caused hyperpolarization of the PM, which reduced the K+ loss mediated by the depolarization-activated channels in the PM of salinized roots. Therefore, we conclude that PeREM6.5 regulated H+-ATPase activity in the PM, thus enhancing the plant capacity to maintain ionic homeostasis under salinity.

Sign in / Sign up

Export Citation Format

Share Document