scholarly journals Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.

1992 ◽  
Vol 118 (1) ◽  
pp. 63-69 ◽  
Author(s):  
W J Chang ◽  
K G Rothberg ◽  
B A Kamen ◽  
R G Anderson
Keyword(s):  
Folate Receptor ◽  
Cholesterol Content ◽  
Cholesterol Depletion ◽  
Morphologic Data

The folate receptor is clustered on the surface of MA104 cells in association with caveolae. This relationship is thought to be essential for the proper internalization and recycling of the receptor during the delivery of 5-methyltetrahydrofolate to the cytoplasm of folate-depleted cells. Both the clustered organization of the receptor and the integrity of caveolae are disrupted when cells are deprived of cholesterol. We now show that cholesterol depletion of MA104 cells markedly reduces the rate of 5-methyltetrahydrofolate internalization and causes a 70% decline in the number of receptors present in the internal, recycling compartment. This effect is consistent with morphologic data showing that cholesterol-depleted MA104 cells have a reduced number of caveolae as well as fewer receptors per caveolae.

scholarly journals The rate of sphingomyelin synthesis de novo is influenced by the level of cholesterol in cultured human skin fibroblasts

1998 ◽  
Vol 335 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Petra LEPPIMÄKI ◽  
Robert KRONQVIST ◽  
J. Peter SLOTTE
Keyword(s):  
Palmitic Acid ◽  
Human Skin ◽  
De Novo ◽  
Cholesterol Content ◽  
Skin Fibroblasts ◽  
Cholesterol Depletion ◽  
Human Skin Fibroblasts ◽  
Cellular Cholesterol ◽  
Cholesterol Levels ◽  
A Cell

Plasma membrane sphingomyelin (SM) is known to affect the cellular distribution of cholesterol. The aim of this work was to examine how SM homoeostasis in human skin fibroblasts is affected by alterations in the level of cholesterol in the cell. The cellular cholesterol level was decreased by exposing cells to 2-hydroxypropyl-β-cyclodextrin, and increased by exposing cells to cholesterol–methyl-β-cyclodextrin inclusion complexes. A lowering of the cellular unesterified cholesterol content by 20% was shown to increase the incorporation of [14C]palmitic acid into SM by 70%. Subsequently, the cellular SM mass was shown to be increased (24% increase after a 24 h period). Since l-cycloserine completely abolished the increased incorporation of [14C]palmitic acid into SM in cholesterol-depleted cells, we concluded that the de novo synthesis of the sphingosine backbone of SM was activated in cholesterol-depleted cells. This conclusion was further verified by performing a cell-free assay of serine C-palmitoyltransferase (SPT) in cholesterol-depleted cells, which showed that the activity of the enzyme was increased by 30% after cholesterol depletion. Most of the newly synthesized SM in cholesterol-depleted cells was susceptible to degradation by sphingomyelinase, indicating that it was transported efficiently to the cell surface. Loading of fibroblasts with cholesterol had essentially the opposite effects on SM homoeostasis to those of cholesterol depletion, i.e. 20–30% decreased incorporation of [14C]palmitic acid into SM and decreased activity of SPT. The results of this study show that cellular cholesterol levels have marked effects on the homoeostasis of SM.

scholarly journals Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1120
Author(s):  
Hélène Pollet ◽  
Anne-Sophie Cloos ◽  
Amaury Stommen ◽  
Juliette Vanderroost ◽  
Louise Conrard ◽  
...  
Keyword(s):  
Cholesterol Content ◽  
Membrane Curvature ◽  
Acid Sphingomyelinase ◽  
Cholesterol Depletion ◽  
Membrane Composition ◽  
Biophysical Properties ◽  
Erythrocyte Morphology ◽  
Lipid Domain ◽  
Functional Consequences

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in OK cells

2005 ◽  
Vol 289 (1) ◽  
pp. F154-F165 ◽  
Author(s):  
Sophia Y. Breusegem ◽  
Nabil Halaihel ◽  
Makoto Inoue ◽  
Hubert Zajicek ◽  
Eleanor Lederer ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Cholesterol Depletion ◽  
Moderate Increase ◽  
Protein Abundance ◽  
Lipid Microdomains ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Per Se

We previously showed an inverse correlation between membrane cholesterol content and Na-Pi cotransport activity during the aging process and adaptation to alterations in dietary Pi in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85–F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-Pi cotransport activity and apical membrane Na-Pi protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with β-methyl cyclodextrin (β-MCD) resulted in a significant increase in Na-Pi cotransport activity accompanied by a moderate increase in apical membrane Na-Pi protein abundance and no alteration of total cellular Na-Pi protein abundance. Conversely, acute cholesterol enrichment achieved with β-MCD/cholesterol resulted in a significant decrease in Na-Pi cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-Pi protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-Pi cotransport activity by diverse mechanisms that also include significant interactions of Na-Pi protein with lipid microdomains.

scholarly journals Cholesterol depletion enhances TGF-β Smad signaling by increasing c-Jun expression through a PKR-dependent mechanism

2018 ◽  
Vol 29 (20) ◽  
pp. 2494-2507 ◽  
Author(s):  
Keren E. Shapira ◽  
Marcelo Ehrlich ◽  
Yoav I. Henis
Keyword(s):  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Cholesterol Content ◽  
Receptor Activation ◽  
Cholesterol Homeostasis ◽  
Cholesterol Depletion ◽  
Enhanced Sensitivity ◽  
Eif2α Phosphorylation ◽  
Mild Reduction ◽  
Free Membrane

Transforming growth factor-β (TGF-β) plays critical roles in numerous physiological and pathological responses. Cholesterol, a major plasma membrane component, can have pronounced effects on signaling responses. Cells continually monitor cholesterol content and activate multilayered transcriptional and translational signaling programs, following perturbations to cholesterol homeostasis (e.g., statins, the commonly used cholesterol-reducing drugs). However, the cross-talk of such programs with ligand-induced signaling responses (e.g., TGF-β signaling) remained unknown. Here, we studied the effects of a mild reduction in free (membrane-associated) cholesterol on distinct components of TGF-β–signaling pathways. Our findings reveal a new regulatory mechanism that enhances TGF-β–signaling responses by acting downstream from receptor activation. Reduced cholesterol results in PKR-dependent eIF2α phosphorylation, which enhances c-Jun translation, leading in turn to higher levels of JNK-mediated c-Jun phosphorylation. Activated c-Jun enhances transcription and expression of Smad2/3. This leads to enhanced sensitivity to TGF-β stimulation, due to increased Smad2/3 expression and phosphorylation. The phospho/total Smad2/3 ratio remains unchanged, indicating that the effect is not due to altered receptor activity. We propose that cholesterol depletion induces overactivation of PKR, JNK, and TGF-β signaling, which together may contribute to the side effects of statins in diverse disease settings.

Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its β-adrenergic enhancement in ventricular myocytes

2008 ◽  
Vol 294 (1) ◽  
pp. H285-H292 ◽  
Author(s):  
Hiroto Tsujikawa ◽  
Yumei Song ◽  
Makino Watanabe ◽  
Haruko Masumiya ◽  
Sachin A. Gupte ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Ventricular Myocytes ◽  
Cholesterol Content ◽  
Cholesterol Depletion ◽  
Signaling Proteins ◽  
External Application ◽  
Pipette Solution ◽  
Rat Myocytes ◽  
G Protein Coupled

Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae, where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl-β-cyclodextrin (MβCD), we studied the role of cholesterol in the modulation of L-type Ca2+ currents ( ICa,L). MβCD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM MβCD for 10 min, a positive shift in membrane potential at half-maximal activation ( V0.5) from −8 to −2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased ICa,L approximately threefold and caused a negative shift in V0.5 in control cells, but it did not increase ICa,L in MβCD-treated myocytes, nor did it shift V0.5. The effect of MβCD (10 or 30 mM) was concentration dependent: 30 mM MβCD suppressed the ISO-induced increase in ICa,L more effectively than 10 mM MβCD. MβCD dialysis also abolished the increase in ICa,L elicited by forskolin or dibutyryl cAMP, but not that elicited by (−)BAY K 8644. External application of MβCD-cholesterol complex to rat myocytes attenuated the MβCD-mediated inhibition of the ISO-induced increase of ICa,L. Biochemical analysis confirmed that the myocytes′ cholesterol content was diminished by MβCD and increased by MβCD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal ICa,L and β-adrenergic cAMP/PKA-mediated increases in ICa,L. We suggest that cholesterol affects the structural coupling between L-type Ca2+ channels and adjacent regulatory proteins.

scholarly journals The Membrane Cholesterol Modulates the Interaction Between 17-βEstradiol and the BK Channel

2021 ◽  
Vol 12 ◽  
Author(s):  
Sara T. Granados ◽  
Ramon Latorre ◽  
Yolima P. Torres
Keyword(s):  
Cholesterol Content ◽  
Bk Channels ◽  
Bk Channel ◽  
Cholesterol Concentration ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Vascular Cells ◽  
Channel Activity ◽  
Channel Activation ◽  
Α Subunit

BK channels are composed by the pore forming α subunit and, in some tissues, is associated with different accessory β subunits. These proteins modify the biophysical properties of the channel, amplifying the range of BK channel activation according to the physiological context. In the vascular cells, the pore forming BKα subunit is expressed with the β1 subunit, where they play an essential role in the modulation of arterial tone and blood pressure. In eukaryotes, cholesterol is a structural lipid of the cellular membrane. Changes in the ratio of cholesterol content in the plasma membrane (PM) regulates the BK channel activation altering its open probability, and hence, vascular contraction. It has been shown that the estrogen 17β-Estradiol (E2) causes a vasodilator effect in vascular cells, inducing a leftward shift in the V0.5 of the GV curve. Here, we evaluate whether changes in the membrane cholesterol concentration modify the effect that E2 induces on the BKα/β1 channel activity. Using binding and electrophysiology assays after cholesterol depletion or enrichment, we show that the cholesterol enrichment significantly decreases the expression of the α subunit, while cholesterol depletion increased the expression of that α subunit. Additionally, we demonstrated that changes in the membrane cholesterol cause the loss of the modulatory effect of E2 on the BKα/β1 channel activity, without affecting the E2 binding to the complex. Our data suggest that changes in membrane cholesterol content could affect channel properties related to the E2 effect on BKα/β1 channel activity. Finally, the results suggest that an optimal membrane cholesterol content is essential for the activation of BK channels through the β1 subunit.

scholarly journals Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

2009 ◽  
Vol 84 (2) ◽  
pp. 983-992 ◽  
Author(s):  
Katrin Schlie ◽  
Anna Maisa ◽  
Frank Lennartz ◽  
Ute Ströher ◽  
Wolfgang Garten ◽  
...  
Keyword(s):  
Cholesterol Content ◽  
Proteolytic Processing ◽  
Viral Envelope ◽  
Surface Glycoprotein ◽  
Cholesterol Depletion ◽  
Virus Infectivity ◽  
Lassa Virus ◽  
Oligomeric State ◽  
Virus Envelope ◽  
The Impact

ABSTRACT Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.

Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin

2006 ◽  
Vol 291 (4) ◽  
pp. C757-C771 ◽  
Author(s):  
Thomas Kjær Klausen ◽  
Charlotte Hougaard ◽  
Else K. Hoffmann ◽  
Stine F. Pedersen
Keyword(s):  
Actin Polymerization ◽  
Rho Kinase ◽  
Cholesterol Content ◽  
Water Soluble ◽  
Cholesterol Depletion ◽  
Blocking Antibody ◽  
Cellular Cholesterol ◽  
Actin Organization ◽  
Activation Rate ◽  
Current Activation

The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by ∼20% in modestly, and ∼50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P2 analogue or a PtdIns(4,5)P2-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P2. It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization.

scholarly journals Cholesterol regulates oxysterol binding protein (OSBP) phosphorylation and Golgi localization in Chinese hamster ovary cells: correlation with stimulation of sphingomyelin synthesis by 25-hydroxycholesterol

1998 ◽  
Vol 336 (1) ◽  
pp. 247-256 ◽  
Author(s):  
Margo K. STOREY ◽  
David M. BYERS ◽  
Harold W. COOK ◽  
Neale D. RIDGWAY
Keyword(s):  
Golgi Apparatus ◽  
Chinese Hamster Ovary ◽  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Cholesterol Content ◽  
Cholesterol Depletion ◽  
Experimental Conditions ◽  
Oxysterol Binding Protein

Sphingomyelin (SM) and cholesterol content is positively correlated in cellular membranes, and in several pathological and experimental conditions there is evidence for coregulation. The potential role of oxysterols and oxysterol binding protein (OSBP) in mediating the coregulation of cholesterol and SM was examined using Chinese hamster ovary (CHO) and cholesterol auxotrophic, sterol regulatory defective (SRD) 6 cells. SRD 6 cells grown in the presence or absence of cholesterol for 24 h displayed a 30–50% reduction in SM synthesis compared with control CHO 7 cells. SM synthesis in CHO 7 and cholesterol-supplemented SRD 6 cells was stimulated 2-fold by 25-hydroxycholesterol, but cholesterol-starved SRD 6 cells were unresponsive. Basal and 25-hydroxycholesterol-stimulated SM synthesis was also inhibited in lovastatin-treated wild-type CHO-K1 cells. Lack of 25-hydroxycholesterol activation of SM synthesis in cholesterol-starved SRD 6 and lovastatin-treated CHO-K1 cells was correlated with dephosphorylation of OSBP. In SRD 6 cells, this was evident after 12 h of cholesterol depletion, it occurred equally at all phosphorylation sites and was exacerbated by 25-hydroxycholesterol. Unlike CHO 7 cells, where OSBP was observed in small vesicles and the cytoplasm, OSBP in cholesterol-starved SRD 6 cells was constitutively localized in the Golgi apparatus. Supplementation with non-lipoprotein cholesterol promoted redistribution to vesicles and the cytoplasm. Similarly, OSBP in CHO-K1 cells grown in delipidated serum was predominantly in the Golgi apparatus. Low-density lipoprotein (LDL) supplementation of CHO-K1 cells caused the redistribution of OSBP to the cytoplasm and small vesicles, and this effect was blocked by pharmacological agents {3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one and progesterone}, which inhibited LDL cholesterol efflux from lysosomes. The results showed that localization of OSBP between the Golgi apparatus and a cytoplasmic/vesicular compartment was responsive to changes in cholesterol content and trafficking. In cholesterol depleted SRD 6 cells, this was accompanied by dephosphorylation of OSBP and attenuation of 25-hydroxycholesterol activation of SM synthesis.

scholarly journals Role of cholesterol in substrate recognition by $$\gamma$$-secretase

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Łukasz Nierzwicki ◽  
Michał Olewniczak ◽  
Paweł Chodnicki ◽  
Jacek Czub
Keyword(s):  
Substrate Binding ◽  
Cholesterol Content ◽  
Cholesterol Depletion ◽  
Membrane Thickness ◽  
Gamma Secretase ◽  
Hydrophobic Mismatch ◽  
Multiple Substrates ◽  
Dynamics Simulations ◽  
Amyloid Polypeptides

Abstract$$\gamma$$ γ -Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer’s Disease among the most prominent examples. The activity of $$\gamma$$ γ -secretase strictly depends on the membrane cholesterol content, yet the mechanistic role of cholesterol in the substrate binding and cleavage remains unclear. In this work, we used all-atom molecular dynamics simulations to examine the role of cholesterol in the initial binding of a direct precursor of $$\beta$$ β -amyloid polypeptides by $$\gamma$$ γ -secretase. We showed that in cholesterol-rich membranes, both the substrate and the enzyme region proximal to the active site induce a local membrane thinning. With the free energy methods we found that in the presence of cholesterol the substrate binds favorably to the identified exosite, while cholesterol depletion completely abolishes the binding. To explain these findings, we directly examined the role of hydrophobic mismatch in the substrate binding to $$\gamma$$ γ -secretase, showing that increased membrane thickness results in higher propensity of the enzyme to bind substrates. Therefore, we propose that cholesterol promotes substrate binding to $$\gamma$$ γ -secretase by increasing the membrane thickness, which leads to the negative hydrophobic mismatch between the membrane and binding partners.

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