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Scientific Reports Dec 2016Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes....
Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-β interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised.
Topics: Amyloid; Animals; Benzothiazoles; Catalytic Domain; Cell Membrane; Cell Survival; Circular Dichroism; Flow Cytometry; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lipid Bilayers; Liposomes; Microscopy, Atomic Force; Microscopy, Confocal; Mitochondria; Molecular Conformation; PC12 Cells; Permeability; Phosphatidylcholines; Phospholipids; Rats; Subcellular Fractions; Thiazoles; Tyrosine 3-Monooxygenase
PubMed: 28004763
DOI: 10.1038/srep39488 -
Methods in Molecular Biology (Clifton,... 2017Releasing sterols to the extracellular milieu is an important part of sterol homeostasis in cells and in the body. ATP-binding cassette transporter A1 (ABCA1) plays an...
Releasing sterols to the extracellular milieu is an important part of sterol homeostasis in cells and in the body. ATP-binding cassette transporter A1 (ABCA1) plays an essential role in cellular phospholipid and sterol release to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), the major apolipoprotein in high-density lipoprotein (HDL), and constitutes the first step in the formation of nascent HDL. Loss-of-function mutations in the ABCA1 gene lead to a rare disease known as Tangier disease that causes severe deficiency in plasma HDL level. Mammalian cells receive exogenous cholesterol mainly from low-density lipoprotein. In addition, they synthesize cholesterol endogenously, as well as multiple precursor sterols that are sterol intermediates en route to be converted to cholesterol. HDL contains phospholipids, cholesterol, and precursor sterols, and ABCA1 has an ability to release phospholipids and various sterol molecules. Recent studies using model cell lines showed that ABCA1 prefers to use sterols newly synthesized endogenously as its preferred substrate, rather than cholesterol derived from LDL or cholesterol being recycled within the cells. Here, we describe several methods at the cell culture level to monitor ABCA1-dependent release of sterol molecules to apoA-I present at the cell exterior. Sterol release can be assessed by using a simple colorimetric enzymatic assay, and/or by monitoring the radioactivities of radiolabeled cholesterol incorporated into the cells, and/or of sterols biosynthesized from radioactive acetate, and/or by using gas chromatography-mass spectrometry analysis of various sterols present in medium and in cells. We also discuss the pros and cons of these methods. Together, these methods allow researchers to detect the release not only of cholesterol but also of other sterols present in minor quantities.
Topics: ATP Binding Cassette Transporter 1; Animals; Apolipoprotein A-I; Cell Line; Cholesterol; Humans; Phospholipids; Tangier Disease
PubMed: 28205180
DOI: 10.1007/978-1-4939-6875-6_19 -
Lipids Jan 2018ABCD1 is a gene responsible for X-linked adrenoleukodystrophy (X-ALD), and is critical for the transport of very long-chain fatty acids (VLCFA) into peroxisomes and...
ABCD1 is a gene responsible for X-linked adrenoleukodystrophy (X-ALD), and is critical for the transport of very long-chain fatty acids (VLCFA) into peroxisomes and subsequent β-oxidation. VLCFA-containing lipids accumulate in X-ALD patients, although the effect of ABCD1-deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1-deficient mice brains were profiled by liquid chromatography-mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1-deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn-1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1-deficient mice brains, odd-numbered saturated or mono-unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1-deficiency causes metabolic alternation of long-chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn-1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X-ALD.
Topics: ATP Binding Cassette Transporter, Subfamily D, Member 1; Adrenoleukodystrophy; Animals; Brain; Disease Models, Animal; Fatty Acids; Fibroblasts; Humans; Lipid Peroxidation; Mice; Oxidation-Reduction; Peroxisomes; Phosphatidylcholines; Phospholipids
PubMed: 29469952
DOI: 10.1002/lipd.12022 -
Biochimica Et Biophysica Acta.... May 2022Flaviviruses include many significant human pathogens, comprising dengue, West Nile, Yellow fever, Japanese encephalitis, Zika and tick-borne encephalitis viruses and...
Flaviviruses include many significant human pathogens, comprising dengue, West Nile, Yellow fever, Japanese encephalitis, Zika and tick-borne encephalitis viruses and many others, affecting millions of people in the world. These viruses have produced important epidemics in the past, they continue to do it and they will undoubtedly continue to do so in the future. Flaviviruses enter into the cells via receptor-mediated endocytosis by fusing its membrane with the endosomal membrane in a pH-dependent manner with the help of the envelope E protein, a prototypical class II membrane fusion protein. The envelope E protein has a conserved fusion peptide at its distal end, which is responsible in the first instance of inserting the protein into the host membrane. Since the participation of other segments of the E protein in the fusion process should not be ruled out, we have used atomistic molecular dynamics to study the binding of the distal end of domain II of the envelope E protein from Dengue virus (DENV) with a complex membrane similar to the late-endosome one. Our work shows that not only the fusion peptide participates directly in the fusion, but also two other sequences of the protein, next to the fusion peptide it in the three-dimensional structure, are jointly wrapped in the fusion process. Overall, these three sequences represent a new target that would make it possible to obtain effective antivirals against DENV in particular and Flaviviruses in general.
Topics: Amino Acid Sequence; Dengue Virus; Endosomes; Hydrogen-Ion Concentration; Molecular Dynamics Simulation; Phospholipids; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Viral Envelope Proteins
PubMed: 35167815
DOI: 10.1016/j.bbamem.2022.183889 -
Current Opinion in Lipidology Oct 2023ATP-binding cassette transporter A1 (ABCA1) plays a key role in high-density lipoprotein (HDL) biogenesis and cholesterol export from artery wall cells. Recent evidence... (Review)
Review
PURPOSE OF REVIEW
ATP-binding cassette transporter A1 (ABCA1) plays a key role in high-density lipoprotein (HDL) biogenesis and cholesterol export from artery wall cells. Recent evidence challenges the generally accepted model for lipid transport by ABCA1, termed the alternating access mechanism, which proposes that phospholipid moves from the inner leaflet to the outer leaflet of the plasma membrane.
RECENT FINDINGS
In contrast to the standard model, our computer simulations of ABCA1 indicate that ABCA1 extracts phospholipid from the plasma membrane's outer leaflet. The lipid then diffuses into the interior of ABCA1 to contact a structure termed the 'gateway'. A conformational change opens the gateway and forces the lipid through a ring-shaped domain, the 'annulus orifice', into the base of an elongated hydrophobic tunnel in the transporter's extracellular domain. Engineered mutations in the gateway and annulus strongly inhibited lipid export by ABCA1 without affecting cell-surface expression levels of the transporter, strongly supporting the proposed model.
SUMMARY
Our demonstration that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it into an elongated hydrophobic tunnel contrasts with the alternating access model, which flops phospholipid from the membrane's inner leaflet to its outer leaflet. These results suggest that ABCA1 is a phospholipid translocase that transports lipids by a mechanism distinct from that of other ABC transporters.
Topics: Humans; Lipoproteins, HDL; ATP Binding Cassette Transporter 1; Biological Transport; Phospholipids; ATP-Binding Cassette Transporters
PubMed: 37548415
DOI: 10.1097/MOL.0000000000000895 -
Biochemistry Jan 2019The outer membrane (OM) of Gram-negative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts. This barrier...
The outer membrane (OM) of Gram-negative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts. This barrier function depends critically on the asymmetric lipid distribution across the bilayer, with lipopolysaccharides (LPS) facing outside and phospholipids (PLs) facing inside. In Escherichia coli, the OmpC-Mla system is believed to maintain OM lipid asymmetry by removing surface exposed PLs and shuttling them back to the inner membrane (IM). How proteins in the pathway interact to mediate PL transport across the periplasm is not known. Evidence for direct transfer of PLs between these proteins is also lacking. In this study, we mapped the interaction surfaces between the two PL-binding proteins, MlaC and MlaD, using site-specific in vivo photo-cross-linking, and obtained a physical picture for how these proteins may transfer PLs. Furthermore, we demonstrated using purified proteins that MlaD spontaneously transfers PLs to MlaC, suggesting that the latter has a higher affinity for PLs. Our work provides insights into the mechanism of bacterial intermembrane lipid transport important for the maintenance of OM lipid asymmetry.
Topics: Biological Transport; Chromatography, Thin Layer; Cross-Linking Reagents; Escherichia coli Proteins; Membrane Proteins; Membrane Transport Proteins; Phospholipid Transfer Proteins; Phospholipids; Porins; Protein Interaction Maps; Tandem Mass Spectrometry
PubMed: 30284446
DOI: 10.1021/acs.biochem.8b00897 -
Scientific Reports Mar 2017Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of...
Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of polymerizable bis-imidazolium and urea hosts were evaluated as cationic and neutral hosts for phosphates and phosphonates, the latter used as mimics of the phospholipid head groups. The bis-imidazolium hosts interacted with the guests in a cooperative manner leading to the presence of tight and well defined 1:2 ternary complexes. Optimized monomer combinations were subsequently used for imprinting of phosphatidic acid as an endotoxin dummy template. Presence of the aforementioned ternary complexes during polymerization resulted in imprinting of lipid dimers - the latter believed to crudely mimic the endotoxin Lipid A motif. The polymers were characterized with respect to template rebinding, binding affinity, capacity and common structural properties, leading to the identification of polymers which were thereafter subjected to an industrially validated endotoxin removal test. Two of the polymers were capable of removing endotoxin down to levels well below the accepted threshold (0.005 EU/mg API) in pharmaceutical production.
Topics: Cross-Linking Reagents; Drug Contamination; Endotoxins; Imidazoles; Methacrylates; Molecular Imprinting; Molecular Mimicry; Organophosphonates; Pharmaceutical Preparations; Phosphates; Phospholipids; Polymerization; Urea
PubMed: 28303896
DOI: 10.1038/srep44299 -
Journal of Molecular Biology Jun 2021The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a...
The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4-3.9 Å. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaFEBD, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.
Topics: Adenosine Diphosphate; Bacterial Proteins; Binding Sites; Biological Transport; Cross-Linking Reagents; Models, Molecular; Multiprotein Complexes; Phospholipids; Protein Conformation; Pseudomonas aeruginosa
PubMed: 33845086
DOI: 10.1016/j.jmb.2021.166986 -
Cellular Physiology and Biochemistry :... 2017The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal...
BACKGROUND/AIMS
The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca2+ entry and/or oxidative stress.
METHODS
Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence.
RESULTS
A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Gefitinib further significantly increased DCFDA fluorescence.
CONCLUSIONS
Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca2+ and paralleled by oxidative stress.
Topics: Calcium; Calcium Signaling; Cell Death; Erythrocyte Membrane; Female; Gefitinib; Humans; Male; Oxidative Stress; Phospholipids; Quinazolines; Reactive Oxygen Species
PubMed: 28359067
DOI: 10.1159/000471823 -
Biochimica Et Biophysica Acta.... Jul 2017The ATP-binding cassette transporter A7 (ABCA7), which is highly expressed in the brain, is associated with the pathogenesis of Alzheimer's disease (AD). However, the...
The ATP-binding cassette transporter A7 (ABCA7), which is highly expressed in the brain, is associated with the pathogenesis of Alzheimer's disease (AD). However, the physiological function of ABCA7 and its transport substrates remain unclear. Immunohistochemical analyses of human brain sections from AD and non-AD subjects revealed that ABCA7 is expressed in neuron and microglia cells in the cerebral cortex. The transport substrates and acceptors were identified in BHK/ABCA7 cells and compared with those of ABCA1. Like ABCA1, ABCA7 exported choline phospholipids in the presence of apoA-I and apoE; however, unlike ABCA1, cholesterol efflux was marginal. Lipid efflux by ABCA7 was saturated by 5μg/ml apoA-I and was not dependent on apoE isoforms, whereas efflux by ABCA1 was dependent on apoA-I up to 20μg/ml and apoE isoforms. Liquid chromatography-tandem mass spectrometry analyses revealed that the two proteins had different preferences for phospholipid export: ABCA7 preferred phosphatidylcholine (PC)≥lysoPC>sphingomyelin (SM)=phosphatidylethanolamine (PE), whereas ABCA1 preferred PC>>SM>PE=lysoPC. The major difference in the pattern of lipid peaks between ABCA7 and ABCA1 was the high lysoPC/PC ratio of ABCA7. These results suggest that lysoPC is one of the major transport substrates for ABCA7 and that lysoPC export may be a physiologically important function of ABCA7 in the brain.
Topics: ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Alzheimer Disease; Animals; Apolipoprotein A-I; Apolipoproteins E; Biological Transport; Cell Line; Cholesterol; Cricetinae; HEK293 Cells; Humans; Lipid Metabolism; Lysophosphatidylcholines; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Sphingomyelins
PubMed: 28373057
DOI: 10.1016/j.bbalip.2017.03.012