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1
Deol, Sundeep Singh. "Analysis of lipid-protein interactions." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424760.
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Rathnayake, Sewwandi S. "A BIOPHYSICAL CHARACTERIZATION OF PROTEIN-LIPID INTERACTIONS OF THE LIPID DROPLET BINDING PROTEIN, PERILIPIN 3." Kent State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=kent1469552680.
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Carr, Neil Owen. "Lipid binding and lipid-protein interaction in wheat flower dough." Thesis, University of Reading, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293285.
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A study of lipid CClIplexirq in wheat floor ck:ujl has been made in an att:en¢ to explain the decrease in lipid extractability occurrin;J on dough developnent. '!be involvement of dough protein in this process has been assessa:i am new concepts have been evaluatai in the light of the known functionality of lipids in breadInakinI. PUblished W'Ork has irx:ticata:l that low IIDlecular \¥eight gluten proteins (ligolins) have a highly specific function in bin:ti.rg lipid. USl.rxJ similar fractionation methods to the plblished \¥Ork, it was possible to confirm this protein-lipid associaticn, although detergent cx:mtamination was d:JseIved followin;J the experimental procedure. It was sham that protein-lipid associaticn developed only in the preserx=e of detergent, whien led to a questionin;J of the pI'O{X)SeCi lipid bi.n::tin;J role of these low mlecular \¥eight proteins. It was also shown that the use of certain organic solvents can be unsatisfactory in the stlrly of protein-lipid interaction in dough. Fractionation by dilute acid provided evi~ that the 'baJnj' lipids of gluten were primarily in high mlecular \¥eight form, represented at least in part by a lip:JSaDal dispersion, whien were reasoned to be eri:e:tied within the gluten J'le'bvork in a oon-specific way. It was corcl.uled that interaction bet\¥een protein am such interactive lipid rmses ccW.d be responsible for the biniin:J of lipid durin;J dough develq:ment. Further sttnies are reported ~ the infll.lelDa of short:eni.n:J fat on lipid biniin;J. While there was sane in:lication that hard fat functionality was linked to an ability to maintain a critical pool of 'free' polar lipid, further work is required to investigate this early tentative CXl'd.usion. 'lhese stmies have been di srussed against a background of published WOrk, which has led to speculations al the nature and signifi~ of lipid b~ in the breadInakinI process. -
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PERISSINOTTO, FABIO. "Lipid raft formation and lipid-protein interactions in model membranes." Doctoral thesis, Università degli Studi di Trieste, 2018. http://hdl.handle.net/11368/2919798.
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The biological membranes of eukaryotic organisms contain functional, highly dynamic nano-domains called "lipid rafts" (LRs) which are enriched in cholesterol, sphingolipids and GPI-anchor proteins. They are involved in several biological processes which implicate or are mediated by the plasma membrane. Moreover, LRs seem to have a critical role in the onset of some neurodegenerative diseases such as the Alzheimer’s disease (AD), Parkinson’s disease (PD) and Prion protein disorders. In the last two decades, the complexity of studying such domains in living cells has caused a growing interest in the use and design of artificial membrane models, which mimic the structure and composition of biological membranes. In this context, I promoted the formation and investigated the properties of lipid raft domains in artificial lipid bilayers by exploiting Atomic Force Microscopy (AFM). I compared two different fabrication methods for the production of artificial lipid bilayers, the drop-casting and the direct vesicle fusion techniques. I started from one-component lipid membranes and I progressively moved towards more complex models, as binary and ternary lipid compositions, in order to study the main LRs features in relation to specific biological phenomena, such as protein-lipid interactions involved in particular pathological diseases. The direct vesicle fusion method appeared to be the most suitable approach in term of reproducibility, stability and control of lipid composition. I took advantage from this method for carrying out a morphological characterization of raft-like model membranes composed by phosphocoline (DOPC), sphingomyelin (SM) and cholesterol focusing in particular on lipid phase behavior. Membranes exhibited the coexistence of two lipid phases, the fluid phase made by DOPC, and the solid-ordered phase made by SM and cholesterol, the latter resembling raft-like domains.
With selected 3-component lipid systems, I then investigated the distribution of GM1 ganglioside, a LR marker, into my system, demonstrating its preferential localization in the nano-domains and highlighting the feasibility and versatility of model membrane technology. For the first time, I studied the binding of synthetic full-length Prion protein (PrPc), carrying a C-terminal membrane anchor (MA), to LRs domains. The conversion of PrPc into the scrapie isoform PrPsc, which displays high propensity to aggregate leading to cytotoxicity, has been reported to take place into LRs and to be influenced by lipid-anchors. I demonstrated with this study the propensity of this protein to specifically target LR domains of my artificial systems, observing an aggregation process occurring even at low protein concentrations. A comparative analysis with PrPc lacking of MA is however required to assess the role of lipid-anchor into the protein distribution and aggregation.
Finally, in the last part of my research I focused on the study of the role of iron ions in the interaction between alpha synuclein (αS) and lipid membranes. αS is the central protein of PD and the presence of amyloid αS fibrils is the main pathological hallmark of the disease. By AFM in combination with attenuated total reflectance infrared (ATR-IR) spectroscopy, I compared the structural behavior of the wild-type (wt) and a mutant form of αS (A53T) in presence of Fe2+ ions and the effect of the iron ions on the interaction with my artificial membrane, and specifically with LRs.
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Morrow, Isabel C. "Protein-lipid interactions within the cell /." [St. Lucia, Qld.], 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18271.pdf.
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Bromley, Emma. "Protein and lipid based bioinorganic composites." Thesis, University of Bristol, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441316.
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Punyamoonwongsa, Patchara. "Synthetic analogues of protein-lipid complexes." Thesis, Aston University, 2007. http://publications.aston.ac.uk/9803/.
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Hypercoiling poly(styrene-alt-maleic anhydride) (PSMA) is known to undergo conformational transition in response to environmental stimuli. The association of PSMA with lipid 2-dilauryl-sn-glycero-3-phosphocholine (DLPC) produces polymer-lipid complex analogues to lipoprotein assemblies found in lung surfactant. These complexes represent a new bio-mimetic delivery vehicle with applications in the cosmetic and pharmaceutical industries. The primary aim of this study was to develop a better understanding of PSMA-DLPC association by using physical and spectroscopic techniques. Ternary phase diagrams were constructed to examine the effects of various factors, such as molecular weight, pH and temperature on PSMA-DLPC association. 31P-NMR spectroscopy was used to investigate the polymorphic changes of DLPC upon associating with PSMA. The Langmuir Trough technique and surface tension measurement were used to explore the association behaviour of PSMA both at the interface and in the bulk of solution, as well as its interaction with DLPC membranes. The ultimate aim of this study was to investigate the potential use of PSMA-DLPC complexes to improve the bioavailability and therapeutic efficacy of a range of drugs. Typical compounds of ophthalmic interest range from new drugs such as Pirenzepine, which has attracted clinical interest for the control of myopia progression, to the well-established family of non-steroid anti-inflammatory drugs. These drugs have widely differing structures, sizes, solubility profiles and pH-sensitivities. In order to understand the ways in which these characteristics influence incorporation and release behaviour, the marker molecules Rhodamine B and Oil Red O were chosen. PSMA-DLPC complexes, incorporated with marker molecules and Pirenzepine, were encapsulated in hydrogels of the types used for soft contact lenses. Release studies were conducted to examine if this smart drug delivery system can retain such compounds and deliver them at a slow rate over a prolonged period of time.
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Botelho, Ana Vitoria. "Lipid-protein interactions: Photoreceptor membrane model." Diss., The University of Arizona, 2005. http://hdl.handle.net/10150/280765.
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G-protein coupled receptors (GPCRs) are transmembrane proteins capable of recognizing an astonishing variety of biological signals, ranging from photons of light to hormones, odorants, and neurotransmitters involved in key biological signaling processes. The aim of this work is to identify how lipid-protein interactions involving the membrane bilayer ultimately affect such vital biological functions. Here the relationship between the bilayer thickness, hydrophobic mismatch, and protein aggregation are investigated by expanding the framework of membrane-receptor interactions in terms of a new flexible surface model. Previously, we have shown how coupling of the elastic stress-strain due to mismatch of the spontaneous curvature and hydrophobic thickness at the lipid/protein interface can govern the conformational transitions of membrane proteins. This approach has now been extended to include coupling of the lateral organization of the GPCR rhodopsin to the curvature and area stress and strain of the proteolipid membrane. Rhodopsin was labeled with site-specific fluorophores, and a FRET technique was employed to probe protein association in different lipid environments. Moreover, UV-visible spectroscopy was used for thermodynamic characterization of the conformational change of rhodopsin. Lastly, the deformation of the lipids with and without rhodopsin was probed in terms of acyl chain order parameters and relaxation rates by solid-state NMR methods, giving insight into the lipid deformation. The results showed that optimal receptor activation occurs in phosphatidylcholine bilayers of 20-carbon acyl chain length, hence one can say that metarhodopsin II is likely to adopt an elongated shape. Lipids promoting aggregation, or below their gel to liquid crystalline transition temperature all favor formation of metarhodopsin I. The data also showed that association and activation of rhodopsin do not always correlate. In terms of the extended flexible surface model, the stress due to hydrophobic mismatch is coupled via the effective number of lipids surrounding the protein due to the lateral organization of the membrane. The measured changes in rhodopsin-rhodopsin interactions and membrane influences on the conformation of the protein after photoisomerization may be crucial to understanding physiological regulation of the rod disk membranes. They are relevant to understanding the complexity of biomembranes involved in many cellular mechanisms, including signal transduction.
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Saeed, Suhur. "Lipid oxidation mechanisms and lipid-protein interactions in frozen mackerel (Scomber scombrus)." Thesis, University of Surrey, 1998. http://epubs.surrey.ac.uk/843251/.
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Atlantic mackerel (Scomber scombrus) is a pelagic fish widely distributed along the Northern coast of Great Britain. The lipid content of mackerel was found to be about 13% of the total body weight and 50% of total fatty acids were eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (fatty acids which are reported to reduce the concentration of plasma triglycerides, LDL (low density lipoproteins) and cholesterol in humans and animals). The proximate analysis also showed that mackerel is a good source of protein (20% w/w). The poly unsaturated fatty acids (PUFA) are prone to oxidation during frozen storage leading to rancidity and protein damage. Thus the objective of this project was to prolong the shelf-life of mackerel by controlling and understanding lipid oxidation mechanisms. HPLC, GCMS and 13C NMR spectroscopy were used for the first time to monitor the production of hydroperoxides and their secondary products in fish matched pairs of mackerel fillets were stored at either -20°C or -30°C. In addition fillets were also stored with or without different antioxidants at -20°C. The development of lipid oxidation products were recorded for up to 24 months. The oxidation products identified were mixtures of alcohol derivatives of hydroperoxides, namely: 13-hydroxy-9-trans, 11-cis-octadecadienoic, 13-hydroxy-9-trans, 11-trans-octadecadienoic, 9-hydroxy-10-cis, 12-transoctadecadienoic and 9-hydroxy-10-trans, 12-transoctadecadienoic acids. The amount of hydroxides produced were higher in fillets stored at -20°C compared with fillets stored at -30°C. Similarly, the hydroperoxides produced were considerably higher in samples stored without antioxidant than in fillets stored with vitamin E. In this study the transfer of radicals from lipid oxidation to proteins and subsequent formation of protein-cross-links has been reported for the first time. The interaction between lipids and proteins were examined by both ESR and fluoroscence spectroscopy. A central esr free radical (g )signal was observed in both simple systems (methyl linoleate and pure amino acids) and complex systems (fish lipid and pure proteins (lysozyme, ovalbumin) or fish protein (myosin)). The esr signal reached a maximum within a week and then started to decline and with a concomitant increase in a pinkish yellow chromogen. This chromogen which was soluble in organic solvent and fluoresced at an excitation wavelength 360 nm and emission wavelength 420 nm and indicated the formation of protein cross-links. Synthetic (BHT, BHA) and natural (vitamins E, C) antioxidants were capable of preventing both the radical transfer and protein cross-linking. In this study lipoxygenase was isolated from mackerel flesh and its involvement in lipid oxidation mechanism was established. The molecular weight of partially purified lipoxygenase was 119,000 Daltons. This enzyme was capable of oxidising arachidonic acid to 12-hydroeicosatetraenoic acid (12-HETE), which was identified by HPLC. This 12-HETE was absent in pure arachidonic acid and in samples to which boiled enzyme was added. Conventional inhibitors, synthetic and natural antioxidants also inhibited the formation of 12-HETE, indicating the importance of lipoxygenase in fish lipid oxidation. During frozen storage, protein solubility decreased and the texture deteriorated in Atlantic mackerel stored for 3, 6, 12 and 24 months at -20°C and -30°C. There was an increase in peroxide value and TBARS; decrease in myosin ATPase activity a decrease in myofibrillar protein solubility in high salt concentration as well as formation of high molecular weight aggregates which showed low thermal stability and high G' and G" modulus values. There were significant differences (P < 0.01) between samples stored at -20°C and -30°C, with greater deterioration evident in samples stored at -20°C. Similarly, there were significant differences (P < 0.01) between samples stored with and without antioxidants; the samples stored without antioxidants deteriorated faster than samples stored with antioxidants. This suggests the involvement of lipid oxidation products in protein deterioration during frozen storage.
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Connell, E. J. "Protein-lipid interactions in synaptic vesicle exocytosis." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597894.
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The fusion of neurotransmitter-filled synaptic vesicles with the pre-synaptic membrane in response to calcium influx is exquisitely regulated. Synaptic vesicle exocytosis is energetically demanding and the neuronal SNARE proteins syntaxin, SNAP25 and synaptobrevin have come to prominence as the driving engines behind this process. Resident on both vesicular and pre-synaptic membranes they form a stable four-helical bundle, the assembly of which contributes to membrane fusion. However, SNAREs do not act in isolation during synaptic vesicle exocytosis but are instead regulated by a complex web of interactions with other proteins including synaptotagmin, a calcium-sensing component of the vesicle itself, and Munc18, a highly-conserved cytosolic protein. In addition, changes in the lipid environment surrounding the SNAREs play a critical role. In this thesis I report the results of two lines of investigation, into both synaptotagmin’s and Munc18’s action. Firstly, I consider the significance of the cytoplasmic double C2 domain structure of synaptotagmin. Using several strategies including a novel real-time absorbance assay, I show that these tandem C2 domains, but neither domain alone, rapidly cross-link lipid membranes in the presence of calcium. This property is conserved. Cross-linking ability can be masked in full-length synaptotagmin, via an electrostatic interaction with the membrane in which it is embedded. Finally, I address the mechanism of arachidonic acid action on syntaxin/Munc18, showing that this lipid activates Munc18-bound syntaxin and that a Munc18/syntaxin/SNAP25 assembly exists in brain. Arachidonic acid also activates free syntaxin, defining a molecular target for the reported role of this lipid in the promotion of vesicle fusion. My data are incorporated into a revised model of the protein-lipid interactions underlying synaptic vesicle exocytosis.
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Pilot, Jeffrey David. "Effects of lipid on membrane protein function." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390717.
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Annangudi, Palani Suresh Babu. "Lipid-based Oxidative Protein Modifications in Glaucoma." Case Western Reserve University School of Graduate Studies / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=case1129558048.
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Malik, Saira. "Protein-protein and protein-lipid interactions of band 3 in native and model membranes." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302943.
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Thurston, Victoria Louise. "Biophysical, structural and protein engineering studies on rabbit ileal lipid binding protein." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517865.
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Ivanova, Vesselka Petrova. "Theoretical and experimental study of protein lipid interactions." Doctoral thesis, [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=961248726.
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Rankin, Saffron Emily. "Lipid-protein interactions and nicotinic acetylcholine receptor function." Thesis, University of Oxford, 1996. https://ora.ox.ac.uk/objects/uuid:3deca85b-9f09-4f72-9db3-e34851e10542.
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The effect of bilayer composition, specifically the presence of cholesterol, upon the function of the reconstituted nicotinic acetylcholine receptor (nAcChoR) was investigated using stopped-flow fluorescence. The nAcChoR was purified and reconstituted from the electroplaques of Torpedo nobiliana, using affinity column chromatography, into bilayers of defined composition and the function of each sample assessed and compared with those of the native receptor. Investigation of the effect of bilayer composition upon the kinetics of agonist binding to the nAcChoR, using the fluorescent acetylcholine analogue, Dns-C6-Cho, established that the receptor pre-existed in equilibrium between the resting and two desensitised states. However, Dns-C6-Cho inhibited channel gating at high concentrations and another fluorescent probe was sought. The kinetics of carbachol induced nAcChoR conformational changes, reported by ethidium bromide (a non-competitive inhibitor) fluorescence, in native membranes were characterised and an assay developed to investigate whether cholesterol mediated rapid conformational changes in reconstituted samples. It was found that ethidium bromide reported on the carbachol-induced development of the fast desensitised state from the open channel state, and that this conformational change was sensitive to changes in bilayer composition. The onset of fast desensitisation from the open channel state was not observed when the receptor was reconstituted into DOPC or DOPC-DOPA bilayers. However, increasing the cholesterol content in these bilayers increased the amplitude of the component reporting this conformational change, while the observed rate at which it occurred was independent of bilayer cholesterol content. This result agrees with the suggestion that cholesterol facilitates channel opening and the onset of fast desensitisation by binding to specific sites on the nAcChoR and that these must be occupied for a functionally viable receptor (Jones and McNamee, 1988).
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Wang, Yuqin. "Protein and lipid interactions of mammalian antibacterial peptides /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4698-1/.
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Suresh, Swetha. "Probing protein-lipid interactions using atomic force microscopy." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609231.
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Hedger, George. "Characterisation of lipid-protein interactions through computational modelling." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:788a8496-fdcc-4962-af19-a10e746e1529.
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Parallel advances in lipidomics and the structural biology of membrane proteins over the past decade have revealed the highly diverse and complex nature of cell membrane composition. These compositional complexities influence the behaviour of embedded proteins, and a number of biomedically important membrane proteins are now known to be modulated via interactions with their lipid environment. This project aims to apply an array of computational tools to probe and characterize the molecular level detail of these interactions, and obtain information to complement experimental studies. In particular the interactions of modulatory lipid species with the transmembrane domain of the epidermal growth factor (EGF) receptor were addressed using coarse-grained potential of mean force (PMF) calculations. The results suggest this approach, widely applied in other areas of computational biology, may be successfully adapted to probe the strength and selectivity of lipid-protein interactions, and the effects of protein mutation. Subsequently these calculations were applied in tandem with equilibrium simulations to explore cardiolipin interactions with a key mitochondrial transporter, the ADP/ATP Translocase. The data show the coarse-grained model employed is capable of accurately identifying three specific cardiolipin binding sites on the protein, in agreement with prior crystallographic and NMR data. These sites were shown to be specific to cardiolipin, rather than general phospholipid interaction sites. In the third project, prospective predictions were made as to the location of PIP2 lipid binding sites on the membrane-exposed surface of a class A GPCR, the Neurotensin Receptor 1 (NTS1). A protein known to specifically bind these lipids from native mass spectrometry (MS) measurements at hitherto unknown regions. The results suggest PIP2 binds to defined loop regions on the intracellular portion of the protein. Finally, atomistic simulations are applied to examine the effect of a crystallographically resolved cholesterol molecule on the dynamics of a class F GPCR, Smoothened. The data suggest a marked influence of bound cholesterol on intra-domain dynamics of the extracellular region of Smoothened.
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Apsel, Beth. "Dual-specificity inhibitors of lipid and protein kinases." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3311357.
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Wilkinson, Dorothy Kate. "Functional characterisation of the lipid raft protein stomatin." Thesis, University of Leeds, 2005. http://etheses.whiterose.ac.uk/448/.
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The function of the integral membrane protein stomatin is as yet poorly understood. Stomatin is deficient from the erythrocyte membrane of patients suffering with Overhydrated Hereditary Stomatocytosis (OHSt). Patient erythrocytes have altered morphology and are known as stomatocytes. It is believed that stomatin is mistrafficked in the developing stomatocyte. These patients suffer grossly abnormal cation fluxes in the stomatocyte membrane which causes increased osmotic fragility of the cell and results in haemolytic anaemia. This study set out to characterise further stomatin and to investigate its role in the cell. The membranes and lipid rafts of stomatocytes were found to have reduced actin levels as compared to erythrocytes, suggesting that stomatin may function as a structural protein linking the cytoskeleton to the membrane. Overexpression of stomatin in nucleated cells caused enhanced actin association with cell membranes and lipid rafts, further confirming the findings from stomatocytes. Calcium-induced vesiculation was found to be significantly enhanced from the stomatocyte as compared to the erythrocyte, with defective partitioning of the flotillin proteins into the vesicles. This suggests that stomatin may function as a negative regulator in this vesiculation, possibly due to its interaction with actin and that the flotillins may substitute for stomatin in this process within the stomatocyte. Mutating the principle cysteine residue for palmitoylation within stomatin caused the protein to show less affinity for the membrane and lipid rafts but an increased affinity for the nucleus. This suggests that palmitoylation of stomatin affects the affinity of stomatin for the membrane and that this modification may be involved in regulating the shuttling of stomatin between the plasma membrane and the nucleus. Prokaryotic stomatin exists in an operon with a serine protease, suggesting a functional link between the two. Using a reporter gene construct approach the potential for mammalian stomatin to be proteolytically processed was investigated. Stomatin was found to be proteolytically processed in the membrane by a serine protease with the subsequent release of a C-terminal fragment.
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Garner, K. L. "An investigation into protein and lipid binding by the phosphatidylinositol transfer protein RdgBβ." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1359853/.
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The phosphatidylinositol transfer proteins (PITPs) are a family of lipid carrier proteins that bind and transfer phosphatidylinositol (PI) and phosphatidylcholine (PC) between membranes. PITPs are commonly involved in phosphoinositide-requiring processes, including phospholipase C and PI 3-kinase signalling, and membrane trafficking. In this study I focus on the uncharacterised soluble PITP, RdgBβ (PITPNC1). The lipid binding and transfer properties of RdgBβ have scarcely been characterised, and the function of RdgBβ is completely unknown. I uncover that RdgBβ interacts with 14-3-3 through its long, disordered C-terminus. RdgBβ is ubiquitinated and subject to rapid degradation in cells, and binding of 14-3-3 via two phosphorylated residues may serve to protect the protein from protease digestion. Whereas RdgBβ binds 14-3-3 under basal conditions, I deduce that, upon stimulation of cells with phorbol ester, RdgBβ binds the Angiotensin II receptor (AT1R)-associated protein, ATRAP, via its N-terminal PITP domain. Others have shown that ATRAP suppresses Angiotensin II signalling by uncoupling AT1R from G proteins and promoting AT1R internalisation. I find that the RdgBβ-ATRAP interaction is blocked by inhibition of protein kinase C or protein synthesis, and may function to re-localise RdgBβ to the membrane in stimulated cells. Unexpectedly, I find that RdgBβ binds PI and phosphatic acid (PA), rather than PI and PC, and that binding of PA is increased by stimulation of cells with GTPγS. Mass spectrometry is used to analyse the molecular species of PI and PA bound by RdgBβ, and reveals that whereas RdgBβ is non-selective in its binding of PI, it selects short-chain monounsaturated or saturated PA species, likely derived from the hydrolysis of PC by phospholipase D.
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Katsiadaki, Ioanna. "Physical and chemical attributes of cod roe." Thesis, University of Lincoln, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387667.
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Klemm, Robin. "Lipid rafts in protein sorting and yeast cell polarity." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1184754346185-43377.
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The major sorting station of biosynthetic material destined for the cell surface or secretion is the trans Golgi Network, TGN. This organelle sorts proteins and lipids into vesicular transport carriers that are targeted via different pathways to distinct membrane compartments of the cell. The molecular principles that operate in cargo sorting at the TGN are still not very well understood. Especially, we know very little about the sorting of lipids. It was postulated that a sorting mechanism based on clustering of lipid rafts, dynamic membrane domains enriched in sphingolipids and sterols, could be an important part of the picture. My thesis study dealt with the elucidation of the molecular sorting principles at the TGN and their exploitation for cell surface polarity in the yeast Saccharomyces cerevisiae. To this end, we conducted a genome wide screen that identified yeast mutants defective in cell surface delivery of the model cargo protein FusMid-GFP. The most striking result of this screen was that mutant strains with defects in ergosterol (the major yeast sterol) and sphingolipid biosynthesis lost sorting competence. To elucidate a direct role for sphingolipids and ergosterol in cargo sorting and secretion we sought to characterize the lipid composition of secretory vesicles. Hence, we established a vesicle purification protocol based on an immunoisolation strategy. Additionally, in collaboration with the group of A. Shevchenko, we developed a mass spectrometry methodology that allows the comprehensive and quantitative lipid analysis of subcellular organelles. Preliminary results corroborate our genetic evidence. The data show that the vesicles are enriched in sphingolipids and decreased in phosphatidylcholine indicating a role for raft clustering in cargo sorting at the TGN. The studies of cell polarity during yeast mating also unraveled a role for raft clustering. We could identify that the lipid bilayer at the tip of the mating projection was more ordered than at the plasma membrane enclosing the cell body and that this was dependent on sphingolipid synthesis. The results of my thesis suggest that in the yeast Saccharomyces cerevisiae fundamental cell biological processes such as cargo sorting and vesicle formation at the TGN as well as cell surface polarity during mating employ raft clustering mechanisms.
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Fraser, Diane Patricia. "Theoretical studies of lipid-protein interactions in biological membranes." Thesis, University of Central Lancashire, 1987. http://clok.uclan.ac.uk/20009/.
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Monte Carlo simulations are used to investigate the conformational and orierjtational properties of lipids and proteins in a bilayer membrane. in the first instance, linear, hard-core tri-atomics are used to represent the two-dimensional projections of the lipid molecules. Studies on lipid only systems show that the average number of gauche rotations and the cross-sectional area of the lipids decrease with increasing density. There is no long range orientational order within the lipids but the local orieritational order increases with increasing density. No first order phase transitions are observed though a glassy solid is observed at high densities. The properties of the bulk lipid are unchanged upon the addition of protein molecules represented by hard discs of varying sizes. The nearest neighbour lipids are unchanged conformationally but are found to exhibit a high degree of orientational ordering around the proteins preferring to have the long axis of their projections parallel to the proteins surface. The degree of ordering increases with increas-ing density and decreasing curvature of the protein. The lateral pressure is almost independent of protein size or concentration if expressed as a function of the bulk lipid density. The hard-core of the lipids is softened to a site-site Lennard-Jones potential. The particles are found to cluster within the periodic cell used. There is a critical density below which the average number of gauche rotations and the cross-sectional area change little and the local orientational order increases. Above this density these properties are the same as for the hard-core systems. The structure, indicated by the radial distribution functions, is much greater than that for the hard-core systems and is almost independent of density. The invariance of the lipid conformations and the observed lipid-protein orientational order resolve the conflict that has arisen in the past regarding the presence or absence of an annulus of lipid around integral protein molecules. The different experimental methods are seen to examine different lipid properties.
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Zech, Tobias Nikolai. "Lipid protein interactions at sites of T cell activation." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531794.
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Tognoloni, Cecilia. "Formation of polymer lipid nanodiscs for membrane protein studies." Thesis, University of Bath, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760950.
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Aivazian, Dikran A. (Dikran Arvid) 1971. "Lipid-protein interactions of immunoreceptor signaling subunit cytoplasmic domains." Thesis, Massachusetts Institute of Technology, 2001. http://hdl.handle.net/1721.1/8583.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.Vita.
Includes bibliographical references (leaves 116-131).
Protein-lipid interactions are emerging as key components of cellular processes such as protein and membrane trafficking and cell-cell signaling. Many proteins bind lipid reversibly, including cytoplasmic proteins involved in signal transduction, such as Ras and Src. Membrane binding is vital for the function of these signaling proteins both through co-localization with other signaling proteins as well as effects of lipid on intrinsic activities. In this thesis, protein-lipid interactions of subunits of key antigen recognition receptors of the immune system are investigated. The proteins studied are the cytoplasmic domains of immunoreceptor signaling subunits that mediate transmembrane signal transduction in response to receptor engagement. The cytoplasmic domains derive from the T cell receptor, the B cell receptor, Fe receptors and Natural Killer cell stimulatory receptors. The TCR, CD3, CD3, CD3, ... and DAP12 cytoplasmic domains all bind lipid, whereas those of B cell receptor Iga and Igp do not. While all of these proteins are unstructured in solution, ... and CD3 undergo extensive increases in secondary structure upon lipid binding. Lipid binding of ... is found to inhibit its accessibility to kinase-mediated phosphorylation. Based on these results it is proposed that interactions with lipid may regulate the function of receptor cytoplasmic domains, as with many cytosolic proteins involved in signaling processes.
by Dikran A. Aivazian.
Ph.D.
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Huster, Daniel. "Solid-state NMR spectroscopy to study protein-lipid interactions." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-190961.
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The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid-protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly
contributed to the understanding of lipid-protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid-protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K+ channel KcsA are discussed.
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Huster, Daniel. "Solid-state NMR spectroscopy to study protein-lipid interactions." Universität Leipzig, 2014. https://ul.qucosa.de/id/qucosa%3A14047.
Full textAbstract:
The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid-protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly
contributed to the understanding of lipid-protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid-protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K+ channel KcsA are discussed.
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Zhu, Xiaochun. "Characterization of Protein Modification by Products of Lipid Peroxidation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1225734704.
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Shi, Chuan. "Protein Modification and Catabolic Fates of Lipid Peroxidation Products." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1481231287497328.
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Gole, A. M. "Synthesis, characterization and possible applications of protein lipid and protein gold colloid biocomposite materials." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2002. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2718.
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Klein, Noreen [Verfasser]. "Protein-Protein- und Protein-Lipid-Wechselwirkungen beeinflussen die Oligomerisierung und Funktion des E. coli Aquaglyceroporins GlpF / Noreen Klein." Mainz : Universitätsbibliothek Mainz, 2015. http://d-nb.info/1070955701/34.
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Bunner-Pilotelle, Anne. "Lipid-protein and protein-protein interactions in the mechanisms of photosynthetic reaction centre and the Na+, K+-ATPase." Paris 11, 2008. http://www.theses.fr/2008PA112163.
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Les interactions lipides -protéines et protéines- protéines jouent un rôle important dans le fonctionnement et la régulatiol1 des protéines de membranes. Cette thèse se concentre sur deux différentes protéines de membrane, les centres réactionne (RC) de bactéries pourpres et l'enzyme Na, K-ATPase. L'influence des lipides et du cholestérol sur le cinetique des RC a été étudié en reconstituant les protéines dans des liposomes contenant du cholestérol ou ses dérivés chimiques connus pour changer la valeur du potentiel dipolaire. Les expériences faites sur l'enzyme Na, K-ATPase ont pour but de mieux comprendre le rôle de l'interaction oligomère dans le fonctionnement de la protéine. Les résultats ont montres que le cholestérol et ses dérivés modulent la cinetique de transfert d'électron. Leurs effets semblent être liés à leur impact sur le potentiel dipolaire. Nous avons interprété ces donnés comme une augmentation de la barrière énergétique pour les protons devant entrer dans la protéine. Des donnés sur la constante de dissociation de l'ATP pour l'enzyme Na, K-ATPase avec des techniques. De cinetique et des techniques de calorimétrie donnent des valeurs différentes. Basé sur des résultats obtenus par ITC calorimétrie et stopped-flow cette thèse propose une explication de ces différence en se proposant d'étudier l'effet du magnésium sur la constante de dissociation de l'ATP. Les résultats ont montrés que le complexe ATP-magnésium se liait à l'enzyme sans aucune intervention de l'enzymeReported Iiterature values of the dissociation constant, Kd, of ATP with the E1 conformation of the Na+. K++ATPase based on equilibrium titrations and kinetic methods disagree. Using isothermal titration calorimetry ((TC) and simulations of the expected equilibrium behaviour for different binding models, this thesis presents an eX~lanation for this apparent discrepancy based on protein-protein interactions. Because of the importanCE of Mg + in ATP hydrolysis, kinetic studies of Mg2+ binding to the protein were also carried out. These studies showed that ATP alone is responsible for Mg2+ complexation, with no significant contribution from the enzyme environ ment
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Pilotelle-Bunner, Anne. "Lipid-protein and protein-protein interactions in the mechanisms of photosynthetic reaction centre and the Na+,K+-ATPase." Thesis, The University of Sydney, 2008. http://hdl.handle.net/2123/7774.
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Lipid-protein and protein-protein interactions are likely to play important roles in the function and regulation of charge-transporting membrane proteins. This thesis focuses on two different membrane proteins, the photosynthetic reaction centre (RC) from purple bacteria and the Na+,K+-ATPase. The influence of the lipid surroundings and cholesterol derivatives on the kinetics of electron transfer of the RC were investigated by reconstituting the protein in phosphatidylcholine vesicles containing cholesterol and derivatives known to modulate the membrane dipole potential. The experiments performed on the Na+,K+-ATPase were designed to contribute to a better understanding of the role that oligomeric protein-protein interactions have in the enzyme’s mechanism. Our results show that the cholesterol derivatives significantly modify the electron transfer kinetics within the RCs and their multiphasic behavior. These effects seem to be associated with the extent of the dipole potential change experienced by the RC within the phospholipid membrane. Indeed, the largest effects on the rates are observed when 6-ketocholestanol and cholesterol are present, consistent by with their previously demonstrated significant increase of the dipole potential. We interpret this data as indicating an increased free energy barrier for protons to enter the protein. The consequences of the increased dipole potential seem to be experienced across the entire protein, since the rates of the P+QA- charge recombination in the presence of AQ- acting as QA are also modified by the same effectors. Also interesting is the effect of the dipole potential on the two conformational states of the RCs (previously reported) as revealed by the biphasic decays of the electron transfer kinetics. In particular, we report for the first time a biphasicity of the P+QA- charge recombination in the WT RCs. This non exponential behaviour, absent in the phospholipid membrane or isolated RCs, is induced by the presence of the cholesterol derivatives, suggesting that the equilibration time between the two RC conformations is slowed down significantly by these molecules. According to this work, the dipole potential seems to be an important parameter that has to be taken into account for a fine understanding of the charge transfer function of the RCs. Reported literature values of the dissociation constant, Kd, of ATP with the E1 conformation of the Na+,K+-ATPase based on equilibrium titrations and kinetic methods disagree. Using isothermal titration calorimetry (ITC) and simulations of the expected equilibrium behaviour for different binding models, this thesis presents an explanation for this apparent discrepancy based on protein-protein interactions. Because of the importance of Mg2+ in ATP hydrolysis, kinetic studies of Mg2+ binding to the protein were also carried out. These studies showed that ATP alone is responsible for Mg2+ complexation, with no significant contribution from the enzyme environment.
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Horton, Margaret R. (Margaret Ruth). "Influence of protein and lipid domains on the structure, fluidity and phase behavior of lipid bilayer membranes." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38982.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2007.Includes bibliographical references (p. 136-148).
The lipid bilayer forms the basic structure of the cell membrane, which is a heterogeneous matrix of proteins and lipids that provides a barrier between the interior of a cell and its outside environment. Protein and lipid domains in cell membranes can facilitate receptor localization, stabilize membranes, and influence membrane fluidity. In this thesis, we study how ordered protein and lipid domains influence the physical properties of lipid bilayers to better understand the roles of membrane domains in biological mechanisms. Model cellular membranes that mimic the behavior of biological membranes offer a controllable environment for systematically studying the isolated effects of protein and lipid ordering on membrane organization. Using fluid and solid-supported lipid bilayers, we study ordered peripheral membrane proteins and lateral lipid phase separation with fluorescence microscopy and X-ray reflectivity. To model cellular protein coatings and peripheral proteins, we prepare biotin-functionalized membranes that bind the proteins streptavidin and avidin. Fluorescence microscopy studies demonstrate that proteins crystallized in a single layer on lipid bilayer surfaces can change the lipid curvature and stabilize lipid vesicles against osmotic collapse.
(cont.) At solid interfaces, we characterize the electron density profiles of protein-coated bilayers to determine how a water layer separates an immobile protein layer from the fluid lipid bilayer. Liquid-ordered lipid phases enriched in cholesterol and sphingomyelin can localize molecules in cell membranes and this lipid phase separation behavior may be influenced by proteins and molecules in the membrane. Caveolae are specialized liquid-ordered domains in the plasma membrane that are enriched in the protein caveolin-1. We demonstrate that caveolin-1 peptides influence the onset of lipid phase separation and bind phase-separated lipid bilayers in solution. On solid surfaces, the formation of liquid-ordered lipid phases is influenced by surface roughness; with reflectivity, we determine that lipid bilayers containing cholesterol and sphingomyelin thicken with increasing cholesterol content. The membrane receptor GM1 also thickens the lipid bilayer when it is incorporated into the bilayer upper leaflet. The diverse experimental platforms that we present are applicable to studying additional and more complex biological systems to elucidate the influence of lipid and protein domains on cell membrane structure, organization and fluidity.
by Margaret R. Horton.
Ph.D.
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Ocampo, Minette C. "Protein-Lipid Interactions with Pulmonary Surfactant Using Atomic Force Microscopy." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1395050693.
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Hermelink, Antje. "Phosphatidylinositol 3-kinase [gamma] characterization of a protein-lipid interaction." Berlin dissertation.de, 2008. http://d-nb.info/994112912/04.
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Heath, George R. "Atomic force microscopy studies of protein interactions with lipid membranes." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/7599/.
Full textAbstract:
The behaviour of biological components in cellular membranes is vital to the function of cells however many vital phenomena associated with membrane functions are not yet fully understood. Supported lipid bilayers provide a model of real cellular membranes. This thesis examines how increasing the complexity of model lipid bilayers through changes in lipid composition, membrane protein content and cytoskeletal interactions can be used to extract significant biological information with biophysical techniques and analysis. The atomic force microscope (AFM) is a powerful tool in the study of biological systems allowing both three dimensional sub-nanometer resolution and mechanical interrogation under physiological conditions. The recent arrival of high-speed atomic force microscopy has transformed the information and processes which can be obtained, enabling direct imaging of biomolecular processes in real time. The work in this thesis shows that the AFM cannot only be used to investigate membranes but also deposit them in situ at lateral scales comparable to their height. Studies of confining lipid and protein diffusion in these quasi-one dimensional systems shows confinement reduces mobility of lipid with important implications on the behaviour of pores and defects cellular membranes. Studies of lipid phase behaviour of compositions thought be simplified models of the cell membrane lipid content show evidence that the small “raft” domains detected in real cells are not stable equilibrium phase separated domains, but non-equilibrium compositional fluctuations. Actin polymerisation induced at positively charged bilayers in non-polymerising conditions provides new insight into polymerisation processes whilst also describing a simple novel method to create “synthetic” robust actin-membrane scaffolds with controllable coverage. This polymerisation process was then applied to coating of lipid microbubbles for combined ultrasound imaging and drug delivery applications. The addition of the actin coating increased bubbles lifetimes, stability, elasticity and stiffness whilst allowing the attachment of model drug carriers.
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Sizer, P. J. H. "Studies of specific molecular interactions within and between membrane bilayers." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233640.
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Harris, Frederick. "Investigation into the membrane interactive properties of the escherichia coli low molecular weight penicillin-binding proteins." Thesis, University of Central Lancashire, 1998. http://clok.uclan.ac.uk/8692/.
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Various results have suggested that in Escherichia coli murein assembly may involve a protein complex(es) which could include low molecular mass penicillin-binding prpteins (PBPs). These proteins include PBP4, PBP5 and PBP6 which are penicillin sensitive enzymes associated with the periplasmic face of the inter membrane. The levels of these associations have been linked to enzymic activity and elucidation of the mechanism(s) involved in these associations may help identify and understand the regulation of this putative protein complex. It is currently accepted that the membrane associations of PBP5 and PBPÔ involve C-terminal amphiphilic cz-helices and such helices are ubiquitously employed in the lipid associations of membrane interactive protein molecules. Whether such helical structure features in the membrane associations of PBP4 or indeed if this protein is membrane bound or soluble, are, as yet, open questions. The focus of this research has been to investigate the lipid and membrane interactions of PBP4, PBP5 and PBP6 and in particular, to investigate the role played by these interactions of the C-terminal region of these proteins. Haemolytic analysis has shown that peptide homologues of the PBP5 and PBP6 C-terminal regions, P5 and P6, are active at the membrane interface and CD analysis has shown that these peptides possess a capacity for a-helix formation. CD and pressure - area isotherm analysis of monolayers formed from PS and P6 have shown that these peptides are able to adopt a-helical structure at an air - water interface. Monolayer studies have shown that P5 and P6 are able to interact with lipids and that these interactions are characterised by minor requirements for anionic lipid and the involvement of predominantly hydrophobic forces which are enhanced by low pH. Similar characteristics were revealed when perturbant washes and Western blotting were used to investigate the interactions of PBP5 with membranes derived from a mutant E. coli strain, HDL 11, in which the level of anionic lipid can be controlled. Overall, these results strongly support the hypothesis that C-terminal amphiphilic a-helices feature in PBP5 and PBP6 membrane anchoring. Molecular area determinations have implied that a peptide homologue of the PBP4 C-terminal region, P4 is able to adopt a-helical structure and this was confirmed by CD analysis. P4 showed no haemolytic activity but the peptide was found to interact generally with lipid monolayers. These monolayer interactions were characterised by a requirement for anionic lipid and involved predominantly electrostatic forces, which were enhanced by low pH. Similar characteristics but with no detectable requirement for anionic lipid were revealed when perturbant washes and chemiluminesence were used to investigate the interactions of PBP4 with membranes of the overproducing strain HB 10 I/pBK4 and those of HDL1 1. It is suggested that the PBP4 C-terminal region may play a role in PBP4 - membrane anchoring. Using chemiluminesence, no soluble form of PBP4 could be detected in the wild type E. coli, MRE600, suggesting that in wild type strains, PBP4 is exclusively membrane bound. It is suggested that PBP4 - membrane anchoring occurs at a specific binding site and overall, may differ fundamentally from that of PBP5 and PBP6.
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Horn, Patrick J., Christopher N. James, Satinder K. Gidda, Aruna Kilaru, John M. Dyer, Robert T. Mullen, John B. Ohlrogge, and Kent D. Chapman. "Identification of a New Class of Lipid Droplet-Associated Proteins in Plants." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/4752.
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Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.
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Breitsamer, Michaela [Verfasser], and Gerhard [Akademischer Betreuer] Winter. "Lipid-based depots : manufacturing, administration and interactions of protein drugs with lipid formulations / Michaela Breitsamer ; Betreuer: Gerhard Winter." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1193049105/34.
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Grachan, Jeremy J. "Characterization of Hypoxia-Inducible Lipid Droplet Associated Protein (HILPDA) Dependent Lipid Droplet Abundance in Pancreatic Cancer Tumors Cells." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586437335477715.
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Temprano, López Ana. "The lipin protein family in human adipocytes: lipid metabolism and obesity." Doctoral thesis, Universitat Rovira i Virgili, 2016. http://hdl.handle.net/10803/398025.
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Les lipins són una família conservada evolutivament de fosfatases de fosfatidat (PAP1) dependents de Mg2+, que generen diacilglicerol per a la síntesi de fosfolípids i triacilglicerol. En mamífers, la família consta de lipina-1, lipina-2 i lipina-3. Mentre en ratolins la mutació del gen Lpin1 causa lipodistròfia, les mutacions deletèries en el gen LPIN1 en humans no afecten la distribució del greix. No obstant, persones amb diabetis tipus 2 mostren nivells reduïts de l'expressió de LPIN1 i de l'activitat PAP1. Aquesta tesi estudia el paper de les lipins en el teixit adipós humà, la adipogènesi i la lipòlisi. Descobrim que la expressió de gens i proteïnes lipin és alterada en el teixit adipós de les persones amb diabetis tipus 2. Silenciant cada membre de la família lipin en la línia cel•lular humana de preadipòcits del síndrome Simpson-Golabi-Behmel (SGBS), mostrem que mentre que els tres membres tenen un paper en el primers estadis de l’adipogènesi, els preadipòcits silenciats de lipin es diferencien i acumulen lípids neutres, la qual cosa condueix a la hipòtesi de l'existència de vies alternatives per a la síntesi de triacilglicerol en adipòcits humans quan es reprimeix l'expressió de les lipin. Les lipin participen també en el reciclatge d'àcids grassos alliberats mitjançant la via lipolítica. Després de la inducció de la lipòlisi, les lipines són defosforilades i es desplacen a la membrana del reticle endoplasmàtic, on exerceixen la seva funció enzimàtica. Aquesta activació és induïda pels àcids grassos alliberats i s'inverteix amb la presència d’albúmina o triacsin C. La inducció d’adipòcits silenciats de cada lipina demostra el seu paper en el metabolisme dels lípids neutres. En resum, les lipin semblen no tenir un paper imprescindible en la adipogènesi humana però sí poden comprometre el reciclatge d'àcids grassos, important per a la homeòstasis lipídica.Las lipinas son una familia de fosfatasas de fosfatidato (PAP1) dependientes de Mg2+ evolutivamente conservadas, que generan diacilglicerol para la síntesis de fosfolípidos y triacilglicerol. En mamíferos, la familia consiste en lipina-1, lipina-2, y lipina-3. Mientras en ratones la mutación del gen Lpin1 causa lipodistrofia, las mutaciones deletéreas en el gen LPIN1 en humanos no afectan a la distribución de grasa. Sin embargo, los individuos con diabetes tipo 2 manifiestan niveles reducidos de expresión de LPIN1 y de actividad PAP1. En esta tesis doctoral se estudia la función de las lipinas en el tejido adiposo humano, la adipogénesis y la lipólisis. Descubrimos que la expresión génica y proteica de las lipinas está alterada en el tejido adiposo de individuos con diabetes tipo 2. La depleción de cada miembro de las lipinas en la línea celular humana de preadipocitos del síndrome Simpson–Golabi–Behmel (SGBS), mostró que, a pesar de que los tres miembros tienen un papel en la adipogénesis temprana, los adipocitos deplecionados de lipinas se diferencian y acumulan lípidos neutros, llevándonos a la hipótesis de la existencia de vías alternativas para la síntesis de triacilglicerol en adipocitos humanos cuando la expresión de las lipinas es reprimida. Las lipinas también intervienen en el reciclaje de los ácidos grasos liberados por la vía lipolítica. Tras la inducción de la lipólisis, las lipinas son defosforiladas y se desplazan a la membrana del retículo endoplásmico, donde ejercen su función. Esta activación es inducida por los ácidos grasos liberados, y revertida con albúmina o triacsin C. La depleción de cada lipina en adipocitos SGBS y posterior inducción de la lipólisis, demuestra su papel en el metabolismo de lípidos neutros. En resumen, las lipinas parecen no tener un papel indispensable en la adipogénesis humana pero sí comprometer el reciclaje de ácidos grasos, importante para la homeostasis lipídica.
Lipins are evolutionarily conserved Mg2+-dependent phosphatidate phosphatases (PAP1) that generate diacylglycerol for phospholipid and triacylglycerol synthesis. In mammals the Lipin family consists of lipin-1, lipin-2 and lipin-3. Whereas mutations in the Lpin1 gene cause lipodystrophy in mouse models, LPIN1 deleterious mutations in humans do not affect fat distribution. However, reduced LPIN1 expression and PAP1 activity have been described in participants with type 2 diabetes. In this doctoral thesis we investigate the roles of all lipin family members in human adipose tissue, adipogenesis and lipolysis. We found that adipose tissue gene and protein expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson–Golabi–Behmel syndrome (SGBS) pre-adipocyte cell line showed that even though all members alter early stages of adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids, pointing to the hypothesis of alternative pathways for triacylglycerol synthesis under repression of lipin expression. Lipins also have a role in the recycling of the fatty acids released by the lipolytic pathway. They become dephosphorylated upon lipolytic induction, and translocate to their active site, the endoplasmic reticulum membrane. This activation is induced by fatty acids and reversed with albumin or triacsin C. Depletion of every lipin member and subsequently stimulation of lipolysis in SGBS adipocytes revealed a role for lipins in neutral lipid metabolism. Overall, our data support that lipins may not have an indispensable role in adipogenesis, but their depletion compromise fatty acid recycling and lipid homeostasis.
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Chandrasekar, Indra. "On the lipid mediated regulation of the cell adhesion protein vinculin." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975657429.
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Ulrich, Anne S. "Deuterium NMR studies of lipid bilayer hydration and membrane protein structure." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334957.
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Andrews, Shantaya. "Localization of SIP470, a Plant Lipid Transfer Protein in Nicotiana tabacum." Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etd/3520.
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SABP2-interacting protein 470 (SIP470), a non-specific lipid transfer protein (nsLTP), was discovered in a yeast two-hybrid screening using SABP2 as bait and tobacco leaf proteins as prey. SABP2 is an important enzyme in systemic acquired resistance that converts salicylic acid to methyl salicylate. Localization studies are an important aspect to understanding the biological function of proteins. nsLTPs are generally considered apoplastic proteins and has been localized intracellularly and extracellularly. Transient expression shows highest expression of SIP470-eGFP at 2 days post infiltration into Nicotiana benthamiana. Confocal microscopy showed localization near the periphery of the cell. Subcellular localization using differential centrifugation showed that SIP470 is localized in the mitochondria. Mitochondria membranes are rich in lipids and have shown lipid exchange with the endoplasmic reticulum in mammalian systems. Co-localization of SIP470-eGFP+mCherry did not express complete co-localization in the targeted organelles. Co-localization pattern suggests possible localization in the endoplasmic reticulum.
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Vadlamudi, Mallika. "Lipid-coated Magnesium Phosphate Nanoparticles for Intrapulmonary Protein Delivery in Mice." Scholarly Commons, 2019. https://scholarlycommons.pacific.edu/uop_etds/3631.
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Proteins are a diverse category of biomolecules with great therapeutic potential. Intracellular delivery of proteins can augment the deficient activities of dysfunctional or poorly expressed innate proteins and therefore represents a promising strategy to treat the associated diseases. One major barrier to intracellular protein delivery is the translocation of the protein across the cellular membrane. Endocytosis provides an important pathway for protein nanocarriers to enter cells across the plasma membrane. However, the cargo protein must then promptly escape from the endosomes to avoid degradation in the lysosome and to exert its cellular function.
Previously, we reported a cationic lipid-coated magnesium phosphate nanoparticle (LPP) system for intracellular protein delivery. The intracellular delivery of catalase, an antioxidant enzyme, by LPP protected MCF-7 cells from a lethal level of exogenous H2O2 and lowered the reactive oxygen species (ROS) levels in EA.hy926 cells. These findings prompted us to further develop LPP to evaluate its protein delivery in animals.
Two categories of LPP formulations, catalase-encapsulated (CE) LPP and catalase-complexed (CC) LPP, were successfully prepared by a modular approach. Catalase-encapsulated liposomes (CE LP) were prepared by hydrating a thin-film of lipids with catalase solution followed by extrusion. However, extrusion of CE LP resulted in substantial loss of catalase activity. Catalase-complexed liposomes (CC LP) were prepared by first extruding cationic liposomes with a LIPEX extruder and then mixing with catalase solution. The resultant CC LP was much smaller than CE LP and preserved all the catalase activity. Magnesium phosphate nanoparticles (MgP NP) were prepared by the microemulsion precipitation technique. CE LP or CC LP were mixed with MgP NP to yield LPP formulations (CE LPP or CC LPP, respectively). The formulations were then rendered isotonic with glucose (5% w/v). Transmission electron microscopy (TEM) confirmed the proposed structure of LPP comprising a shell of lipid bilayers with a core of MgP NP. Furthermore, TEM showed drastic morphological changes of LPP formulations at acidic pH, consistent with an osmotic explosion.
The LPP formulations were administered by intravenous or intranasal routes to CD-1 mice. LPP formulations of fluorescently labeled catalase distributed substantially into the lung following intranasal administration, whereas intravenous administration of the same formulations caused catalase distribution mainly into the liver. In addition, intranasal administration of both the LPP formulations yielded higher pulmonary catalase activity and lowered the ROS levels in the healthy lung compared to free catalase solution. Based on these results, LPP’s antioxidant effects were further evaluated in mice with lipopolysaccharide-induced acute lung injury (ALI).
Lack of LPP distribution into the lung following intranasal administration indicated that intranasal dosing did not deliver catalase substantially into inflamed lungs. In corroboration, the inflammatory biomarker tumor necrosis factor-alpha (TNF-α) remained unchanged after intranasal dosing of LPP formulations. Intratracheal dosing of LPP formulations delivered the fluorescently labeled catalase deep into the lung and significantly reduced TNF-α production in the inflamed lungs compared to free catalase solution. CC LPP, which was smaller and which better preserved catalase activity than CE LPP, showed greater intrapulmonary catalase activity compared to CE LPP in both healthy and inflamed lungs. Taken together, LPP represents a promising nanocarrier for intracellular protein delivery.