Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease

This study compared the molecular lipidomic profi le of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identifi ed 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profi le in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N -acyltaurines were signifi cantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were signifi cantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identifi ed individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These fi ndings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profi le of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease. -Reis, A., A. Rudnitskaya, P. Chariyavilaskul, N. Dhaun, V. Melville, J. Goddard, D. J. Webb, A. R. Pitt, and C. M. Spickett. Topdown lipidomics of low density lipoprotein reveal altered lipid profi les in advanced chronic kidney disease. J. Lipid Res. 2015.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

Polymer-lipid interactions:biomimetic self-assembly behaviour and surface properties of poly(styrene-alt-maleic acid) with diacylphosphatidylcholines

Abstract Various lubricating body fluids at tissue interfaces are composed mainly of combinations of phospholipids and amphipathic apoproteins. The challenge in producing synthetic replacements for them is not replacing the phospholipid, which is readily available in synthetic form, but replacing the apoprotein component, more specifically, its unique biophysical properties rather than its chemistry. The potential of amphiphilic reactive hypercoiling behaviour of poly(styrene-alt-maleic acid) (PSMA) was studied in combination with two diacylphosphatidylcholines (PC) of different chain lengths in aqueous solution. The surface properties of the mixtures were characterized by conventional Langmuir-Wilhelmy balance (surface pressure under compression) and the du Noüy tensiometer (surface tension of the non-compressed mixtures). Surface tension values and 31P NMR demonstrated that self-assembly of polymer-phospholipid mixtures were pH and concentration-dependent. Finally, the particle size and zeta potential measurements of this self-assembly showed that it can form negatively charged nanosized structures that might find use as drug or lipids release systems on interfaces such as the tear film or lung interfacial layers. The structural reorganization was sensitive to the alkyl chain length of the PC.

Biomimetic synthetic lubricants (Biosurfactants)

Poly(styrene-co-maleic anhydride) (PSMA) based copolymers are known to undergo conformational transition in response to environmental stimuli. This smart behaviour makes it possible to mimic the behaviour of native apoproteins. The primary aim of this study was to develop a better understanding of the structure-property relationships of various PSMA-based copolymers sought. The work undertaken in this thesis has revealed that the responsive behaviour of PSMA-based copolymers can be tailored by varying the molecular weight, hydrophobic (styrene) and hydrophilic (maleic acid) balance, and more so in the presence of additional hydrophobic, mono-partial ester moieties. Novel hydrophilic and hydrophobic synthetic surfactant protein analogues have successfully been prepared. These novel lipid solubilising agents possess a broad range of HLB (hydrophilic-lipophilic balance) values that have been estimated. NMR spectroscopy was utilised to confirm the structures for PSMA-based copolymers sought and proved useful in furthering understanding of the structure-property relationships of PSMA-based copolymers. The association of PSMA with the polar phospholipid, 2-dilauryl-sn-glycero-3- phosphocholine (DLPC) produces polymer-lipid complexes analogous to lipoprotein assemblies present in the blood plasma. NMR analysis reveals that the PSMA-based copolymers are not perfectly alternating. Regio-irregular structures, atactic and random monomer sequence distribution have been identified for all materials studied. Novel lipid solubilising agents (polyanionic surfactants) have successfully been synthesised from a broad range of PSMA-based copolymers with desired estimated HLB values that interact with polar phospholipids (DLPC/DPPC) uniquely. Very low static and dynamic surface tensions have been observed via the du Noϋy ring method and Langmuir techniques and correlate well with the estimated HLB values. Synthetic protein-lipid analogues have been successfully synthesised, that mimic the unique surface properties of native biological lubricants without the use of solvents. The novel PSMA-DLPC complexes have successfully been combined with hyaluronan (hyaluronic acid, HA). Today, the employment of HA is economically feasible, because it is readily available from bacterial fermentation processes in a thermally stable form - HyaCare®. The work undertaken in this thesis highlights the usage of HA in biolubrication applications and how this can be optimised and thus justified by carefully selecting the biological source, concentration, molecular weight, purity and most importantly by combining it with compatible boundary lubricating agents (polar phospholipids). Experimental evidence supports the belief that the combined HA and PSMA-DLPC complexes provide a balance of rheological, biotribological and surface properties that are composition dependent, and show competitive advantage as novel synthetic biological lubricants (biosurfactants).

Protein modification and phospholipid oxidation

Phospholipid oxidation can generate reactive and electrophilic products that are capable of modifying proteins, especially at cysteine, lysine and histidine residues. Such lipoxidation reactions are known to alter protein structure and function, both with gain of function and loss of activity effects. As well as potential importance in the redox regulation of cell behaviour, lipoxidation products in plasma could also be useful biomarkers for stress conditions. Although studies with antibodies suggested the occurrence of lipoxidation adducts on ApoB-100, these products had not previously been characterized at a molecular level. We have developed new mass spectrometry-based approaches to detect and locate adducts of oxidized phospholipids in plasma proteins, as well as direct oxidation modifications of proteins, which avoid some of the problems typically encountered with database search engines leading to erroneous identifications of oxidative PTMs. This approach uses accurate mass extracted ion chromatograms (XICs) of fragment ions from peptides containing oxPTMs, and allows multiple modifications to be examined regardless of the protein that contains them. For example, a reporter ion at 184.074 Da/e corresponding to phosphocholine indicated the presence of oxidized phosphatidylcholine adducts, while 2 reporter ions at 100.078 and 82.025 Da/e were selective for allysine. ApoB-100-oxidized phospholipid adducts were detected even in healthy human samples, as well as LDL from patients with inflammatory disease. Lipidomic studies showed that more than 350 different species of lipid were present in LDL, and were altered in disease conditions. LDL clearly represents a very complex carrier system and one that offers a rich source of information about systemic conditions, with potential as indicators of oxidative damage in ageing or inflammatory diseases.

Biochemical investigation of multicorer sediment profile PS74/129-3

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/106-3

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/108-2

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/116-2

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/119-2

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/120-2

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/121-1

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/127-2

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Biochemical investigation of multicorer sediment profile PS74/128-2

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).