The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

The interaction of sodium chlorite with phospholipids and glutathione:a comparison of effects in vitro, in mammalian and in microbial cells

In this study the interaction of the preservative sodium chlorite with unsaturated lipids and glutathione was investigated, in comparison with peroxides, sodium hypochlorite, and benzalkonium chloride. The aim was to determine whether the action of sodium chlorite could involve membrane lipid damage or antioxidant depletion, and how this related to toxicity in both mammalian and microbial cells. The treatment of phospholipids with chlorite yielded low levels of hydroperoxides, but sodium chlorite oxidized the thiol-containing antioxidant glutathione to its disulfide form very readily in vitro, with a 1:4 oxidant:GSH stoichiometry. In cultured cells, sodium chlorite also caused a substantial depletion of intracellular glutathione, whereas lipid oxidation was not very prominent. Sodium chlorite had a lower toxicity to ocular mammalian cells than benzalkonium chloride, which could be responsible for the different effects of long-term application in the eye. The fungal cells, which were most resistant to sodium chlorite, maintained higher percentage levels of intracellular glutathione during treatment than the mammalian cells. The results show that sodium chlorite can cause oxidative stress in cells, and suggest that cell damage is more likely to be due to interaction with thiol compounds than with cell membrane lipids. The study also provides important information about the differential resistance of ocular cells and microbes to various preservatives and oxidants.

Unique reporter-based sensor platforms to monitor signalling in cells

Introduction - In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. Methods - Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. Findings - We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. Conclusions - We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters.

The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. A shared feature of fab1Delta yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P(2), in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.

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.

Urinary 8-oxo-2′-deoxyguanosine:Redox regulation of DNA repair in vivo?

DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2′-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G → T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically. © 2002 Elsevier Science Inc.

Eukaryotic expression:Developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed. © International Union of Crystallography, 2006.

Phosphorylation and membrane dissociation of the ARF exchange factor GBF1 in mitosis

Secretory protein trafficking is arrested and the Golgi apparatus fragmented when mammalian cells enter mitosis. These changes are thought to facilitate cell cycle progression and Golgi inheritance, and are brought about through the actions of mitotically active protein kinases. To better understand how the Golgi apparatus undergoes mitotic fragmentation we have sought to identify novel Golgi targets for mitotic kinases. We report here the identification of the ARF exchange factor GBF1 as a Golgi phosphoprotein. GBF1 is phosphorylated by CDK1-cyclin B in mitosis, which results in its dissociation from Golgi membranes. Consistent with a reduced level of GBF1 activity at the Golgi membrane there is a reduction in levels of membrane-associated GTP-bound ARF in mitotic cells. Despite the reduced levels of membrane bound GBF1 and ARF, COPI binding to the Golgi membrane appears unaffected in mitotic cells. Surprisingly, this pool of COPI is dependent upon GBF1 for its recruitment to the membrane, suggesting a low level of GBF1 activity persists in mitosis. We propose that the phosphorylation and membrane dissociation of GBF1 and the consequent reduction in ARF-GTP levels in mitosis are important for changes in Golgi dynamics and possibly other mitotic events mediated through effectors other than the COPI vesicle coat.

Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic

In a genome-wide RNA-mediated interference screen for genes required in membrane traffic - including endocytic uptake, recycling from endosomes to the plasma membrane, and secretion - we identified 168 candidate endocytosis regulators and 100 candidate secretion regulators. Many of these candidates are highly conserved among metazoans but have not been previously implicated in these processes. Among the positives from the screen, we identified PAR-3, PAR-6, PKC-3 and CDC-42, proteins that are well known for their importance in the generation of embryonic and epithelial-cell polarity. Further analysis showed that endocytic transport in Caenorhabditis elegans coelomocytes and human HeLa cells was also compromised after perturbation of CDC-42/Cdc42 or PAR-6/Par6 function, indicating a general requirement for these proteins in regulating endocytic traffic. Consistent with these results, we found that tagged CDC-42/Cdc42 is enriched on recycling endosomes in C. elegans and mammalian cells, suggesting a direct function in the regulation of transport.

Effect of phosphatidylcholine chlorohydrins on human erythrocytes

Hypochlorite generated in vivo under pathological conditions is a known oxidant and chlorinating agent, able to react with proteins and lipids, which affects the stability of biological membranes. Reaction with unsaturated fatty acyl chains in glycerophospholipids such as phosphatidylcholine results in the formation of chlorohydrins. The aim of this study was to determine the effects of chlorohydrins formed by the reaction of hypochlorite with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonylphosphatidylcholine on biophysical properties of bilayers and their effects on human erythrocytes. Using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in a decrease in the rotational correlation time and an increase in the order parameter of liposomes. Unilamellar chlorohydrin liposomes had a lower permeation coefficient for calcein than liposomes made of parent lipids. Flow cytometry demonstrated fast incorporation of uni and multilamellar chlorohydrin liposomes labeled with NBD-phosphatidylethanolamine into erythrocytes. This effect was accompanied by changes in erythrocyte shape (echinocyte formation) and aggregation. Similar but less pronounced effects were noticed for parent lipids only after longer incubation. Chlorohydrins showed also a stronger hemolytic action, proportional to the lipid:erythrocyte ratio. These results are important for understanding the effects of HOCl on mammalian cells, such as might occur in inflammatory pathology.

Cellular interaction with novel biomaterials

The objective of this thesis is to report the behaviour of mammalian cells with biocompatible synthetic polymers with potential for applications to the human body. Composite hydrogel materials were tested as possible keratoprosthetic devices. It was found that surface topography is an important consideration, pores, channels and fibres exposed on the surface of the hydrogels tested can have significant effects on the extent of cell adheson and proliferation. It is recommended that the core component is fabricated out of one of the following to provide a non cell adhesive base; A8, A11, A13, A22, A23. The haptic periphery fabricated out of one of the following would provide a cell adhesive composite; A16, A30, A33, A37, A38, A42, A43, A44. The presence of vitronectin in the ocular tissue appears to lead to higher cell adhesion to the posterior surface of a contact lens when compared to the anterior surface. Group IV contact lenses adhere more cells than Group II contact lenses - this may indicate that more protein (including vitronectin) is able to adhere to the contact lens due to the Group IV contact lenses high water content and ionic hydrogel matrix. Artificial lung surfactant analogues were found to be non cytotoxic but also decreased cell proliferation when tested at higher concentrations. Poly(lysine ethyl ester adipamide) [PLETESA] had the most favourable response on cell proliferation and commercial styrene/maleic anhydride (pMA/STY sp2) the most pronounced inhibitory response. The mode of action that decreases cell proliferation appears to be through membrane destabilization. Tissue culture well plates coated with PLETESA allowed cells to adhere in a concentration dependent manner, multilaminar liposomes possibly of PLETESA were observed in solution in PLETESA coated wells. Polyhydroxybutryate (PHB) and polyhydroxyvalerate (PHV) blends that contained hydroxyapatite were found to be the most cell adhesive material of those materials tested. The blends that were most susceptible to degradation adhered the most cells in initial stages of degradation. The initial slight increase in cell adhesion may be due to the increased rugosity of the material. As the degradation continued the number of cells adhering to the samples decreased, this may indicate that the polarity was inhibitory to cell adhesion during the later stages of degradation.

Improved production of industrially relevant recombinant proteins: application of multi-factorial design of experiments and scalable process modelling

The work described in this thesis focuses on the use of a design-of-experiments approach in a multi-well mini-bioreactor to enable the rapid establishments of high yielding production phase conditions in yeast, which is an increasingly popular host system in both academic and industrial laboratories. Using green fluorescent protein secreted from the yeast, Pichia pastoris, a scalable predictive model of protein yield per cell was derived from 13 sets of conditions each with three factors (temperature, pH and dissolved oxygen) at 3 levels and was directly transferable to a 7 L bioreactor. This was in clear contrast to the situation in shake flasks, where the process parameters cannot be tightly controlled. By further optimisating both the accumulation of cell density in batch and improving the fed-batch induction regime, additional yield improvement was found to be additive to the per cell yield of the model. A separate study also demonstrated that improving biomass improved product yield in a second yeast species, Saccharomyces cerevisiae. Investigations of cell wall hydrophobicity in high cell density P. pastoris cultures indicated that cell wall hydrophobin (protein) compositional changes with growth phase becoming more hydrophobic in log growth than in lag or stationary phases. This is possibly due to an increased occurrence of proteins associated with cell division. Finally, the modelling approach was validated in mammalian cells, showing its flexibility and robustness. In summary, the strategy presented in this thesis has the benefit of reducing process development time in recombinant protein production, directly from bench to bioreactor.

Scale-up of human mesenchymal stem cell culture:current technologies and future challenges

Regenerative medicine technologies have the potential to revolutionise human healthcare. However, whilst science has revealed the potential, and early products have shown the power of such therapies, there is now a need for the long-term supply of human stem cells in sufficient numbers to create reproducible and cost effective therapeutic products. The industrial platforms to be developed for human cell culture are in some ways analogous to those already developed for biopharmaceutical production using mammalian cells at large scales. However, there are a number of unique challenges that need to be addressed, largely because the quality of the cell is paramount, rather than the proteins that they express. © 2013 Elsevier Ltd.

Rapid aquaporin translocation regulates cellular water flow:the mechanism of hypotonicity-induced sub-cellular localization of the aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The family's structural features and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this has only been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations, through transient receptor potential channels, that trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly-changing local cellular water availability. Moreover, since calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

GPCR production in a novel yeast strain that makes cholesterol-like sterols

The activities of many mammalian membrane proteins including G-protein coupled receptors are cholesterol-dependent. Unlike higher eukaryotes, yeast do not make cholesterol. Rather they make a related molecule called ergosterol. As cholesterol and ergosterol are biologically non-equivalent, the potential of yeast as hosts for overproducing mammalian membrane proteins has never been fully realised. To address this problem, we are trying to engineer a novel strain of Saccharomyces cerevisiae in which the cholesterol biosynthetic pathway of mammalian cells has been fully reconstituted. Thus far, we have created a modified strain that makes cholesterol-like sterols which has an increased capacity to make G-protein coupled receptors compared to control yeast.