1,721,017 research outputs found
Use of Membrane Proteins as Antifungal Drug Targets
Get PDFFungal infections represent a much-overlooked threat that has yet to receive its due consideration. Their invasive branch has an underestimated impact on human morbidity and mortality. Despite this, research on antifungal therapies has been stalling for almost two decades now, while resistant strains have emerged on essentially every class of drugs that has been commercially available. Therefore, developing ways to counteract this emerging resistance is of paramount importance if we wish to remain capable of treating invasive fungal infections. New classes of drugs using new mechanisms of action would be highly desirable, especially if they are targeting fungal markers that have not been identified as potential targets before. This thesis project has been carried out in partnership with F2G Ltd, Manchester, UK, regarding the expression of new potential drug targets for antifungal treatments. It focuses on membrane proteins, which are a crucial gateway to the cell and an important source of untargeted markers that could represent very promising alternatives. Two main targets, both enzymatic membrane proteins, have been expressed in Pichia Pastoris yeast cells and solubilised using poly (styrene-co-maleic acid) lipid particles or SMALPs, which enables to retain the membrane protein with its surrounding lipids so that the protein stays in its native conformation. This allowed the protein to remain functional, so that a functional assay could be developed later on in order to test its activity. The final step was to test potential antifungal compounds developed by F2G to inhibit the enzymatic reaction, which was the key for the antifungal activity detected in earlier studies by F2G. Because membrane proteins are much harder to work with than soluble ones, they can sometimes be more difficult to obtain in sufficient amount and purity. Therefore, an attempt at engineering a contaminant-free P. pastoris cell line was carried out, with the goal of removing the main contaminant found during membrane protein production and purification
Membrane phospholipids as regulators of tetraspanin oligomerisation
Get PDFTetraspanins are integral membrane proteins that play a key role in organising multi-molecular complexes. Tetraspanin CD81 is involved in cellular processes such as cell adhesion, cell proliferation, and mediating infection of medically important pathogens, including the hepatitis C virus. Despite these important roles, the comprehensive structural organisation, membrane distribution, and CD81 interaction with surrounding proteins and lipids are not known. To enable these studies, CD81 was solubilised and purified from Pichia pastoris membranes in poly (styrene-co-maleic-acid) lipid particles or SMALPs, to retain its surrounding membrane environment. Biophysical characterisation was conducted by circular dichroism spectroscopy and antigen-antibody ELISA. This indicated that SMALP-CD81 retains its secondary structure and is functionally stable, even at higher temperatures, in marked contrast with detergent-purified CD81. Subsequently, gel filtration conditions have been optimised to isolate functionally active SMALP-CD81 fractions. The native CD81 membrane distribution in HEK 293 and Huh-7 cell-lines was also studied using electron microscopy (EM). The EM images of cell sections indicated that CD81 is organised in isolated monomers as well as in clusters of potentially higher-order structures in both cell-lines. This data agrees with the general consensus that tetraspanins exist as tetraspanin-rich microdomains that modulate the function of interacting proteins
Optimisation of a novel antibody format targeting ion channels involved in autoimmune diseases
No full textAutoimmune diseases affect 3 % to 6 % of the world’s population but current immunotherapies cause detrimental side-effects or cannot be used effectively for the treatment of most patients. There is a huge unmet need of novel therapeutic drugs for the specific treatment of autoimmune diseases. The potassium voltage-gated ion channel Kv1.3 became a prominent target for this cause. Inhibiting Kv1.3 results in the specific suppression of effector memory T-lymphocytes that mediate autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and type-1-diabetes. However, due to their complexity and high sequence homology, the selective targeting of ion channels is a huge challenge. To provide remedy, the KnotBody technology was created. It combines the high potency of venom peptides with the engineerability and pharmacokinetics of monoclonal antibodies. During the course of this project a panel of anti-Kv1.3 KnotBodies was developed. A significant improvement of the KnotBody biophysical properties was achieved through mammalian display while retaining KnotBody inhibitory activity against Kv1.3. Sequence analysis gave first insights into the interplay between specific amino acid residues of the antibody framework, complementarity determining regions, and the venom peptide. To enable future in vitro selections and screening campaigns for lead candidate optimisation, Kv1.3 and the chimera KcsA1.3 were recombinantly produced using either a traditional detergent approach or through the SMALP technology. Furthermore, the selective binding of an anti-Kv1.3 KnotBody against the chimera was proven through a newly established DELFIA-TRF binding assay
Determining the cytotoxic properties of a Fagonia indica extract on breast and colon cancer
Get PDFFagonia indica is an herbaceous plant common to dry arid environments, including those across Eastern Europe and the Middle East. Traditional communities; particularly those in small villages of Pakistan, utilise Fagonia indica as an alternative medicine for a number of ailments, including breast cancer. Previous research has demonstrated several lines of antineoplastic activity from an aqueous extract of Fagonia indica against breast cancer cell lines, in vitro. Cytotoxicity in these cell lines was associated with activation of p53 and FOXO3a which were able to inhibit cell cycle progression and induce apoptosis in breast cancer. Despite this, there is still a limited understanding on the breadth of effects of Fagonia indica treatment, particularly against other aspects of tumorigenesis. The present study aimed to determine the mechanisms of action of an aqueous extract of Fagonia indica against phenotypically distinct breast and colon cancer cell lines. One of the most confounding issues in cancer treatment, is the acquisition of resistance mechanisms against commonly used chemotherapies. For the first time, this study also investigated the effect of Fagonia indica treatment against multi-drug resistant breast and colon cancer cell lines. An aqueous extract of Fagonia indica was able to induce cell death in wild-type and chemotherapy resistant breast and colon cancer cell lines, in correlation with a dysregulation of metabolism/ ATP production. Other associated mechanisms of action of Fagonia indica included down-regulation of VEGF, ICAM-1 and NF-kB expression, related to angiogenic and inflammatory processes. The effect of Fagonia indica on chemotherapy resistant breast and colon cancer cell lines was associated with down-regulation of ABC transporters; ABCC4 and AGCG2. Investigation into the chemical properties of Fagonia indica revealed that a methanolic extract of Fagonia indica had increased total flavonoid, triterpene and antioxidant contents which was associated with increased antineoplastic activity in vitro. Further work is required to separate and identify individual compounds from this methanolic fraction for development into a therapeutic treatment
De novo design of membrane protein channels
Get PDFAdvances in the field of synthetic biology and de novo protein design come in the aid of the existing methods, and aim to contribute with providing essential answers referring to the sequence-structure- function relationship problem. Advances in the implementation of computational techniques in these research fields, promoted the speed of research, however, the computer-only based studies cannot provide sufficient data, especially due to insufficient real-life-based training information. For this reason, the need of interdisciplinary studies and combining computational techniques with laboratory-based experiments are required. De novo protein design aims to contribute to the design of small building blocks, which can self-associate into known or new structures, stabilising existing scaffolds. Thus, it enables the possibility of creating a library of small building blocks and their influence on structure formation and stability. In this cross-disciplinary study, we aimed to contribute to de novo membrane protein design with a novel, repetitive sequence (CC1), able to associate into an antiparallel homotetrameric helical bundle. In this study it is also proposed an additional computational framework, for de novo membrane protein design, combining Crick parametrisation tools and simulations in implicit and explicit membranes. The design pathway combined minimal and rational approaches, completed by knowledge-based and statistical studies. Molecular dynamics simulations in GROMACS using explicit POPC, POPE and POPG-containing membranes, showed that the antiparallel homotetrameric bundle formed by the repetitive CC1 sequence, appeared to be stable across all types of lipid compositions tested, showing great stability, as predicted by the initial energy score functions. Moreover, in POPE and POPG- explicit membranes, the CC1 antiparallel tetramer showed structural conformations which guide towards a potential mechanosensitive channel-like function. The experiments were compared to controls represented by the designed REAMP tetramer and poly-leucine antiparallel bundle. The CC1 antiparallel homotetramer has also proven biocompatibility, when expressed in E. coli C41 (DE3) cells, having in the structure the mistic protein and the split-variant of the superfolder yellow fluorescent protein, as “internal chaperone”, to overcome the challenges associated with the translocation machinery. The expression levels have shown to be comparable to the REAMP control, having attached the same tags, and the expression levels have shown temperature and medium composition dependence, essential for future considerations. CC1 was also successfully cell-free synthesised. The efficiency of insertion into liposomes of the folded state and the fluorescence of the sfYFP have shown liposome composition dependence. CC1 have shown to also stabilise POPC: DPhPC containing bilayers in droplet-interface bilayers assays. The present work comprises a novel finding in the field of de novo design, highlighting a novel, biocompatible sequence, with a special highlight on the importance of the lipid composition in de novo protein stability and membrane insertion
Solubilisation and stabilisation of MRP4 using novel detergents for function and structural studies
Get PDFKnowledge of membrane protein function and structure is limited due to the complications in the production of stable, functional membrane proteins that can be used in functional and structural studies. These limitations are due to their location within a lipid bilayer and this creates challenges at almost every stage including expression, solubilisation, stabilisation and purification of membrane proteins. This study aimed to combat some of these limitations for the human membrane protein ABCC4/MRP4 (multidrug resistance protein 4) from the ABC superfamily, in particular focusing on the solubilisation and stabilisation aspects. MRP4 was expressed in Spodoptera frugiperda (Sf 9) insect cells, Pichia pastoris yeast cells and Human Embryonic Kidney (HEK) cells. It was found that the highest level of expression was achieved using Sf 9 insect cells. MRP4 was then tested for functionality using a fluorescent vesicular transport assay that was developed within this study and shown to be functional. It was found that Calixarene C4C7 and the polymer SMA 2000 were both more efficient at solubilising and stabilising MRP4 than conventional detergents. Conditions for purification with both C4C7 and SMA 2000 were optimised, and were able to produce protein at ~40% and ~70% purity and concentrations of ~400 μg/mL and ~25 μg/mL respectively. Following purification it was determined, using tryptophan fluorescence quenching binding assays, that the SMA purified MRP4 bound ligands and retained increased stability compared to conventional detergents. Preliminary studies were performed using electron microscopy, which showed potential for this approach in the future. The techniques and methods developed in this study can be used by others in the future to gain better structural and functional knowledge of MRP4 and can also be applied to the study of other membrane proteins
Structural characterisation of human tetraspanins and their interaction with cholesterol and gangliosides
Get PDFTetraspanins are a family of membrane proteins that play a role in various functions, such as cell migration, signal transduction and intracellular trafficking. They are organisers within the membrane, forming tetraspanin-enriched microdomains (TEMs) comprised of tetraspanins, partner proteins, cholesterol and gangliosides. Knowledge of the precise interactions between these molecules is limited. Understanding more about these interactions would add to the body of knowledge about how TEMs are formed. With this knowledge these interactions could be disrupted to prevent negative biological events associated with TEMs, such as microbial infections. To study these interactions CD81, which interacts with hepatitis C virus, was used as a model tetraspanin. The purification of CD81 expressed in Pichia pastoris in styrene-maleic acid lipid particles (SMALPs) was optimised by including imidazole in the binding buffer and increasing the concentration of imidazole in the purification wash steps. CD81 mutants were created to study cholesterol-dependent conformational change using electron paramagnetic resonance. Protein-ligand docking was used to investigate the interaction between the two tetraspanins, CD81 and CD82, and the sugar residues in gangliosides. Asp122 in CD81 and Asp37 and Ser135 in CD82 were identified as interaction sites. The basic structure of tetraspanins appears to be ubiquitous but a variable region in the large extracellular loop (LEL) requires further research because this is a crucial area for tetraspanin interactions. LELs of some human tetraspanins were modelled and their disulfide bond arrangement was analysed, finding a different arrangement in the subset of tetraspanins referred to as TspanC6-CxCs. Sequence and structural alignment of human tetraspanins highlighted conserved residues within different structural regions that were used as anchor residues to build a universal amino acid numbering system
A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model
Get PDFTransmembrane 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
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