This thesis describes the synthesis of new [Ni]- and [NiFe]-containing complexes as analogues of the active site of the [NiFe] hydrogenases and which have the potential as catalysis for the electro- or photochemical production of H2. The research described in the thesis focuses on two key aspects: (i) the modulation of the potential at which proton reduction may occur through the nature of the ligand about the metal centres and (ii) the incorporation of photosensitiser units into [Ni]- and [NiFe]-containing complexes. Chapter 1 provides an introduction to the [FeFe] and [NiFe] hydrogenases and provides the context for the research carried out in this thesis. Relevant coordination chemistry is surveyed with a focus on mononuclear and heteronuclear coordination compounds containing Ni-centres in S-rich coordination environments. The electro- and photocatalytic properties of some of the centres are discussed. Chapter 2 describes the attempted synthesis and characterisation of the novel [Ni(p-qdtR1,R2)] (R1 = R2 = Me ; R1= H and R = tBu) and [Ni(p-q6,7dtR1,R2)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) complexes. The complexes contain quinoxaline units that (i) may shift the reduction potentials to more positive for each complex and (ii) contain N atoms that may serve as protonation sites that may facilitate the reduction of protons. [Ni(p-q6,7dtR1,R2)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) undergo an electrochemically reversible reduction process at ca. -1.47 V vs Fc+/Fc to generate the EPR-active [Ni(p-q6,7dtR1,R2)]- anions. The spin Hamiltonian parameters derived from simulations of the EPR spectra are consistent with a SOMO that is principally metal-centred. Density functional calculations provide a framework for the interpretation of the spectroscopic properties of [Ni(p-q6,7dtR1,R2)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) and suggest that the redox-active LUMO is delocalised over the NiS4 centre. Cyclic voltammetric measurements of [Ni(p-q6,7dtR1,R2)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) in acidic solutions show that these complexes are potential electrocatalysts for the reduction of H+ and operate at ca. -1.7 V vs Fc+/Fc at a comparable potential to [Ni(LA)]. Comparisons of the cathodic currents produced by [Ni(p-q6,7dtR1,R2)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu), [Ni(LA)] and [Ni(LA)Fe2(CO)6] suggest that the catalytic activities of [Ni(p-q6,7dtR1,R2)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) and [Ni(LA)] are comparable and higher than that for the benchmark [Ni(LA)Fe2(CO)6] compound. Chapter 3 describes the syntheses and characterisations of [Ni(qdtR1,R2)(dppe)] (R1 = R2 = Me; R1 =H and R2 = tBu) and [Ni(q6,7dtR1,R2)(dppe)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) complexes [Ni(qdtR1,R2)(dppe)] (R1 = R2 = Me; R1 = H and R2 = tBu) and [Ni(q6,7dtR1,R2)(dppe)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) undergo an electrochemically reversible reduction process at ca. -1.79 and ca. -1.86 V vs Fc+/Fc, respectively, to generate the EPR-active [Ni(qdtR1,R2)(dppe)]- and [Ni(q6,7dtR1,R2)(dppe)]- anions. The EPR spectra are consistent with DFT calculations that reveal SOMOs that involve contributions from Ni 3dxy and S/P-σ combinations. Cyclic voltammetric studies show that [Ni(qdtR1,R2)(dppe)] (R1 = R2 = Me; R1 = H and R2 = tBu) and [Ni(q6,7dtR1,R2)(dppe)] (R1 = R2 = Me, Et and Ph; R1 = H and R tBu) are electrocatalysts that function at ca. -1.8 V vs Fc+/Fc, and comparisons of the cathodic currents produced by [Ni(qdtR1,R2)(dppe)] (R1 = R2 = Me; R1 = H and R2 = tBu), [Ni(q6,7dtR1,R2)(dppe)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu), [Ni(p-q6,7dtEt,Et)] and [Ni(LA)Fe2(CO)6] suggested that the catalytic activities of [Ni(qdtR1,R2)(dppe)] (R1 = R2 = Me; R1 = H and R2 = tBu) and [Ni(q6,7dtR1,R2)(dppe)] (R1 = R2 = Me, Et and Ph; R1 = H and R = tBu) are comparable to [Ni(LA)Fe2(CO)6] and lower than that for [Ni(p-q6,7dtEt,Et)]. Chapter 4 discusses the reactions of the NiII precursor complexes [Ni(p-qdtR1,R2)] [Ni(p-q6,7dtR1,R2)], [Ni(qdtR1,R2)(dppe)] and [Ni(q6,7dtR1,R2)(dppe)] described in Chapters 2 and 3 with [Fe]-containing fragments to isolate [NiFe] compounds as analogues of the active sites of the [NiFe] hydrogenases. The syntheses of [Ni(q6,7dtR1,R2)2Fe2(CO)6] and [Ni(q6,7dtR1,R2)2FeCp(CO)2](BF4) were confirmed by FTIR spectroscopy and mass spectrometry and [Ni(q6,7dtMe,Me)2Fe2(CO)6] was characterised by X-ray crystallography. In addition, [Ni(q6,7dtEt,Et)2Fe2(CO)6] and [Ni(q6,7dtH,tBu)2Fe2(CO)6] undergo an electrochemically reversible reduction process at ca.-1.22 V vs Fc+/Fc. FTIR spectroscopy confirms the successful synthesis of [Ni(q6,7dtR1,R2)(dppe)FeCp(CO)2](BF4) and [Ni(q6,7dtEt,Et)(dppe)FeCp(CO)2](BF4) and [Ni(q6,7dtH,tBu)(dppe)FeCp(CO)2](BF4) were characterised by X-ray crystallography. Chapter 5 describes research towards the incorporation of photosensitising units into [Ni]- and [NiFe]-containing complexes. A novel dithiol substituted dppz complex, [Ni(dppz(S)2)(dppe)], was characterised by X-ray crystallography and represents the first example of a NiII centre bound to S-donors from a dppz ligand. Additional reactions between this unit and [ReCl(CO)3] fragments, and between [ReCl(CO)3(dppz(S)2)] and NiII-precursors are described, together with attempts to synthesise a NiS4 complex containing two dppz units. Chapter 6 draws together the main conclusions of the thesis and suggests areas for future work.

The mechanisms underlying the development of insulin resistance in skeletal muscle are very complex and are not completely understood. Insulin resistance in skeletal muscle is of particular importance because muscle is the major site of insulin stimulated glucose uptake. Skeletal muscle is one of the major insulin sensitive organs and it is responsible for 80% of insulin stimulated glucose disposal as well as fatty acid oxidation. Elevated circulating free fatty acids (FFAs) and their derivatives such as endocannabinoids (ECs) have been described in obesity and are thought to be influential in the development of muscle insulin resistance. While several hypotheses have been put forward to explain the mechanisms by which FFAs and ECs may cause insulin resistance, there are still many potential signalling pathways which may be involved that have not yet been examined. The main aim of this thesis was to characterize the role of the cannabinoid receptors and free fatty acid receptor 1 (GPR40) in cell signalling in human and rat skeletal muscle tissue and primary cultured myotubes. Gene expression profiling of human skeletal muscle and cultured myotubes and myoblasts indicated that the cannabinoid receptor CB1 and GPR40 were expressed at low levels and these results were confirmed using Taqman QRTPCR. CB2 receptor expression was only detected in rat tissue and as a result was not further studied in cell culture systems. When global gene expression profiles were further examined it was evident that whilst cultured myotubes retained many characteristics of skeletal muscle tissue, the phenotype appeared to be closer to fetal than adult muscle. Furthermore, when metabolic gene expression networks were analysed using pathway based analysis, it was apparent that expression of genes involved in oxidative phosphorylation, insulin signalling and glucose transport were markedly reduced in cultured cells. The most striking example being GLUT4 which was expressed at approximately 3000-fold lower levels in cell culture as compared to tissue. The functionality of CB1 and GPR40 receptors was demonstrated using selective agonists and antagonists. CB1 activation by both synthetic and endogenous ligands was confirmed using phosphorylation of ERK1 and 2 as was the presence of functional GPR40 protein in myotubes. Neither agonists nor antagonists of CB1 or GPR40 receptors were found to modulate insulin signalling as determined by phosphorylation of downstream targets Akt and GSK3alpha/beta. Global expression profiling was also carried out on myotubes treated with GPR40 agonists and antagonists obtained from AstraZeneca. No changes in metabolic or insulin signalling genes were observed. Rather, antagonists of GPR40 appear to activate gene expression networks involved in cell proliferation – in particular an elevation in the ERBB2 signalling pathway.

In the developing embryo the heart is the first organ to develop and thus supply the rest of the developing embryo with a good blood supply. Regulation of cardiogenesis in these early stages of development is key as any dysregulation will result in defects in the heart. Dysregulation of sarcomeric proteins has been associated with a range of cardiomyopathies and septal defects. This demonstrates the importance of structure on development. However, structural genes have not been linked to conduction disorders in the heart. Myosin heavy chain genes (MYH) encode sarcomeric structural proteins (MHC). Previous work by Rutland et al. (2011) showed that alpha myosin heavy chain (αMHC), beta myosin heavy chain (βMHC) and embryonic myosin heavy chain (eMHC) are necessary for correct Ca2+ transients, with eMHC also required for a normal action potential and normal intracellular K+. The thesis uses a chick model to analyse the effect of structure on the conduction system. The first part of the thesis utilises antisense oligonucleotide morpholino technology for gene knockdown (KD) of the mRNA of αMCH, βMHC and eMHC proteins to analyse the effect on structure and conduction in the heart. Cultures of atrial and ventricular KD HH29 cells showed no differences in beating rate, though 2 out of 9 samples of αMYH and eMYH culture failed to form beating syncytiums, compared to all controls that did. The structural maturity of KD cultures was assessed through Z-disc integrity by immunocytochemistry. Decreased maturity of both the atria and ventricular culture was found KDs. Expression of selected conduction genes was also assed with the pace maker cell potassium channel HCN4 showing decreased levels at the sinus venosus region by in situ hybridisation and significant decreases by RT-qPCR in whole chick heart embryos at HH20. The decrease in expression of such genes could be caused by disruption to internal cell architecture that organises expression of proteins at the cell membrane. Structure at the Z-disc, which show immaturity in KD hearts, is key for this process. Altered haemodynamics and cardiomyopathy is known to effect internal heart structure and the next phase of the project utilised an out flow tract banding (OFT-banding) technique that has been shown previously to alter haemodynamics, effect heart structure and show features of cardiomyopathy. Global sequencing was carried out in order to assess the effect that OFT-banding may have on the conduction system in HH29 chick embryos. In order to carry out global gene expression on OFT-banded hearts, a library preparation method was optimised that removed excess haemoglobin from the hearts and an >99% reduction in all embryonic globin genes was seen, this then allowed detailed gene analysis even of low read genes by RNA sequencing. Sequencing revealed differential expression of calcium sequestering genes in what appears to be a conductive cardioprotective mechanism to maintain coordinated contraction. Interestingly, sequencing also revealed a gene profile that would be expected to alter AMPK signalling that could lead to a multitude of disorders or affects such glycogen storage cardiomyopathy or increased inhibition of myosin expression.

Ubiquitin Specific Protease (USP) 4 is a deubiquitinating enzyme (DUB) which is an important regulator of different cellular pathways, such as Wnt signalling, and A2A-adenosine receptor signalling. USP4 can remove ubiquitin from RIP1, PDK-1, and Ro52, interacts with SART3 at the spliceosome and regulates TNFα and IL-1β in cancer. The full-length structure of USP4 remains to be investigated. The structure of USP4 consists of an N-terminal DUSP (domain in USPs), two Ubl (Ubiquitin-like) domains, and two subdomains that form one catalytic domain. Only the structure of the DUSP-Ubl and catalytic core lacking the Ubl2 domain has so far been determined. Six constructs were cloned based on the USP4 domain architecture to investigate the structure and function of individual USP4 domains. The constructs were tested by enzymatic activity assays. Site directed mutagenesis of the catalytic site, namely mutants H881N and C311S were generated to form complexes with Ubiquitin, Ubiquitin-GGG, diubiquitin and diubiquitin-L73X. The 6 constructs were: USP4FL (full-length), USP4htt (head-to-tail), USP4httΔUbl2, USP4C1C2 (catalytic core), USP4ΔDU (lacking the N-terminal DUSP-Ubl domains), and USP4DU (N-terminal domains). Expression and purification was generally done by the Ni column and gel filtration method. Some further purification was performed by anion exchange chromatography. Enzymatic activity assays were conducted using Ub-AMC (Ubiquitin-7-amido-4-methylcoumarin) as the fluorogenic substrate. The mutagenesis was done by exchange of Histidine (H) to Asparagine (N) and Cysteine (C) to Serine (S) in the catalytic triad and protein complexes were created by combining the mutants with Ubiquitin variants. These complexes were set up for crystallization trials. The protein complexes of active site mutant USP4 with Ubiquitin variants were analysed using ESI-MS and ITC. All the protein constructs were successfully cloned, expressed and purified. USP4httΔUbl2 was well expressed with high yields and USP4htt needed optimisation to increase the yield. Enzymatic activity assays showed that the highest specific activity was obtained for USP4C1C2 whereas USP4htt was the lowest. Some protein crystals were obtained from complexes of active site mutant USP4 with Ubiquitin variants. Only the complex USPFL-H881N with diubiquitin produced crystals that diffracted to 3 Å resolution although the structure has not yet been solved. Binding interaction between various active site mutant constructs of USP4-C311S with ubiquitin variants showed that Ubiquitin and Ub-GGG have the highest affinity for USP4C1C2-C311S. Diubiquitin had the highest affinity for USP4FL-C311S. Together these data provide novel insights into USP4 structure and ubiquitin recognition.

Metabolic complications of obesity are mainly linked to body fat distribution. Since body fat distribution is sex-linked, understanding the role of sex hormones in body fat distribution and storage is important. The acylation stimulating protein (ASP) is a novel lipogenic factor that is suggested to be influenced by hormonal changes. The aim of the present study was to investigate the effect of sex steroid hormones on ASP production and related gene expression, by using adipose tissue explants isolated from ovariectomized rats and the 3T3-L1 cell line generated from mouse embryonic fibroblast cells. In ex vivo studies, 3-way ANOVA analysis showed an effect of hormones, fat depots and chylomicrons interactions on ASP production (p=0.005). Further analysis demonstrated that progesterone and estrogen (P&E) treatment stimulated ASP production (p=0.006) in comparison to estrogen group in subcutaneous adipose tissue explants. In the presence of chylomicrons, visceral adipose tissue explants incubated with P&E showed increased ASP secretion by 54% (p=0.003). The same treatment led to a significant reduction of ASP concentration in subcutaneous explant media (32% lower, P=0.03). C5L2 protein expression were more affected by hormone and chylomicrons interaction (p=0.025). The expression of C5L2 protein increased significantly by P&E treatment (p=0.04) in comparison to the control group in subcutaneous tissue explants. Fat depots and chylomicrons interaction showed a significant effects on effect on ASP’s precursors C3 (p=0.003), factor B (p=0.009) and adipsin (p=0.001) mRNA expressions. DGAT1 have shown to be influenced by hormone and chylomicrons interactions (p=0.3) with no interaction effect on LPL mRNA expression. Using 3T3-L1 cells, high doses of progesterone in the presence of estrogen significantly induced ASP production (p< 0.001). C5L2 protein expression increased in dose-dependent manner with progesterone or estrogen treatments. Treatment of 3T3-L1 adipocyte cells with P&E and chylomicrons had no effect on ASP production. Estrogen receptors (ERs) blockade increased the gene expression of C5L2 and PLC significantly by 5-fold. In summary, female steroid hormones (P&E) may regulate fat distribution through their effects on ASP and C5L2 in subcutaneous adipose tissue. Chylomicrons modulated the ASP responses to P&E treatment in this tissue. P&E interaction with ASP was proven to be regulated through their receptors (PR and ERs) in 3T3-L1 adipocytes. These results demonstrated an important role of female sex steroid in fat distribution and adipocyte lipid metabolism by mediating ASP- C5L2 pathway. Therefore ASP is an important lipogenic factor that, under normal female hormonal states, may reduce the metabolic health risk in women.

The transcription factor ELK-1 is associated with numerous cellular processes, notably in cell proliferation and lineage determination. ELK-1 forms a ternary complex with a dimer of Serum Response Factor at Serum Response Elements associated with immediate-early genes, such as CFOS. Following mitogen-stimulated activation of the ERK cascade, ELK-1 is phosphorylated to mediate transcriptional activation of its target genes. Further to phosphorylation, ELK-1 is covalently modified post-translationally with both monoubiquitin and polyubiquitin chains. This study sought to understand the functional significance and regulation of ELK-1 ubiquitination. Sites of ubiquitination of ELK-1 ectopically expressed in HEK293T cells were mapped and screened for the signature diglycine motif associated with ubiquitinated tryptic peptides using liquid chromatography - tandem mass spectrometry (LC-MS/MS). This revealed that lysine residues within the amino-terminal DNA-binding domain of ELK-1 are monoubiquitinated, including those involved in DNA binding. Ubiquitination is reversed through the action of deubiquitinating enzymes (DUBs), which are target specific, allowing dynamic control over the protein modification state. USP17, one of several candidate DUBs, was shown to deubiquitinate ELK-1 when ectopically expressed in HEK293T cells. This was reversed when USP17 expression was knocked down, which also downregulated transcription of ELK-1 responsive genes CFOS and EGR1. Furthermore, USP17 knock-down in HEK293T cells reduced cell proliferation, an effect that was partially rescued by expression of a hypo-ubiquitinated ELK-1 mutant. Taken together, these results reveal monoubiquitination to be a key regulator of ELK-1 transcriptional potency and mitogen-driven proliferation.

Cyclin dependent kinases (CDKs) belong to a family of serine/threonine protein kinases that play a key role in cell cycle and transcriptional regulation. CDK9, in complex with its regulatory partner cyclin T1, is involved in RNA transcriptional regulation through phosphorylating the RNA polymerase II. Inhibition of CDK9 can target multiple cancer-relevant pathways by downregulating the transcriptionally inducible genes of cancer cells, such as cell cycle regulators and antiapoptotic factors. Although many CDK inhibitors have been in clinical trials, there is only one CDK9-selective compound (BAY1143572) in trials. Therefore, it is still necessary to study other highly selective CDK9 inhibitors. Through the analysis of the cocrystal structure of lead compound 5-(2-((3-(1,4-diazepan-1-yl)phenyl)amino)-pyrimidin-4-yl)-N,4-dimethyl-thiazol-2-amine (5) bound to CDK2 and CDK9, compound (5) favours the “inward” conformation in CDK9, while it adopts both the “inward” and “outward” conformations in CDK2. Aurora A and aurora B (ARKs) inhibitors are also found to adopt the similar “outward” conformation” in ATP binding site of ARKs. Therefore, an “inward” conformation macrocyclic structure was designed to improve the selectivity against CDK9 over CDK2 and ARKs. Three series of macrocyclic compounds were designed, synthesised and tested against CDK9, CDK2, and ARKs. The first series macrocyclic compounds with amide linker shows poor inhibitory activity against CDK9, and the molecular docking study shows these macrocyclic compounds fail to reside in the ATP binding site of CDK9. Following that, ring closing metathesis (RCM) was exploited as an alternative cyclisation strategy. Although the synthesis work was unsuccessful at the first onset, compound 6-methyl-12-oxa-3,6-diaza-2(4,2)-pyrimidina-1,4(1,3)-dibenzenacyclodo-decaphan-8-ene (43) with a similar structure as designed compounds was introduced as the model compound to optimise the RCM reaction. The thiazole group was then demonstrated to be the interfering factor in RCM and compound 11-oxa-3-aza-2(4,2)-pyrimidina-5(1,4)-piperazina-1,4(1,3)-dibenzenacyclo-undecaphan-7-ene (44) with a replacing phenyl group was successfully synthesised and showed poor activity against CDK2 and ARKs (>219 nM for these three kinases). According to the published paper regarding lead compound (5), extensive substituents modification (-CH3, -CN, -F) of compound (44) afforded 11-oxa-3-aza-2(4,2)-pyrimidina-5(1,4)-piperazina-1,4(1,3)-dibenzenacyclo-undecaphan-7-ene-1-carbonitrile (79) as the most selective CDK9 inhibitor. It shows 60-fold selectivity for CDK9 over CDK2 and 90-fold selectivity for CDK9 over ARKs. A further Diversity Kinase Profile Screening demonstrates the high selectivity of compound (79) that only tyrosine-protein kinase Lyn(h) appeared as the off-target. To improve the inhibitory activity against CDK9, alternative macrocyclic pyrimidine systems were designed, and compound 1-methoxy-4-(4-methylpiperazine-1-carbonyl)-3,8-diaza-2(4,2)-pyrimidina-1(1,3),4(1,2)-dibenzenacyclononaphan-7-one (89) showed an attractive Glide score based on molecular docking studies. Because of the time limit, the synthetic work for compound (89) has not been finished.

MiRNAs (miRNAs) are important for the proper regulation of translation, with aberrant miRNA expression contributing to diseases such as cancer. MiRNAs are expressed from longer primary-miRNA transcripts and found in a variety of genomic locations, including introns and exons of coding or long noncoding RNAs. MiRNA biogenesis begins with excision of the precursor miRNA hairpin by the Microprocessor complex which consists of two proteins: Drosha and DGCR8. Both Microprocessor cleavage and splicing occur co-transcriptionally. Splicing and Microprocessing coexist when a miRNA hairpin is located within an intron without detriment to mature mRNA or miRNA production, but little is known about how these two processes interact when a miRNA hairpin is located within an exon. Similarly, little is known about how a miRNA is processed from a long non-coding (lnc)RNA, which is an RNA transcript longer than 200 nucleotides that does not code for a protein. Intronic miRNAs are processed without detriment to the splicing of the host transcript and production of functional mRNA and protein; however, exonic miRNA processing would be expected to lead to cleavage of the exon, interfering with production of mature mRNA. To understand how the genomic location of a miRNA hairpin affects the splicing of the host gene and how splicing affects miRNA production from different locations, two different approaches were used. Firstly, liver-specific miR-122 was inserted at a series of locations within a β-globin plasmid and transfected into HeLa cells, which do not express miR-122. qPCR and northern blotting show that mature miR-122 expression is increased when located within either an intron or exon compared to its endogenous context within a long noncoding RNA. Importantly, the levels of spliced β-globin was shown to decrease when miR-122 was expressed from an exon. This suggests that there is some competition between miRNA biogenesis and splicing. Secondly, the effect of the splice inhibitor pla B was used to understand the effect of splicing on endogenous pri-miR-122. Splice inhibition led to a decrease in the levels of spliced pri-miR-122, but interestingly also led to a decrease in the levels of unspliced transcript indicating that splice inhibition strongly reduces the transcription of pri-miR-122. This was found to be unique to the endogenous gene, as it was not seen when pri-miR-122 was ectopically expressed in HeLa cells. Analysis of the chromatin-associated RNA further confirmed that splice inhibition was having a strong negative effect on transcription. Transcription of pri-miR-122 is known to be terminated by Microprocessing rather than the canonical pathway, and analysis of other lnc-pri-miRNAs which are Microprocessor terminated showed that the effect of splice inhibition on transcription was not unique to pri-miR-122, but was also shared by the important oncogene pri-miR-17~92a. The results in this thesis show that intronic or exonic miRNA hairpins do have an effect on splicing, and unexpectedly that splicing is important for efficient transcription of specific lnc-pri-miRNAs. This work shows that the relationship between Microprocessing and splicing, as well as transcription, is complex. By elucidating how these co-transcriptional processes occur on the same transcript, the understanding of how miRNA biogenesis is regulated from different genomic locations will be improved. This could further the current knowledge of how miRNA disregulation occurs in disease and may lead to the development of new drugs to treat them.

DEF6 (Differentially Expressed in FDCP 6, also known as IBP and SLAT) is critical for the development of autoimmune disease and cancer. In T cells, DEF6 participates in TCR-mediated signalling determining T helper cell-mediated immune responses. In addition, DEF6 acts as a guanine nucleotide exchange factor (GEF) for Rho GTPases facilitating F-actin assembly and stabilisation of the immunological synapse (IS). However, DEF6 is also a component of mRNA processing bodies (P-bodies) linking it to mRNA metabolism. DEF6 and its only relative switch-associated protein-70 (SWAP70) share a common domain structure that is highly conserved from human to trichoplax adhaerens suggesting an ancestral function that is distinct from its role in T cell immunity. To further dissect the structure-function relationship of DEF6, a comprehensive analysis of wild type and mutant DEF6 proteins expressed in either COS7 or Jurkat T cells was conducted. The results demonstrate that DEF6 can adopt multiple conformations that result in different cellular localisations and functions. Post translational modifications such as phosphorylation result in conformational change liberating functional domains that are masked in the native state of DEF6. ITK phosphorylation of Try210/222 liberates the N- terminal end and to a certain extend also the C-terminal coiled coil domain of DEF6 resulting in P-body colocalisation. In fact, the N-terminal 45 amino acids of DEF6 that forms two Ca2+-binding EF hands are sufficient to target P-bodies. The coiled coil domain in conjunction with the N-terminal end is facilitating dimerisation and oligomerisation of DEF6 likely to be essential for GEF activity which was mapped to amino acids 537-590, a region that includes the C-terminal end of the coiled coil domain. Mutant proteins that unleashed the coiled coil domain spontaneously aggregated forming large structures in the cytoplasm. These aggregates trapped proteins such as the P-body component DCP1. In some cases, aggregates appeared to function like prions enforcing conformational change onto wild type as well as mutant DEF6 proteins. Ectopically expressed DEF6 colocalised with F-actin in cell protrusions as well as with P-bodies in resting Jurkat T cells. Upon TCR-mediated activation, wild type and several mutant DEF6 proteins were recruited to the IS. However, while wild type DEF6 localised to the central supramolecular activation cluster (cSMAC), mutant DEF6 with disrupted coiled coil domain were present in the outer ring of IS suggesting that recruitment of DEF6 to the cSMAC depends upon a functional coiled coil domain. In contrast, recruitment to the IS of DEF6 that seemed to be mediated through cellular protrusion contacting the antigen presenting cell was independent of both the N-terminal end and a functional coiled coil domain. Taken together, the data presented provide evidence that DEF6 is more than a GEF linking TCR-mediated signalling with F-actin organisation and control of mRNA metabolism.

GPR18 receptor is a candidate cannabinoid receptor with potential of being a novel therapeutic target due to its wide distribution in various tissues. NAGly which is a metabolite of the first isolated endocannabinoid anandamide, had been suggested by several studies as an endogenous high potency ligand of GPR18 receptor. Yet, some studies have reported the lack of activation of GPR18 by NAGly and some other putative GPR18 cannabinoid ligands. Identifying the signalling mechanism of GPR18 will advance our understanding of the complexity of the endocannabinoid system and may have important implications in determination of the molecular mechanism of action of phytocannabinoids. The rationale of this study was to characterize the pharmacology of GPR18 by investigating the effects of NAGly and other putative GPR18 cannabinoid ligands like THC, Abn-CBD, O-1918 and NARAS on the intracellular signalling mediators, extracellular signal-regulated kinases (ERK1/2) and Ca2+ ions in HEK293TR heterologously expressing GPR18 receptor as well as the cell lines that have been reported in previous studies to express GPR18 endogenously or show biological responses to NAGly namely, mouse microglial cells (BV-2), rat insulinoma β-cell line (INS-1 832/13 β-cells) and human colorectal cancer cells (Caco-2). Changes in the intracellular Ca2+ were also measured following co-expression of GPR18 in Chinese hamster ovarian cell line (CHO) heterologously expressing CB2 and exposure to the GPR18 putative ligands. SNAP-tagged human GPR18 receptor under the control of a tetracycline regulated expression system was heterologously expressed in HEK293TR cells. The effect of putative GPR18 agonists on intracellular Ca2+ mobilisation was assessed using FlexStation and single cell Ca2+ imaging techniques with different Ca2+ probes (fluo-4 and fura-2). NAGly induced ERK phosphorylation was quantified by immunoblotting. The tested panel of cannabinoid ligands did not observed to elicit a significant ligand-mediated phosphorylation of ERK1/2 or mobilisation of intracellular Ca2+ in GPR18-expressing HEK293TR cells, BV-2 cells, INS-1 832/13 β-cells, Caco-2 cells and GPR18-transfected CHO cells heterologously expressing CB2 receptors. In this study recombinant human GPR18 receptor was not activated by NAGly or other cannabinoid ligand suggested by previous studies which indicated that NAGly may be not the natural endogenous ligand of GPR18 and argue against the deorphanization of GPR18 and assignment as a candidate cannabinoid receptor. Also it can concluded that the activation of GPR18 signalling mechanism may involve prodigious pathways not examined in the current study.