Chemokines are the principle regulators of leukocyte migration in vivo and function during both normal (homeostatic) and inflammatory conditions to direct leukocytes to appropriate tissue locales. Chemokines mediate their affects by binding to their cognate G-protein coupled receptors (GPCRs) which are expressed on the surface of cells, and generate a signal upon ligand binding resulting in the initiation of a response such as chemotaxis. As well as the classical chemokine receptors which generate a conventional GPCR signal upon ligand binding, there exists a small family of atypical chemokine receptors that are characterised by an inability to mount classical receptor signalling. One of the most prominent members of this family is the atypical chemokine receptor, D6, which can bind at least 14 inflammatory CC chemokines with high affinity, but instead of the generation of a classical G-protein signalling response, D6 internalises ligands and targets them for lysosomal-mediated degradation. This functional attribute makes D6 a highly efficient binder, internaliser and scavenger of inflammatory CC chemokines that has been shown to be important for the resolution of inflammatory responses in vivo. Despite its well-studied biological role, very little is known about the structure/function relationships within and around D6 which regulate ligand binding and scavenging. Glycosaminoglycans have been demonstrated to be important for chemokine sequestration and presentation to many of the conventional chemokine receptors. Consequently, the role of glycosaminoglycans (GAGs) in chemokine presentation to D6 was studied using a cell line which is deficient in the synthesis of proteoglycans (CHO 745). Transfection of these cells with D6 and comparison to transfected WT CHO cells revealed that D6-mediated uptake and internalisation of chemokine is significantly reduced in the absence of GAGs. The N-terminus of D6 is thought to be the principle site for ligand binding, and the ability of D6 to bind all inflammatory CC chemokines makes this region an attractive target for therapeutic manipulation. Therefore a sulphated peptide representing the first 35 amino acids of D6 (D6-N (s)) was synthesised and investigated for its ability to bind D6 ligands. D6-N (s) was shown to neutralise the activity of the inflammatory CC chemokine CCL2 and prevent its interaction with its cognate receptor CCR2 in vitro. Importantly D6-N (s) was active, only in a specifically sulphated form, highlighting the importance of sulphated tyrosines for ligand binding. Considering the functional significance of the synthetic D6 peptide, attempts were made to identify a naturally ‘shed’ D6 N-terminal peptide which had been reported previously. Further study demonstrated the ability of the bacterial protease staphopain A, released from Staphylococcus aureus, to cleave the N-terminus of D6 and suppress its ligand internalisation activity. Finally, the conserved tyrosine motif present on the N-terminus of D6 was investigated more closely. Site-directed mutagenesis and sulphation inhibition of this region revealed the importance of post-translational tyrosine sulphation for ligand binding, internalisation and scavenging of inflammatory chemokines and alluded to the existence of an optimal sulphation pattern for ligand binding. Overall the results presented in this thesis shed new light on the nature of the molecules around, and the structural features within D6 that contribute to ligand binding and function of this extraordinary receptor. Furthermore, it was shown that a sulphated peptide derived from the N-terminus of D6 has the potential to be used therapeutically as a broad-based chemokine scavenger, which may be useful for dampening the effects of excessive chemokine production in chronic inflammatory conditions.

NF-κB is a key transcription factor involved in the regulation of inflammation. The transcriptional activity of NF-κB is regulated by a number of post-translational modifications, including phosphorylation. Phosphorylation of NF-κB subunits may regulate transcription in a gene selective manner. The NF-κB p50 subunit is an important dual regulator of inflammatory responses, which can either promote or repress gene expression depending on the formation of heterodimer or homodimer complexes, respectively. Although p50 is a critical regulator of the immune system, phosphorylation of this subunit is largely understudied. In this thesis, the role of phosphorylation of NF-κB p50 on serine 80 (S80) in regulating transcriptional responses is investigated, using two human NFKB1S80A knock-in cell lines generated by CRISPR/Cas9 genome editing techniques. Transcriptomic analyses reveal a critical role for S80 in selectively regulating the expression of a subset of NF-κB target genes in response to TNFα and LPS, including pro-inflammatory chemokines and cytokines. DNA binding analyses demonstrate that S80 regulates the binding of p50 to NF-κB binding sites in a sequence-specific manner. Specifically, phosphorylation of S80 reduces the affinity of p50 for κB sites that have an adenine at the -1 position. These data indicate that p50 S80 phosphorylation predominantly regulates transcription through the p50:p65 heterodimer, where S80 phosphorylation acts in trans to limit the NF-κB mediated transcription of pro-inflammatory genes. This advances our understanding of the regulation of transcriptional programmes by the NF-ĸB p50 subunit.