Worth, A.J.J.; (2010) Analysis of the domain specific function of the Wiskott Aldrich Syndrome Protein, in vitro and in vivo. Doctoral thesis, UCL (University College London).
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Wiskott Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterised by low numbers of low volume platelets, eczema, severe immunodeficiency and increased susceptibility to malignant and autoimmune diseases. Wiskott Aldrich Syndrome protein (WASp), the product of the gene mutated in WAS is a multidomain cytosolic protein which acts by integrating downstream signals from cell surface receptor signalling cascades and transduces these signals into remodelling of the actin cytoskeleton. WASp is involved in cellular processes as diverse as cell polarisation, migration, phagocytosis and T cell synapse formation. Dendritic cell function and migration is known to be impaired in WAS. Specifically, dendritic cells lacking WASp in both mice and humans show an impaired ability to form transient actin rich adhesive structures called podosomes. In this thesis, I am presenting the characterisation of a panel of domain deletion WASp proteins and a series of point mutations which, in children give rise to a range of disease phenotypes. I have expressed these constructs as GST tagged proteins and developed a bead based in vitro assay to asses the protein activity. I have compared the ability of these mutants to bind key regulators of WASp function, WIP and Cdc42, using a variety of immunoprecipitation techniques. Finally I have expressed EGFP tagged WASp proteins in myeloid cell lines to assess expression and stability of these constructs and in WASp null murine dendritic cells to assess their ability reconstitute podosomes. Point mutations which cause WAS have slightly increased actin polymerisation activity compared to wild type WASp (WT) and have a normal affinity for Cdc42. Although they have a reduced affinity for WIP compared to WT, there is still significant association of WIP to mutant WASp in cells. These mutants have reduced stability and different patterns of phosphorylation compared to WT, when expressed in myeloid cells lines. The EVH1, Polyproline and VCA domains of WASp are all essential for optimal actin polymerisation activity, whereas the Basic domain and the point mutation H246D (which inhibits Cdc42 binding) could be deleted with no loss of molecular activity. The EVH1 domain is essential for WIP binding and the Basic domain is essential for Cdc42 binding. Deletion of the EVH1 domain resulted in protein which had greater stability than WT WASp, suggesting that residues within the EVH1 domain are essential for the physiological degradation of WASp. This work demonstrates that mutant WASp which causes WAS in patients has normal molecular activity, but the regulation of mutant WASp degradation is impaired in myeloid cells. Results from this project also suggest that Cdc42 is not essential for normal WASp functioning, but does appear to be essential for the phosphorylation of the Y291 residue, which is known to be critical for the regulation of WASp activity. Further work will aim to elucidate the functional (in vivo) significance of these findings.
|Title:||Analysis of the domain specific function of the Wiskott Aldrich Syndrome Protein, in vitro and in vivo|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Child Health > Department of Infection and Immunity > ICH - Molecular Immunology Unit|
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