Kindler syndrome (KS) is an autosomal recessive genodermatosis resulting from pathogenic mutations in the FERMT1 (KIND) gene. This gene encodes kindlin-1 (also known as fermitin family homologue 1), a focal adhesion protein involved in activation of the integrin family of extracellular matrix receptors. Most cases of KS show a marked reduction or complete absence of the kindlin-1 protein in keratinocytes, resulting in defective integrin activation and abnormal cell adhesion and migration. However, currently very little is known about the way in which different FERMT1 mutations found in patients impact upon keratinocyte behaviour. The aim of this thesis is to screen for novel binding partners of kindlin-1 to elucidate possible new functions for this protein in epidermal cells. The main approaches taken were to assess differentially expressed proteins in normal vs. KS patient keratinocytes and also to use recombinant wild type and mutant kindlin-1 for pulldown assays to identify novel binding partners. Mass spectrometry analysis revealed a number of downregulated proteins in KS keratinocytes including epidermal growth factor receptor (EGFR) and thrombospondin-1 (TSP-1). Immunoblotting confirmed a significant reduction in both of these proteins. Further investigation showed changes in localisation of both TSP-1 and EGFR, and also defective signalling and response following EGF stimulation in KS keratinocytes. Moreover, EGFR levels in KS keratinocytes were partially restored following treatment with lysosomal inhibitors, suggesting that kindlin-1 can regulate the recycling of this receptor. Mass spectrometry analysis also identified the potential interaction partners of kindlin-1 to be talin and integrin linked kinase (ILK). Biochemical analysis revealed that kindlin-1 interacts specifically with the talin head domain. This interaction was significantly reduced with a mutant form of kindlin-1 dentified in a KS patient that lacks part of the F3 subdomain. Taken together, these studies have identified novel proteins that are regulated by kindlin-1 and may be important regulators of keratinocyte adhesion and migration.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:762262 |
| Date | January 2013 |
| Creators | Begum, Rumena |
| Contributors | Parsons, Madeline ; McGrath, John Alexander |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/kindlin1-proteinprotein-interactions-and-functional-relevance-to-kindler-syndrome(6c122016-c55d-4cbe-b4c3-99be4def7e9e).html |
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