Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420645
Title: A proteomic investigation of the effect of endothelin-1 on gene expression and protein phosphorylation
Author: Predic, Jelena
ISNI:       0000 0001 3498 8828
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2005
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Abstract:
This thesis describes the investigation of the effect of endothelin-1 (ET-1) on gene expression and protein phosphorylation states in two different fibroblast cell lines using proteomics tools. Protein synthesis was monitored during the first 4 hours following stimulation of human lung fibroblasts with ET-1 using pulsed S methionine labelling. Out of the total proteome visible on 2D gel electrophoresis 22 proteins that are differentially expressed were identified using MALDI-TOF peptide fingerprinting methodology. Changes in protein expression were temporally distinct and proteins could be classified into several different groups according to their similar kinetic behaviour possibly implying functional correlations between them. The presence of ET receptors in NRK-49F (normal rat kidney fibroblast) cells was confirmed by calcium imaging and radioactive labelled specific ET ligand binding studies. Changes in the phosphorylation states of proteins as well as the kinetics of these changes were assessed in NRK-49F cells following brief stimulation with ET-1. For these studies the enriched phosphoproteome was used. Enrichment was achieved by an immobilized metal affinity chromatography methodology developed in our laboratory. Individual proteins were detected with immunostaining or mass spectrometry. ET-1 stimulation was associated with phosphorylation of multiple proteins including: the members of ERK 1/2 and p38 MAPK pathways and small GTP-ase pathway an enzyme regulating calcium levels a transcription factor and a number of cytoskeletal, chaperone and metabolic proteins. Nearly all of the identified proteins were represented in multiple phosporylated forms that individually showed different kinetic behaviour during the examined period of time. The multiplicity of phosphorylations in the identified proteins has an impact on their function. The results widen the current knowledge about ET-1 signalling in NRK fibroblasts and provide a framework for further studies. From the list of the detected phosphoproteins the phosphorylation of RhoGDI was analyzed further. By using SILAC methodology and mass spectrometry a RhoGDI peptide phosphorylated after ET-1 stimulation was detected and phosphorylation sites were mapped. This finding gives an insight into the role RhoGDI phosphorylation may have in regulating the activity of some small GTPases as downstream effectors of ET-1 signalling.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.420645  DOI: Not available
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