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Title: A chemical proteomic approach to investigate Rab prenylation in living systems
Author: Berry, Alexandra Fay Helen
ISNI:       0000 0004 2732 5032
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Protein prenylation is an important post-translational modification that occurs in all eukaryotes; defects in the prenylation machinery can lead to toxicity or pathogenesis. Prenylation is the modification of a protein with a farnesyl or geranylgeranyl isoprenoid, and it facilitates protein-membrane and protein-protein interactions. Proteins of the Ras superfamily of small GTPases are almost all prenylated and of these the Rab family of proteins forms the largest group. Rab proteins are geranylgeranylated with up to two geranylgeranyl groups by the enzyme Rab geranylgeranyltransferase (RGGT). Prenylation of Rabs allows them to locate to the correct intracellular membranes and carry out their roles in vesicle trafficking. Traditional methods for probing prenylation involve the use of tritiated geranylgeranyl pyrophosphate which is hazardous, has lengthy detection times, and is insufficiently sensitive. The work described in this thesis developed systems for labelling Rabs and other geranylgeranylated proteins using a technique known as tagging-by-substrate, enabling rapid analysis of defective Rab prenylation in cells and tissues. An azide analogue of the geranylgeranyl pyrophosphate substrate of RGGT (AzGGpp) was applied for in vitro prenylation of Rabs by recombinant enzyme. Alternatively, geranylgeranylated proteins (including Rabs) were labelled with AzGG via metabolic labelling of live cells with AzGGOH. Once Rabs were tagged with an azide moiety they could be labelled via a bioorthogonal ligation reaction with a trifunctional alkyne probe. The probe contained a fluorophore for in-gel fluorescence analysis and a biotin affinity label for affinity purification of labelled proteins. The conditions for protein tagging, labelling, affinity purification and LC-MS/MS analysis were optimised significantly during this work. Affinity purified proteins were identified and in some cases quantified using LC-MS/MS techniques, with iTRAQ labelling for quantification. Rab prenylation was probed in cell culture, in cells treated with the drug Mevastatin and in tissue from mouse models with defects in the prenylation machinery.
Supervisor: Seabra, Miguel ; Tate, Ed Sponsor: Institute of Chemical Biology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral