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Title: Development of chemical tools to study protein palmitoylation and palmitoyl acyltransferases
Author: Masumoto, Naoko
ISNI:       0000 0004 6420 9567
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Protein palmitoylation is an attachment of palmitate which is one of the most common post-translational modifications (PTMs) of proteins. It is important in various cellular events, and dysregulation has been observed in many diseases including cancer and neurological/neuropsychiatric disorders. There is increased interest in the development of novel therapeutic agents targeting palmitoyl acyltransferases (PATs); however, progress in understanding palmitoylated proteins/PATs has been slow due to the nature of modification and the lack of reliable assays until the recent development of the bioorthogonal ligation technique. Within the rich array of palmitoylated proteins, the main focus of this thesis was on Hedgehog (Hh) proteins which are irreversibly palmitoylated unlike the majority of reversibly palmitoilated proteins. Hh proteins act as morphogens in embryonic development and play a crucial role in tissue homeostasis in adult organisms. Mutations in various Hh signal transduction effectors and aberrant Hh signalling results in developmental malformation and tumourigenesis in adults. Hh proteins are dual lipidated (N-terminal cysteine by palmitate and C terminal glycine by cholesterol) in order to produce mature, fully-functional Hh signalling ligands. Irreversible N-palmitoylation of Hh proteins is catalysed by a member of membrane bound O-acyltransferases (MBOATs) called Hedgehog acyltransferase (HHAT). Efforts have been made to find pharmacologic agents to control the Hh signalling pathway; however, inhibition of downstream Hh signal components has led to the emergence of drug resistance, thus there being an urgent need to circumvent such issues. RU-SKI candidate HHAT inhibitors have opened new avenues to control Hh signalling by inhibiting palmitoylation of Hh ligands, hence attenuating the activation of the Hh pathway. This thesis describes the utilisation of the bio-orthogonal ligation technique to profile palmitoylated proteins, particularly the most abundant Hh homologue, Sonic Hedgehog (Shh) protein, using a palmitate mimic, YnPalm, in cell-based assays. After profiling Shh palmitoylation in depth, detailed characterisation of RU-SKI inhibitors has been performed to understand the effect of Shh secretion and cell signal transduction upon HHAT inhibition. Known PAT inhibitors were derived from chemical reporters in order to elucidate the target proteins by chemical proteomics approach. In short, RU-SKI inhibitors are highly selective against HHAT in terms of profiling their effects in global palmitoylation; however, their toxic effects towards signal reporter cell lines have interrogated the accurate read out in cell signalling activity. From the PAT inhibitor characterisation work, it has been reiterated that there are no specific PAT inhibitors available to date.
Supervisor: Tate, Edward ; Magee, Anthony Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral