Browsing by Subject "Farnesylation"

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    Studies and applications of protein prenylation using isoprenoid analogues and site-directed mutagenesis
    (2014-06) Dozier, Jonathan Kramer
    The site-specific enzymatic modification of proteins offers a powerful tool both for the development of new research techniques and for the development of more effective therapeutics. Protein farnesyltransferase (PFTase) is a promising tool in the field of site-specific protein modification due to its substrate promiscuity, small recognition domain and mild reaction conditions. PFTase is an enzyme that attaches a farnesyl moiety, from farnesyl diphosphate (FPP), to a cysteine residue located at the C-terminus of proteins in a CaaX box consensus sequence. This dissertation seeks to used PFTase as a tool to develop new methods for both scientific research and biotechnologcical development. PFTase has been shown to utilize a number of non-natural FPP analogues that can be used to create covalent conjugates between proteins and other biologically relevant molecules. One of the main disadvantages of this approach, however, is that PFTase has a much lower activity when using FPP analogues that contain large non-natural functional moieties. We sought to develop and analyze different mutations of PFTase that increase the activity of the enzyme toward these FPP analogues. We found several PFTase mutants that can catalyze the addition of large FPP analogues to proteins at much faster rate than the wild-type enzyme. Additionally we showed that we could use PFTase to create a site-specific conjugate of Ciliary Neurotropic Factor (CNTF); a promising drug candidate for a number of different neurological conditions. In addition, we have begun analyzing its biological activity in vivo. This conjugate is a promising new development in the creation of protein therapeutics, that have a similar activity but a longer retention time in the body than previously drugs. Taken together this dissertation will demonstrate the value and utility of using PFTase for a number of different scientific applications.
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    Synthesis and applications of caged thiols for studying protein prenylation.
    (2012-02) Abate Pella, Daniel
    Ras proteins are a subfamily of small GTP-binding proteins that are involved in various critical cellular processes including cell growth, survival and nuclear transport. It has been reported that roughly 30% of human cancers are derived from mutations of Ras, and prenylation is a key step that activates their oncogenicity. Commercial inhibitors of prenylation have been successful at arresting Ras activation and can be categorized into two families: farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase inhibitors (GGTIs). The focus of this thesis is to explore the use of photoremovable protecting groups (caging groups) to better understand the process of prenylation by caging the critical thiol residues of FTIs, GGTIs and peptides. The caging group bromohydroxy coumarin (bhc) was covalently bound to the thiol of the FTI L-744,832 in order to inactivate the inhibitor. This caged FTI was evaluated with respect to its one- and two-photon uncaging kinetics and ability to release FTI upon photolysis. Analysis shows that bhc photolysis occurs more rapidly compared to the most frequently used family of nitrobenzyl-based cages, and that FTI is produced with good yields upon one- and two-photon excitation. Bhc-FTI was then tested on different cell lines in order to show that upon irradiation FTI is released that inhibits Ras farnesylation (observed via Western blot analysis), Ras membrane localization (detected by confocal microscopy), and downstream signaling (fibroblast morphology). This same approach was utilized to cage FTI with bromohydroxy quinoline (BHQ). The covalent inactivation of FTI with BHQ was employed to cage the active site thiol (BHQ-FTI) and active site amine (BHQ-FTI urethane). Kinetic evaluation suggests that BHQ-FTI uncages faster than bhc-FTI but it produces little FTI upon photolysis due to the formation of unreactive photoproducts. Despite its poor yield, one photon cell experiments with BHQ-FTI resulted in the inhibition of Ras farnesylation, Ras membrane localization and downstream signaling. Quantitation and biological experiments with BHQ-FTI urethane are ongoing. Peptides that are substrates of protein farnesyltransferase (PFTase) were caged with bhc and BHQ at their crucial thiol that is targeted for farnesylation. Upon one-photon photolysis peptides caged with BHQ show poor yields of free peptide while bhc-caged ones result in good peptide production. One of these caged peptides was subjected to an in vitro farnesylation assay to show that no farnesylation occurs, but upon one- and two-photon irradiation farnesylated peptide can be detected. Application of this caged peptide to study the mechanism of farnesylation via X-ray crystallography is under way. Certain Ras proteins are alternatively geranylgeranylated and retain full function when farnesylation has been inhibited; as a result, GGTI-286 was caged with bhc to study this phenomenon. The synthesis of this GGTI and the inactivation of its thiol via covalent bonding with bhc is described here. The kinetic analysis of bhc-GGTI as well as its quantitation and biological testing are a work still in progress.