Elucidating regulatory mechanisms of protein prenyltransferases

Kubala, Marta Helena (2012). Elucidating regulatory mechanisms of protein prenyltransferases PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Kubala, Marta Helena
Thesis Title Elucidating regulatory mechanisms of protein prenyltransferases
Formatted title
Elucidating Regulatory Mechanisms of Protein Prenyltransferases
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2012-12
Thesis type PhD Thesis
Supervisor Kirill Alexandrov
Brett Collins
Mike Waters
Total pages 201
Total colour pages 59
Total black and white pages 142
Language eng
Subjects 060199 Biochemistry and Cell Biology not elsewhere classified
060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
060112 Structural Biology (incl. Macromolecular Modelling)
Formatted abstract
Prenylation is post-translational lipidation of proteins where farnesyl or geranylgeranyl isoprenoid groups are attached via thioether linkage to one or two cysteines near to C terminus of protein substrates (Casey, 1996). Addition of a prenyl-group mediates protein association with membranes (Casey, 1994) and modulates protein – protein interactions (Marshall, 1993) together with protein activity. The fact that mutations within prenylated Ras proteins could be identified in 10–15% of all human cancers (Downward, 2003) has made prenylation a popular cancer target and a number of prenyltransferase inhibitors have been identified and are undergoing clinical trials (Agrawal, 2009). The three enzymes responsible for isoprenoid addition to proteins are called prenyltransferases and include farnesyltransferase, geranylgeranyltransferase type I and geranylgeranyltransferase type II (Casey, 1996). All three enzymes are heterodimers consisting of α and β subunits. The β subunit comprises the active site of the enzyme while the role of α subunit is still unclear. Despite extensive research, it is still not known whether protein prenyltransferases are regulated and how it is exerted. In this project we use a combination of structural and biochemical analysis to understand the role of the α subunit in the regulation of protein prenylation. It appears that the α subunit plays the role of a chaperone protein, facilitating folding of the β subunit. We suggest that α subunits of prenyltransferases may also regulate activity of heterodimers via interactions with other proteins. To investigate proteins potentially interacting with α subunits we have performed co-immunoprecipitation experiments followed by mass spectrometry analysis yielding a list of potentially interacting proteins. Combining three technologies: Leishmania tarentolae cell free system, in vitro pull downs and Two Color Coincidence Detection, we established a method to rapidly assess the authenticity of identified interactions. Further the significance of novel interaction of the FTase α with Vps4A was investigated by Fluorescence Recovery After Photobleaching and live cell imaging. The results suggest that FTase α regulates cellular trafficking upstream of Vps4A. To understand structure-function relationship between prenyltransferase subunits we study structure and activity of the enzyme by Small Angle X-ray Scattering, Crosslinking, Multi Angle Laser Light Scattering and fluorescence-based activity assays. The yeast-based Ras recruitment genetic screening system is used for the purpose of identification of activated K164 and Q167 FTase mutants. The active monomeric fusions of FTase are created for the purpose of structure-function studies. In experiments involving L.tarentolae Cell Free System, in co-immunoprecipitation experiments as well as in Alpha Screen a GFP specific antibody also known as GFP-nanobody was used. By means of X-ray crystallography and Isothermal Titration Calorimetry we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding.
Keyword Prenylation
FTase α subunit
GFP-nanobody
Trafficking

 
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Created: Sun, 09 Jun 2013, 23:01:06 EST by Marta Kubala on behalf of Scholarly Communication and Digitisation Service