student profile: Miss Narjis Fatima Rizwan Fatima


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Thesis work

Thesis title: AMPylation of proteins in melanoma cells and chronic lymphoid leukaemia cells maintained in the presence of adenosine and cAMP.

Supervisors: Stuart CORDWELL , Giles BEST, Richard CHRISTOPHERSON

Thesis abstract:

The covalent coupling of an AMP residue to proteins has been well known through examples of the AMPylation of glutamine synthetase isolated from Escherichia coli, AMPylation of host signalling protein GTPases by fic domains of VopS and IbpA in prokaryotes. The catalytic activity of AMPylator is regulated by addition of an AMP moiety through covalent linkage to the protein. Studies of AMPylation in eukaryotes are in initial stages. Research from the laboratory of Dr Vivian Whittaker (University of Sydney) over more than 10 years up to 2001 provided direct and indirect evidence for the covalent addition of nucleotides to proteins in mouse B16 melanoma cells. One of the main novel observations was the formation of proteins with a wavelength of maximal absorbance at 260 nm rather than 280 nm (normally due to tyrosine, phenylalanine and tryptophan residues). This striking difference in the absorption of ultra-violet light in proteins from B16 cells incubated with cyclic AMP (cAMP) was attributed to addition of a chemical derivative of cAMP to the proteins. To take this project further, powerful techniques of proteomics were required that were not available in 2001 in the School of Molecular Bioscience as it was then known.

More recently in 2013, we have commenced the analysis of these proteins using two-dimensional gel electrophoresis, 2D autoradiography, and 2D Western blotting. The results have been extraordinary, with at least 36 different proteins identified that have been radio-labelled from [14C]cAMP. The investigation would extend further to proteins that may be ‘tagged’ with [3H]retinol, 62 such proteins have been detected so far. The PhD project that is available in our laboratory would involve identification of these labelled proteins using liquid chromatography, mass spectrometry and a variety of other techniques. This AMPylation of proteins in human cells is virtually unknown and the initial results obtained with mouse melanoma cells will be translated to several human melanoma cell lines. The functional significance of these covalent modifications in the regulation of cellular growth and metabolism is a key question that will be addressed in this project. These post translational modifications will also be studied in Chronic Lymphocytic Leukaemia cells and our project will try to identify the relation between AMPylation and mechanism of action of key drugs used to treat CLL.

Note: This profile is for a student at the University of Sydney. Views presented here are not necessarily those of the University.