The regulation of cellular activity by intracellular proteins often involves post translational modifications (PTM) with small charged groups, such as PO43-, or small proteins, such as ubiquitin. This thesis explores the role of PTM of proteins with the 9.1kDa protein ubiquitin-fold modifier 1 (UFM1) in a process called UFMylation, within Fanconi Anaemia (FA) cell lines. This follows preliminary data produced by Pierce et al., (Unpublished) which suggested that UFMylation activity of the target protein RPL26, takes place more predominantly in FA mutant cell lines compared to non-FA cell lines.
UFM1 is added to a protein in a similar addition cascade to ubiquitin with a non-Ubiquitin (non-canonical) E1 activating step, E2 conjugating step and E3 ligating step (UBA5, UFC1 and UFL1 respectively). Where Ubiquitination has been extensively identified throughout the cell to upregulate protein production or mark proteins for degradation, UFMylation has a limited set of targets though their roles are widespread. UFMylation has been credited to be involved in endoplasmic reticulum-homology (with RPL26), homologous recombination (with MRE11) and protein regulation (with DDGRK1) amongst other things. The contributing role that the addition of UFM1 has on these targets remains unknown.
FA is a rare autosomal recessive disease characterised by biallelic mutation of any gene within the Fanconi pathway which is crucially is involved with the repair of inter-strand crosslinked (ICL) deoxyribonucleic acid (DNA). Failure in ICL repair, particularly in haematopoietic stem cells, leads to bone marrow failure, haematological defects and increase leukemic risk.
Using western blot analysis to explore UFMylation activity we demonstrated UFMylation activity with RPL26, and the presence of UFMylation cascade proteins, across both FA and non-FA cell lines. Preliminary investigation was commenced to explore the impact of cell flask growth conditions and subsequent cell pellet density on RPL26 UFMylation. The initial study suggested that confluency influenced UFMylation activity; potentially reflecting the cell cycle or cell stress dependent nature of UFMylation activity with RPL26.
Contrary to initial observations, UFMylation of RPL26 is not exclusive to FA cell lines, suggesting a broader regulatory role. We speculate UFMylation’s involvement in non-catalytic signal transduction pathways and propose further exploration into UFMylation’s cellular responses to stress and its implications for protein regulation.