Molecular analysis of the Ccr4-Not deadenylase: relevance to human diseaseTools Airhihen, Blessing (2017) Molecular analysis of the Ccr4-Not deadenylase: relevance to human disease. PhD thesis, University of Nottingham.
AbstractIn eukaryotes, the removal of the poly (A) tail of cytoplasmic mRNA (deadenylation) is a crucial step in post-transcriptional gene regulation. A major enzyme involved in regulated mRNA deadenylation is the Ccr4-Not deadenylase, which contains two catalytic subunits: the Caf1 and Ccr4 ribonucleases. For both enzymes, two Mg2+ ions are required in the active site for activity. These enzymes in addition to six other non-catalytic subunits of the Ccr4-Not complex are possible drug targets in diseases such as metastatic cancer, osteoporosis and obesity. To facilitate the discovery, development and characterisation of small drug-like inhibitors of these enzymes, a biochemical approach was used. First, we investigated the biochemical characteristics of the highly similar CNOT6 and CNOT6L enzymes. Next, we evaluated two biochemical assays for characterisation of Ccr4-Not catalytic subunits and evaluation of N-hydroxyimide inhibitors of CNOT7. A chemiluminescence-based detection assay of AMP was used as the basis of a method and thermal shift assays were also used to characterize binding of compounds to deadenylase enzymes. In addition, cell based assays were used to study interactions between BTG2 variants and CNOT7 and CNOT8 in lymphoma.
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