Project

1. Identifying novel interacting partners of human biotin protein ligase

Biotin, also known as Vitamin H, is an essential micronutrient that is required for normal cellular function and is particularly important during early mammalian development. It has long been recognised that biotin functions as a protein bound cofactor for a family of enzymes known as the biotin-dependent enzymes. In mammalian cells these enzymes are localised in both the cytoplasm and mitochondria and are involved in a number of essential metabolic processes. Biotin is covalently attached to these enzymes through a specific reaction catalysed by biotin protein ligase (BPL). Recently, it has been shown that BPL can also localise in the nucleus where it is proposed to modify histones. This has provoked interest in the field as the expression of approximately 300 genes can be regulated by biotin. Although the mechanism by which gene expression is not understood, BPL has been implicated giving reason to believe that the enzyme has novel roles in addition to its catalytic function. Together these observations suggest that BPL interacts with a number of partner proteins in order to traffic to and function in multiple cellular compartments. Our research aims to identify these interacting proteins and understand how they regulate the activity of BPL and, therefore, biotin metabolism. Importantly, we wish to identify how cells respond to biotin-deprivation, as is observed in some disease states and during early development.

Recent work by our collaborator Prof. Cronan, The University of Illinois, has identified a useful technique for identifying proteins that interact with BPL. By mutating a specific amino acid in the active site of bacterial BPL, the enzyme slowly releases adenylated-biotin (naturally produced by the first of two partial reactions catalysed by the enzyme) into the immediate environment. This species reacts readily with amine groups, thus “tagging” nearby proteins with biotin. We will use this technique to biotinylate potential interacting proteins in vivo in mammalian cells. This project will give the honours student valuable skills in molecular biology, recombinant protein expression and tissue culture. In addition proteomic based techniques will be employed to identify the biotinylated partner proteins, giving the student experience in an exciting and important field of biological science. The information obtained from this work will further our knowledge on the biology of BPL in human health and disease.