Multimodal analysis of proteins of unknown function:MiCATs1

A significant limitation of high-throughput mass spectrometric approaches to establish datasets of interacting proteins is that they only identify stable, multi-protein complexes that do not dissociate during extraction & work-up for analysis. Recently, the bioID system - in which proteins of interest may be tagged in vivo with the enzyme biotin ligase (birA) - allows for identification of more transient, low-affinity interactions that make up the majority of cellular events. Polypeptides that interact with birA-tagged proteins become selectively biotinylated, following addition of biotin to the system. These polypeptides may be subsequently identified by selective chromatographic enrichment on streptavidin-agarose, and analysis by mass spectrometry. One limitation of bioID is sensitivity, current methods require 6 to 24 hours of incubation with biotin and proteins may interact with a large range of targets over time. Differences in the nature and identity of target proteins arising from spatial or temporal changes in cell behaviour - where a sub-population of protein A interacts with X in one location, followed by interaction with Y in another - often make such interaction data difficult to interpret.

The use of high-resolution microscopy for co-localisation analyses of small sub-populations of 2 or more protein species is also challenging. Recently, we have utilised proximity ligation as a tool for the detection, by microscopy, of low-frequency protein-protein interactions in cells (Turton,D., 2014, PhD thesis, University of Sheffield; Zhou, Z., 2017, PhD Thesis, University of Sheffield).

This project will exploit recent emergence of methodology for rapid biotinylation of interacting proteins in vivo (Turbo-ID), to develop molecular tools to undertake large-scale proximity ligation experiments. These will be utilised in in a high-throughput microscopy format, to establish the significance of molecular interactions of proteins of unknown function. The project will utilise low-cost polypeptide expression systems (E.coli) to develop and characterise a bi-functional molecular system (to be called avi-TAG) for detection, using microscopy, of a protein of unknown function in close proximity to a target interacting protein in vivo. The project will involve cloning and soluble expression of a modified monomeric avidin in conjunction with expression of the minimal functional immunoglobulin-binding domain of protein G (PrG). Each of these proteins will be coupled using hetero-bifunctional cross-linking chemistry to unique oligonucleotides capable of enabling rolling circle DNA amplification using fluorescent nucleotides, on microscopy slides, for subsequent high-resolution imaging. In cells expressing a birA-tagged polypeptide, interacting partners will become biotinylated, and the project will determine the efficacy with which the cellular location of such low frequency events may be detected by high-resolution microscopy. The system, once validated using known interacting proteins will be used to investigate the identity of interaction partners, and the sub-cellular localisation of interactions of, a protein of unknown function, here termed MiCats1.

The latter has recently been shown to be involved in two apparently unrelated cellular processes - a checkpoint in DNA-damaged cells (Bowen E., 2013, PhD thesis, University of Sheffield) and regulation of autophagy. MiCats1 shows very distinct subcellular localisations (Interphase:lysosomes; mitosis:Metaphase spindle) depending on cell division cycle status, and a clear prediction is that MiCats1 interaction partners, and their cellular locale, will differ as a function of cell cycle status. This project is expected to validate the effectiveness of the avidin-prG system, which would have widespread utility in conjunction with Turbo-ID, for in-depth characterisation of protein-protein interactions in vivo.