Characterising TRIB1 Nanobodies using a Yeast Surface Display Platform with Flow Cytometry

The mammalian tribble (TRIB) pseudokinase family has three homologues, which act as an adaptor protein to regulate cell signalling pathways. TRIB proteins can bind the E3 ligase, Constitutive Photomorphogenesis Protein 1 (COP1), allowing TRIB regulatory function over essential proteins via the ubiquitin (Ub)-proteasomal pathway. For example, TRIB can cause the degradation of the transcription factor C/EBP-α which, regulates the differentiation of haematopoietic cells and adipocytes. TRIB also regulates critical signalling pathways such as the mitogen-activated protein kinase (MAPK) and serine/threonine-specific protein kinase (Akt) pathways, that controls mitosis, cell differentiation and survival. With different TRIB activities, TRIB proteins have various conformations with its substrates. Dysregulation of TRIB expression has been related to some cancer pathology such as Acute Myeloid Leukaemia (AML) and adenocarcinoma; however, the full molecular mechanism of how TRIB leads to pathogenesis is yet understood. Single-domain antibodies called, nanobodies (Nbs), currently show promise in furthering discoveries in protein function and structure due to their ability to stabilise protein conformations with their small size of ~15 kDa, which allows; Nbs the ability to bind smaller epitopes that conventional antibodies are incapable of doing, and Nbs to be expressed intracellularly. Therefore, it is hypothesised that TRIB-specific Nbs as a research tool, may be used to discover further TRIB proteins’ functional mechanisms that lead to the pathology of cancers. Understanding how TRIB-substrate conformations link with its cell signalling consequences, may reveal new therapeutic avenues. Focusing on TRIB1 alone, TRIB1-specific Nbs previously screened from the McMahon Library, were expressed on the surface of the cell walls of S. cerevesiae (yeast). This allowed for the use of flow cytometry to characterise TRIB-1 Nbs affinity and specificity to its target based on the mean fluorescence intensity of the labelled Nbs and TRIB proteins: This flow cytometry method was able to be used to obtain a binding affinity (KD = 315 nM) similar to isothermal titration calorimetry (KD = 366 nM) for our positive TRIB1-Nb binding control, Nb2.011. Results showed that none of the eight TRIB1-Nbs tested had better affinities to TRIB1 than Nb2.011 (KD > 366 nM). Although no promising TRIB1-Nbs was identified from the library samples, this project did elucidate limitations and important considerations in the use of the yeast surface display system, for the rapid, initial characterisation screening of TRIB-Nbs after panning a Nb library for potential binders at nanomolar concentrations. Overall, we were able to conclude that this method of Nb characterisation, using a yeast display system with flow cytometry, may improve the discovery pipeline of TRIB-Nbs by narrowing down the panel of potential Nbs with desirable KD estimations and specificity to its target