Protein-protein interactions of AIP (Aryl Hydrocarbon Receptor Interacting Protein)

Abstract

Aryl hydrocarbon receptor interacting protein (AIP) is a cytoplasmic molecular co-chaperone and tumour suppressor that assists in protein stability and complex formation. It is involved in biochemical pathways, such as xenobiotic metabolism, cyclic AMP signalling, mitochondrial import and apoptosis. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. This study was focused on the structural and functional characterisation of AIP to investigate whether N-domain mutations, of clinical relevance, affect the ability of the protein to interact with client binding partners. A purification protocol for AIP was successfully devised that maintains the protein in a stable homogenous state. Similarly, variants of full length AIP harbouring N-domain mutations were purified from E.coli to the same level of purity. Circular dichroism showed that the mutations did not significantly affect the thermal stability of the protein and caused no overall disruptive effect in the protein structure. However, through ITC and Biacore experiments, these mutations lowered, to different extents, the binding affinity of AIP towards two of its binding partners, Hsp90β(513-724) and PDE4A5, also purified in this study. The possible biological implications of such disruptions are the destabilisation of complexes requiring Hsp90 and changes in cellular cAMP levels respectively. The latter was further demonstrated through a PDE enzymatic assay in which the mutants failed to attenuate the enzymatic activity of PDE to the same degree as the wild type protein. Through the use of small angle X-ray scattering, the full length model of AIP was obtained and provided valuable information on protein shape, flexibility and inter-domain distances. This study provides clear evidence that AIP N-domain mutations have a significant role in protein-protein interactions and although they may not necessarily contribute directly to pituitary tumourigenesis, the complex interactome of AIP suggests that any observable change in one or more of its binding partners cannot be disregarded as it may have repercussions on other biochemical pathways. To further investigate the AIP protein interactome, a novel protocol for the expression of soluble AhR in E.coli has also been established. AhR is another binding partner of AIP that displays both oncogenic and tumour suppressor abilities and is currently at the focus of various studies.