Aryl hydrocarbon receptor interacting protein (AIP) : effects of N-domain mutations relevant to pituitary adenomas

Abstract

Introduction: Aryl hydrocarbon receptor interacting protein (AIP) is a two-domain, cytoplasmic chaperone and tumour suppressor. It is involved in various biochemical pathways, cyclicAMP signalling, mitochondrial import and apoptosis. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. The aim of this research is to obtain structural and functional data on AIP and investigate how clinically relevant N-domain mutations affect the ability of AIP to bind to its client partners, previously presumed to involve only the C-domain.

Methods: Recombinant, full-length human AIP was sub-cloned and four known N-domain mutants were produced. After overexpression in Escherichia coli, all proteins were purified to homogeneity. Two AIP binding protein partners, heat shock protein 90 (Hsp90) and phosphodiesterase 4A5 (PDE4A5), were also cloned, expressed and purified. Protein folding and thermal stability were analysed through circular dichroism spectrophotometry and binding studies were performed using both isothermal titration calorimetry and surface plasmon resonance. The interaction of AIP and PDE4A5 was further investigated enzymatically. Structural information has been obtained using small angle x-ray scattering (SAXS) and crystallography.