Objective: The aim of this project is to characterise a proposed drug and metabolite binding site on Fanconi Anaemia associated protein-100 (FAAP100). FAAP100 is a part of the Fanconi Anaemia (FA) E3 ubiquitin ligase complex. The FA E3 ligase complex is essential for the function FA DNA repair pathway and is an anti-cancer target. FAAP100 contains a motif that shares amino acid sequence similarity to a drug and metabolite binding site on AMP-activated protein kinase (AMPK) which is allosterically activated by long chain fatty acyl-CoA’s and small molecules.
Methods: The effect of long chain fatty acyl-CoAs and AMPK activating small molecules on FAAP100 and the FA core complex was investigated. FA core complex activity was measured by a high throughput recombinant FA core complex assay which is capable of mono-ubiquitinating its substrate, FANCD2. Detection of FANCD2 mono-ubiquitination is via a proximity assay. A panel of fatty acyl-CoA’s, ranging from 2 to 18 carbons in length, were tested in this assay, as well as a panel of AMPK activators that are predicted to interact with FAAP100. To investigate the predicted binding site, we have characterised FAAP100 with the binding site residues mutated.
Results: We show that long chain fatty acyl-CoA’s inhibit FANCD2-ubiquitination in our recombinant assay. The 16 carbon long fatty acyl-CoA, palmitoyl-CoA, is the most potent inhibitor. Using a non-hydrolysable palmitoyl-CoA, we demonstrate the inhibition is not via palmitoylation, rather through an allosteric binding site. Further, only the E3 ligase subcomplex (FANCB-FANCL-FAAP100) activity in the FA ubiquitination cascade is affected by palmitoyl-CoA, and not the E1 or E2. The AMPK activating small molecules also inhibit FANCD2 monoubiquitination in our recombinant protein assay. Mutating key residues in the FAAP100 binding site eliminates the small molecule’s ability to inhibit the assay, confirming the drug and metabolite binding site in FAAP100.
Conclusion: We have demonstrated that long chain fatty acyl-CoA’s and a group of AMPK activators can inhibit FA core complex activity via an allosteric binding site on FAAP100. Further investigation is needed into the interaction between long chain fatty acyl CoA’s and FAAP100 to completely understand the biological implications and therapeutic potential.