Endocrine Abstracts

Background: Germline mutations of ARMC5 (Armadillo repeat containing 5 gene) were identified as a frequent cause of primary bilateral macronodular adrenal hyperplasia (PBMAH). ARMC5 is considered as a tumor suppressor gene regulating apoptosis and steroidogenesis by unknown mechanisms. The ARMC5 protein contains a N-terminal domain made of Armadillo (ARM) repeats and a C-terminal ‘Bric-a-Brac, Tramtrack, Broad-complex/Pox virus and Zinc finger (BTB/POZ)’ domain. Both domains are important for protein-protein interactions, suggesting that the study of ARMC5 partners might help to understand its cellular function. By co-immunoprecipitation coupled with mass spectrometry, we identified a potential interaction between ARMC5 and Cullin3 (CUL3), also suggested in online databases and in a Yeast-2-Hybrid assay. CUL3 serves as a scaffold protein and assembles a large number of ubiquitin ligase complexes which mediate ubiquitination and degradation of specific substrates. Therefore, our aim was to confirm this interaction and investigate its mechanisms.

Methods: We performed immunoprecipitation experiments, bioluminescence resonance energy transfer (BRET) proximity assays, protein stability experiments and ubiquitination assays in order to investigate the interaction of ARMC5 with CUL3.

Results: ARMC5 co-immunoprecipitated with CUL3 and a hyperbolic BRET saturation curve was observed with YFP-CUL3 and ARMC5-Luc indicating a specific proximity between these proteins. We also observed that the ARMC5 missense mutation in the BTB domain (p.L754P) identified in a patient diagnosed with PBMAH disrupts the interaction with CUL3 and increases ARMC5 protein stability. Moreover, CUL3 overexpression increased ubiquitination of ARMC5 while no effect was observed on ARMC5-p.L754P mutant. Finally, inhibition of the ubiquitin-dependent proteasome system with MG132 induced accumulation of endogenous ARMC5 in secondary cell culture of PBMAH (without ARMC5 alteration), while no effect was observed in a secondary PBMAH cell culture carrying a mutation in the BTB domain (p.H808P) of ARMC5.

Conclusion: In this study we demonstrated the interaction between ARMC5 and CUL3. This interaction leads to the ubiquitination of ARMC5, suggesting that ARMC5 is a substrate for a CUL3-based ubiquitin complex. Moreover, missense mutants in the BTB domain of ARMC5, identified in patients diagnosed with PBMAH, alters the interaction with CUL3 and the ubiquitination by CUL3-based ubiquitin/proteasome system. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives in the understanding of the pathophysiology of PBMAH.