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Endocrine Abstracts (2019) 63 OC10.4 | DOI: 10.1530/endoabs.63.OC10.4

ECE2019 Oral Communications Adrenal 2 (5 abstracts)

Cullin 3 is a partner of armadillo repeat containing 5 (ARMC5), the product of the gene responsible for primary bilateral macronodular adrenal hyperplasia

Isadora Cavalcante 1, , Anna Vaczlavik 3 , Ludivine Drougat 3 , Claudimara Lotfi 2 , Maria Fragoso 4 , Eric Clauser 5 , Marthe Rizk-Rabin 3 , Jérôme Bertherat 3 & Bruno Ragazzon 3

1INSERM U1016 – Institute Cochin, Paris, France; 2Institute of Biomedical Sciences, Sao Paulo, Brazil; 3INSERM U1016 – Institute Cochin, Paris, France; 4Clinicas Hospital – Adrenal Unit, Sao Paulo, Brazil; 5Inserm U970, Paris Cardiovascular Center, Paris, France.

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.

Volume 63

21st European Congress of Endocrinology

Lyon, France
18 May 2019 - 21 May 2019

European Society of Endocrinology 

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