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Endocrine Abstracts (2018) 56 OC5.5 | DOI: 10.1530/endoabs.56.OC5.5

ECE2018 Oral Communications Diving deep into adrenal cortex diseases (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, , Eric Clauser 3 , Anna Vaczlavik 1 , Ludivine Drougat 1 , Claudimara Lotfi 2 , Maria Fragoso 4 , Marthe Rizk-Rabin 1 , Jérôme Bertherat 1, & Bruno Ragazzon 1


1Inserm U1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, France; 2Department of Anatomy, Institute of Biomedical Sciences, São Paulo, Brazil; 3Inserm U970, Paris Cardiovascular Center, Paris Descartes University, Paris, France; 4Adrenal Unit, Laboratory of Hormones and Molecular Genetics LIM/42, University of Sao Paulo, São Paulo, Brazil; 5Department of Endocrinology, Cochin Hospital, Assistance Publique Hôpitaux de Paris, Centre de référence des maladies rares de la surrénale, Centre de référence des cancers rares de la surrénale, Paris, France.


Background: ARMC5 (armadillo repeat containing 5) has been identified as the gene responsible for PBMAH (Primary Bilateral Macronodular Adrenal Hyperplasia). ARMC5 inactivating mutations are reported in 20 to 25% of PBMAH patients. ARMC5, is considered as a tumor suppressor gene controlling apoptosis and regulating steroidogenesis. The mechanisms of action of ARMC5 are unknown. The structure of the ARMC5 protein contains ARM repeats and a BTB domain, patterns known to play a role in protein-protein interactions. Therefore identification of proteins that interact with ARMC5 and study of the mechanisms of this interaction will help to understand its function. By co-immunoprecipitation followed by mass spectrometry in HEK293 cells we identified a potential interaction between ARMC5 and Cullin3 (Cul3), also suggested in online databases and by 2 Hybrid Assay (Hu et al, Nat Com 2017). Cul3 is an E3 ligase that mediates the ubiquitination process and subsequent degradation of specific protein substrates. Therefore, the aim of this study was to confirm this interaction and to investigate its mechanisms.

Methods: We used immunoprecipitation experiments with HA-tagged Cul3 and the bioluminescence resonance energy transfer (BRET) proximity assay in HEK293 cells in order to confirm and investigate the interaction of ARMC5 with Cul3.

Results: ARMC5 co-immunoprecipitated with HA-Cul3 and a hyperbolic BRET saturation curve was observed with YFP-Cul3 and ARMC5-Luc indicating a specific close proximity between these two proteins. We have also observed that a missense mutation in the BTB domain (p.L754P) of ARMC5 causes the loss of its interaction with Cul3. Altogether, these complementary approaches demonstrate that ARMC5 and Cul3 form a complex involving the BTB domain of ARMC5.

Conclusion: These data demonstrate that Cul3 is an ARMC5 partner. A likely direct interaction involves the BTB domain of ARMC5 and can be altered by pathogenic ARMC5 missense mutations. This suggests that ARMC5 participates in the ubiquitination process and open new perspectives in the pathophysiology of PBMAH.

Volume 56

20th European Congress of Endocrinology

Barcelona, Spain
19 May 2018 - 22 May 2018

European Society of Endocrinology 

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