The E3 ubiquitin-protein ligase MDM2 is a novel interactor of the von Hippel\u2013Lindau tumor suppressor

Antonella Falconieri,Raffaele Lopreiato,Geppo Sartori,Maria Pennuto,Silvio C E Tosatto,Giovanni Minervini,Raissa Bortolotto,Damiano Piovesan

doi:10.1038/s41598-020-72683-3

Antonella Falconieri, Raffaele Lopreiato + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-72683-3

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Abstract

Mutations of the von Hippel–Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1 α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein–protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). We found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2. Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoform-specific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already shown for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell metabolic activity and necrosis, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data show an isoform-specific interaction of pVHL with MDM2, suggesting an interplay between these two E3 ubiquitin ligases.

Highlights

Oxygen availability regulates a plethora of physiological processes, such as cell metabolism, mitochondrial ATP production, embryo development and inflammation, and it plays a prominent role in the pathophysiology of cancer[1,2,3]
We previously documented a binary interaction between pVHL30 and p14ARF sustained by the acidic N-terminal tail of pVHL30, suggesting a functional asymmetry among pVHL isoforms22. p14ARF tumor suppressor is well known for its role in regulating p53 s tability[24], sequestering mouse double minute 2 homolog (MDM2) into a subnuclear compartment[25], while inhibiting p53 degradation24. p53 is stabilized through pVHL26
The MDM2 region that we found to participate in this association is immediately downstream of two overlapping binding sites for p14ARF and USP7, a deubiquitinating enzyme involved in p53 stabilization[43]

Summary

Introduction

Oxygen availability regulates a plethora of physiological processes, such as cell metabolism, mitochondrial ATP production, embryo development and inflammation, and it plays a prominent role in the pathophysiology of cancer[1,2,3]. The levels of HIF-1α are regulated by the von Hippel–Lindau (pVHL) tumor suppressor, which continuously targets HIF-1α for proteasomal degradation. The activity of PHDs is inhibited, resulting in HIF-1α stabilization and activation of the hypoxia-response gene expression program. Three biologically active pVHL isoforms have been described so far, namely pVHL30, pVHL19, and pVHL172 Both pVHL19 and pVHL30 act as tumor suppressors and have a redundant function to promote HIF-1α degradation, yet they have a different sub-cellular localization[21] and isoform-specific. PVHL30 was proposed to interact with p 14ARF22, suggesting a further connection between oxygen sensing and apoptosis. These observations suggest that the different pVHL isoforms have redundant as well as isoform-specific functions in the cells, little is known about pVHL isoform-specific protein–protein structural and functional interactions. We showed that a short intrinsically disordered segment of MDM2 is responsible for its association with pVHL in an isoformspecific fashion restricted to pVHL30

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