Germline inactivation of the von Hippel-Lindau (VHL) tumor suppressor predisposes patients to develop different highly vascularized cancers. pVHL targets the hypoxia-inducible transcription factor (HIF-1α) for degradation, modulating the activation of various genes involved in hypoxia response. Hypoxia plays a relevant role in regulating cell cycle progression, inducing growth arrest in cells exposed to prolonged oxygen deprivation. However, the exact molecular details driving this transition are far from understood. Here, we present novel interactions between pVHL and the cyclin-dependent kinase inhibitor family CDKN1 (p21, p27 and p57). Bioinformatics analysis, yeast two-hybrid screening and co-immunoprecipitation assays were used to predict, dissect and validate the interactions. We found that the CDKN1 proteins share a conserved region mimicking the HIF-1α motif responsible for pVHL binding. Intriguingly, a p27 site-specific mutation associated to cancer is shown to modulate this novel interaction. Our findings suggest a new connection between the pathways regulating hypoxia and cell cycle progression.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.

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.

Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle-regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.

Functional impairment of the von Hippel-Lindau tumor suppressor (pVHL) is causative of a familiar increased risk of developing cancer. As an E3 substrate recognition particle, pVHL marks the hypoxia inducible factor 1α (HIF-1α) for degradation in normoxic conditions, thus acting as a key regulator of both acute and chronic cell adaptation to hypoxia. The male mice model carrying VHL gene conditional knockout presents significant abnormalities in testis development paired with defects in spermatogenesis and infertility, indicating that pVHL exerts testis-specific roles. Here we aimed to explore whether pVHL could have a similar role in humans by performing a testis-tissue library screening complemented with in-depth bioinformatics analysis. We identified 55 novel pVHL binding proteins directly involved in spermatogenesis, cell differentiation and reproductive metabolism. In addition, computational investigation of these new interactors identified multiple pVHL-specific binding motifs and demonstrated that somatic mutations described in human cancers reside in these binding regions. Collectively, these findings suggest that, in addition to its role in cancer formation, pVHL may also be pivotal in normal gonadal development in humans.