PR-619

Hypoxia inducible factor 2α (HIF2α) is a major oncogenic driver in clear cell renal cell carcinoma (ccRCC). HIF2α is upregulated by von Hippel-Lindau (VHL) gene loss-of-function mutations in ccRCC. Deubiquitinating enzymes (DUBs) have emerged as key regulators of oncoprotein stability, but which DUBs sustain HIF2α accumulation in ccRCC remains unknown. Our study sought to systematically identify DUBs that stabilize HIF2α regardless of VHL status.

We selected three ccRCC cell lines with distinct VHL gene statuses, and 15 DUB inhibitors were applied to screen for DUBs that regulate the degradation of the HIF2α protein in ccRCC regardless of VHL gene status. Coimmunoprecipitation (Co-IP) and mass spectrometry (MS) were used to verify the interactions between DUBs and the HIF2α protein.

In our study, the PR-619 inhibitor caused a decrease in the HIF2α protein level in 786-O, OS-RC-2, and SW839 cells, and MG132 treatment increased HIF2α protein expression after PR-619 treatment. Furthermore, the MS, Co-IP and Western blotting results revealed that ubiquitin-specific peptidase 10 (USP10) is a DUB that binds to HIF2α and that depletion of USP10 leads to HIF2α downregulation through promotion of HIF2α ubiquitination. We also confirmed that USP10 expression was upregulated in kidney cancer and had a positive correlation with HIF2α expression in RCC patient samples.

Our results suggest that USP10 promotes HIF2α stability.

Ubiquitination influences a myriad of biological processes, such as the trafficking or degradation of ubiquitin-tagged target proteins. This posttranslational modification can be reversed by deubiquitinating enzymes (DUBs) that counterbalance the action of E3 ubiquitin ligases. We investigated the impact of PR-619, a membrane-permeable and broad-spectrum DUB inhibitor, on the entry of Ca²⁺ through native voltage-gated Ca²⁺ channels (VGCCs) of cultured embryonic cortical neurons. Fura-2-based Ca²⁺ imaging experiments showed that PR-619 reduced the cytosolic Ca²⁺ rises induced by depolarization by affecting mainly dihydropyridine-sensitive (L-type) VGCCs. This inhibition was sensitive to dynamin inhibitor Myr-Dip and lysosomal agents chloroquine and bafilomycin-A. PR-619 also reduced the amount of Ca_v1.2 proteins. A pharmacological approach was set out to better delineate the identity of the DUB responsible for this inhibitory action of PR-619. Since UCH-L1 and USP19 are two highly expressed neuronal DUBs, we investigated the effects of selective UCH-L1 (IMP1710, GK13S) and USP19 (ADC141) inhibitors. IMP1710 and GK13S depressed the Ca²⁺ uptake through L-type VGCCs, whereas ADC141 and the UCH-L3 inhibitor TCID had no effects. In addition, UCH-L1 inhibition impaired the neuronal Ca²⁺ storage capacities of neurons and reduced the Ca_v1.2 protein levels. Thus, UCH-L1 influences the neuronal uptake and storage of Ca²⁺, which is likely to have important physiological implications. Altogether, these results posit UCH-L1, the main DUB of the brain, as an important regulator of neuronal Ca²⁺ homeostasis and add to our understanding of its cellular functions.