Post-Translational Regulation of Hypoxia-Inducible Factor 1α by a Novel Deubiquitinase USP52

 Hypoxia-inducible factor 1α (HIF-1α) is the master transcriptional regulator of oxygen homeostasis, typically degraded via von Hippel-Lindau (VHL)-mediated ubiquitination under normoxia. Here we identify ubiquitin-specific protease 52 (USP52) as a previously unrecognized deubiquitinase that stabilizes HIF-1α independently of prolyl hydroxylation. Using a genome-wide siRNA screen in HEK293T cells under normoxic conditions (21% O₂), we ranked deubiquitinases by their effect on a luciferase reporter driven by hypoxia response elements. USP52 knockdown reduced basal HIF-1α protein half-life from 12 minutes to 3 minutes without affecting HIF-1α mRNA levels. Co-immunoprecipitation and GST pull-down assays confirmed direct binding between USP52 and the oxygen-dependent degradation domain (ODD) of HIF-1α (amino acids 401–603). Mass spectrometry identified Lys532 as the primary ubiquitination site targeted by USP52; mutation of Lys532 to arginine abolished USP52-mediated stabilization. Surprisingly, USP52 activity is oxygen-insensitive, yet its expression itself is induced by moderate hypoxia (1% O₂) via an NF-κB-dependent mechanism. Chromatin immunoprecipitation revealed that USP52 binds to the HIF1A promoter under prolonged hypoxia (24 hours), creating a positive feedback loop. In USP52-knockout HeLa cells generated via CRISPR-Cas9, HIF-1α target genes (VEGF, GLUT1, LDHA) showed 80% reduced expression under hypoxia, leading to impaired metabolic shift toward glycolysis (measured by extracellular acidification rate). Xenograft tumors from USP52-null cells grew 60% slower in nude mice and exhibited reduced microvessel density (CD31 staining). Proteomic profiling of USP52 interactors identified a second substrate: the prolyl hydroxylase PHD2. USP52 deubiquitinates PHD2 at Lys298, targeting it for proteasomal degradation, thus indirectly increasing HIF-1α by removing its negative regulator. This dual mechanism—direct HIF-1α stabilization and indirect PHD2 destabilization—amplifies hypoxic signaling. Pharmacological inhibition of USP52 with a newly synthesized small molecule (compound 7b, IC₅₀ = 120 nM) suppressed tumor growth in patient-derived glioblastoma organoids. Clinical data from The Cancer Genome Atlas show that USP52 amplification correlates with poor prognosis in renal clear cell carcinoma (hazard ratio = 2.4, p < 0.001). We propose USP52 as a therapeutic target for hypoxia-driven cancers.