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, 287 (3), 1962-9

Ubiquitin-specific Protease 19 (USP19) Regulates Hypoxia-Inducible Factor 1α (HIF-1α) During Hypoxia

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Ubiquitin-specific Protease 19 (USP19) Regulates Hypoxia-Inducible Factor 1α (HIF-1α) During Hypoxia

Mikael Altun et al. J Biol Chem.

Abstract

A proper cellular adaptation to low oxygen levels is essential for processes such as development, growth, metabolism, and angiogenesis. The response to decrease in oxygen supply, referred to as hypoxia, is also involved in numerous human diseases including cancer, inflammatory conditions, and vascular disease. The hypoxia-inducible factor 1-α (HIF-1α), a key player in the hypoxic response, is kept under stringent regulation. At normoxia, the levels are kept low as a consequence of the efficient degradation by the ubiquitin-proteasome system, and in response to hypoxia, the degradation is blocked and the accumulating HIF-1α promotes a transcriptional response essential for proper adaptation and survival. Here we show that the ubiquitin-specific protease-19 (USP19) interacts with components of the hypoxia pathway including HIF-1α and rescues it from degradation independent of its catalytic activity. In the absence of USP19, cells fail to mount an appropriate response to hypoxia, indicating an important role for this enzyme in normal or pathological conditions.

Figures

FIGURE 1.
FIGURE 1.
USP19 interacts with HIF-1α. A, schematic representation of the full-length and the 1–495-amino acid long USP19, which was used as bait in a yeast two-hybrid screen. CS domain, p23 protein domain (p23), USP domain, ubiquitin-like domain (UBL), myeloid translocation protein 8, Nervy protein, Deaf-1 zinc finger (MYND Zn-finger), transmembrane domain (TMD), and the positions of the amino acids Cys, His, and Asp in the catalytic triad are indicated (DNA binding domain (DBD)). B, immunoprecipitations (IP) using FLAG(M2) affinity gel from HEK293T cells transfected with FLAG-tagged components of the hypoxia pathway as indicated. The co-immunoprecipitated endogenous USP19 was detected with the anti-USP19(A301-587A) antibody (Bethyl Laboratories) as indicated. Note that USP19 appears in multiple forms, indicated by arrows, likely representing splice variants or processed forms. MW, molecular weight markers. C, co-immunoprecipitation of endogenous proteins from HeLa cells in normoxia and hypoxia using the anti-USP19(A301-587A) antibody as indicated. D, co-immunoprecipitation using anti-FLAG(M2) affinity gel showing selective interaction between Myc-USP19 and FLAG-HIF-1α but not FLAG-HIF-2α and FLAG-HIF-3α.
FIGURE 2.
FIGURE 2.
Mapping HIF-1α interaction domain. A, schematic illustration of truncated, FLAG-tagged, HIF-1α constructs. bHLH, PAS domain, ODD, N/C-terminal transactivation domain (N-TAD and C-TAD), nuclear localization signal (NLS), and nuclear export signal (NES) are indicated. B, co-immunoprecipitations (IP) using a FLAG(M2) affinity gel from lysates of HEK293T cells co-transfected with the truncated forms of HIF-1α or FLAG-GAL4 as control, together with Myc-USP19.
FIGURE 3.
FIGURE 3.
USP19 stabilizes HIF-1α independent of catalytic activity. A, active site labeling with the HA-ubiquitin-VME probe in lysates from cells expressing Myc-USP19 and the mutant Myc-USP19(C506S). The upper blot illustrates the enzymatically active Myc-USP19 covalently linked to the probe as detected using an anti-HA antibody. The lower blot illustrates the expression of both Myc-USP19 and Myc-USP19(C506S) using anti-Myc(9E10) antibody. B, Western blot to test the effect of overexpressed USP19 on co-transfected FLAG-HIF-1α steady-state levels as indicated. GFP was included as a co-transfection control. C, Western blot analysis of U2OS cells overexpressing Myc-USP19 and Myc-USP19(C506S), probed as indicated. D, micrographs of cells transfected with Myc-USP19, Myc-USP19(C506S), or GFP as control. Immunostainings are shown with anti-HIF-1α (red) and anti-Myc(A14) (green) and nuclear counterstaining using DAPI as indicated. E, quantification of results in D where 100 USP19-positive cells were scored for positive HIF-1α co-staining. Values show mean ± S.D. of triplicates.
FIGURE 4.
FIGURE 4.
USP19 stabilizes HIF-1α independent of ER localization. A, graphs illustrating HeLa cells, the melanoma cell line M2, and HEK293T cells overexpressing Myc-USP19, Myc-USP19(C506S), and Myc-USP19ΔTM, which lacks ER localization. The number of cells accumulating HIF-1α in USP19-overexpressing cells was scored by counting cells positive for HIF-1α immunostaining. B, Western blot of HeLa cells transiently overexpressing Myc-USP19, Myc-USP19(C506S), and Myc-USP19ΔTM in normoxia. The blots were probed against Myc, HIF-1α, and β-actin as indicated.
FIGURE 5.
FIGURE 5.
Loss of USP19 impairs hypoxic response. A, Western blot experiment assessing the efficiency of two different shRNA-expressing vectors, pRETRO-SUPER-USP19A (indicated by A) and pRETRO-SUPER-USP19D (indicated by D), in suppressing USP19 protein expression. ctrl, control; kd, knockdown. B, 3 days after transfection, HeLa cells transfected with the empty control plasmid pRETRO-SUPER or pRETRO-SUPER-USP19A were exposed to hypoxia or kept in normoxia as indicated. Western blots were probed anti-HIF-1α (short and long exposure (exp.)) to investigate the effect by USP19 knockdown on HIF-1α accumulation. β-Actin was included as control. C, quantification by densitometry from short exposure Western blots of three independent experiments performed as in Fig. 4B. Values represent relative induction of HIF-1α during hypoxia as compared with normoxia, mean ± S.D. D, same experimental setup as in Fig. 4B, but cells exposed to hypoxia for 8 h were treated in parallel with the proteasome inhibitor MG132 (10 μm). The results illustrate a continuous proteasomal degradation of HIF-1α during hypoxia in cells with suppressed USP19 expression, E, knockdown of USP19 impairs the HIF-1α transcriptional response during hypoxia. Relative mRNA expression was assessed by qPCR of the HIF-1α target genes GLUT1 and VEGF in cells with or without USP19 knockdown ± hypoxia. Values represent expression levels relative to β-actin mRNA ± S.D. from four independent experiments. *, p values < 0.05 for indicated comparisons.

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