Hypoxia-inducible factor 1 (HIF-1) 3 is a key regulator of hypoxic adaptation that functions by activating the transcription of several genes involved in angiogenesis, erythropoiesis, and glycolysis (1, 2). It consists of two subunits; HIF-1␣ is rapidly degraded under normoxic condition by the ubiquitin-proteasome system, whereas HIF-1␤ is stable. Under normoxic conditions the proline 564 and/or 402 residues of HIF-1␣ are hydroxylated by HIF-1␣-specific prolyl-4 hydroxylases (PHDs), which need O 2 , ␣-ketoglutarate, vitamin C, and Fe(II) (3-6). The hydroxylated prolines interact with von Hippel-Lindau (VHL) protein, a component of E3 ubiquitin ligase, and the HIF-1␣ is ubiquitinated by the VCB E3 ubiquitin-ligase complex, consisting of VHL protein, ElonginB, ElonginC, Cul2, and Rbx1 (7,8). In hypoxic conditions, proline hydroxylation decreases and HIF-1␣ accumulates. Oxygen molecules inhibit not only the stabilization of HIF-1␣ but also its transactivation activity, because the protein, factor-inhibiting HIF-1␣ (FIH-1), catalyzes hydroxylation of its asparagine residue using O 2 , ␣-ketoglutarate, vitamin C, and Fe(II). Hydroxylation of this asparagine residue in the transactivation domain of HIF-1␣ prevents it from recruiting its coactivator, cAMP-response element-binding protein (CBP) (3, 9).Thus, activation of HIF-1␣ is censored by two systems, proline hydroxylation and asparagine hydroxylation. HIF-1␣ and its targets, such as EPO and VEGF, are being evaluated as therapeutic agents for cerebral and myocardial infarctions, and a small lipophilic HIF-1␣-activating compound is being sought as a treatment for these diseases. However, to generate fully functional HIF-1␣, a putative HIF-1␣ activator should suppress both proline hydroxylation-dependent ubiquitination and asparagine hydroxylation.We showed previously that the zinc chelator N,N,NЈ,NЈ-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) enhances the activity of PHD2 but that the level of HIF-1␣ protein does not fall because TPEN also inhibits its ubiquitination. Because TPEN does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1␣, it leads the accumulation of nonfunctional HIF-1␣ (10, 11). Here, we report that another zinc chelator, Clioquinol, which has relatively low affinity but high selectivity for Zn(II) and Cu(II), has a different effect on the activity of HIF-1␣. Both TPEN and Clioquinol inhibit ubiquitination of HIF-1␣ and cause its accumulation. However, in contrast to TPEN, Clioquinol prevents FIH-1 from hydroxylating HIF-1␣. It therefore stabilizes functional HIF-1␣, leading to expression of its target genes in normoxic cells.Clioquinol has been used in Alzheimer, Parkinson, and Huntington diseases as a Cu(II)-and Zn(II) chelator that reverses Zn(II)-or Cu(II)-induced metalloprotein precipitation (12). It

Hypoxia-induced gene expression is initiated when the hypoxia-inducible factor-1 (HIF-1) alpha subunit is stabilized in response to a lack of oxygen. An HIF-1alpha-specific prolyl-hydroxylase (PHD) catalyzes hydroxylation of the proline-564 and/or -402 residues of HIF-1alpha by an oxygen molecule. The hydroxyproline then interacts with the ubiquitin E3 ligase von Hippel Lindau protein and is degraded by an ubiquitin-dependent proteasome. PHD2 is the most active of three PHD isoforms in hydroxylating HIF-1alpha. Structural analysis showed that the N-terminal region of PHD2 contains a Myeloid translocation protein 8, Nervy, and DEAF1 (MYND)-type zinc finger domain, whereas the catalytic domain is located in its C-terminal region. We found that deletion of the MYND domain increased the activity of both recombinant PHD2 protein and in vitro-translated PHD2. The zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine augmented the activity of wild-type PHD2-F but not that of PHD2 lacking the MYND domain, confirming that the zinc finger domain is inhibitory. Overexpression of PHD2 lacking the MYND domain caused a greater reduction in the stability and function of HIF-1alpha than did overexpression of wild-type PHD2, indicating that the MYND domain also inhibits the catalytic activity of PHD2 in vivo.