CITED2 controls the hypoxic signaling by snatching p300 from the two distinct activation domains of HIF-1α

HIF-1α plays a central role in cellular adaptation to hypoxia, and is closely related to the pathogeneses of life-threatening disorders. HIF-1α induces the expressions of numerous hypoxia-induced genes through two transactivation domains; N-terminal TAD (NAD) and C-terminal TAD (CAD). Furthermore, p300 is known to boost CAD-dependent transactivation, and CBP/p300-interacting transactivator with an ED-rich tail 2 (CITED2) inhibits HIF-1α-driven gene expression by interfering with the interaction between CAD and p300. However, few researches have focused on the role of CITED2 in the regulation of NAD activity, and thus, we addressed this point. CITED2 was found to attenuate the hypoxic activations of NAD-dependent and CAD-dependent genes, suggesting that CITED2 negatively regulates both CAD and NAD. Immunoprecipitation analyses showed that NAD interacts with the Cystein/Histidine region (CH) 1 and CH3 domains of p300. Moreover, CH1 and CH3 both were required for NAD-dependent transactivation. Furthermore, CITED2 was found to inactivate NAD by interfering with NAD binding to CH1, but not to CH3. These results indicate that CITED2 inactivates HIF-1α by blocking p300 recruitment by both NAD and CAD. We also found that pVHL inhibits NAD activity regardless of NAD degradation by blocking the interaction between p300 and NAD. Summarizing, NAD was activated by binding to p300, and this was blocked by either CITED2 or pVHL. We propose that pVHL controls NAD during normoxia and that CITED2 controls NAD during hypoxia. Our results provide a new strategy for controlling HIF-1α.     

Metal-free and Ca2+-bound structures of a multidomain EF-hand protein,CBP40, from the lower eukaryote Physarum polycephalum

Acellular slime mold, Physarum polycephalum, has a unique wound-healing system. When cytoplasm of plasmodia is exposed to extracellular fluid, calcium binding protein 40 (CBP40) seals damaged areas, forming large aggregates Ca(2+) dependently. Part of the CBP40 is truncated at the N terminus by a proteinase in plasmodia (CBP40delta), which does not aggregate in the Ca(2+)-bound form. Here we report the crystal structures of CBP40delta in both the metal-free and the Ca(2+)-bound states. Both structures consist of three domains: coiled-coil, intervening, and EF-hand. The topology of the EF-hand domain is similar to that of calpain. The N-terminal half of CBP40Delta interacts with the C-terminal EF-hands through a large hydrophobic interface, necessary for high Ca(2+) affinity. Conformational change upon Ca(2+) binding is small; however, the structure of CBP40delta provides novel insights into the mechanism of Ca(2+)-dependent oligomerization.     

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

The zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, increases the level of nonfunctional HIF-1alpha protein in normoxic cells

The hypoxia-inducible factor-1alpha (HIF-1alpha) subunit is activated in response to lack of oxygen. HIF-1alpha-specific prolyl hydroxylase and factor inhibiting HIF-1alpha (FIH-1) catalyze hydroxylation of the proline and asparagine residues of HIF-1alpha, respectively. The hydroxyproline then interacts with ubiquitin E3 ligase, the von Hippel-Lindau protein, leading to degradation of HIF-1alpha by ubiquitin-dependent proteasomes, while the hydroxylation of the asparagine residue prevents recruitment of the coactivator, cAMP-response element-binding protein (CBP), thereby decreasing the transactivation ability of HIF-1alpha. We found that the Zn-specific chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), enhances the activity of HIF-1alpha-proline hydroxylase 2 but the level of HIF-1alpha protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1alpha, its presence leads to the accumulation of HIF-1alpha that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1alpha from interacting with CBP, so reducing expression of HIF-1alpha target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1alpha protein in both normoxia and hypoxia.