Endothelin-1 (ET-1T he endothelium plays a central role in the regulation of vascular tone both under normal circumstances and in cirrhosis by releasing endotheliumderived vasodilators and vasoconstrictors in response to a variety of biochemical and physical stimuli. 1 Nitric oxide (NO) and endothelin-1 (ET-1) are two important endothelial mediators that modulate vascular tone. Endothelial NO production is catalyzed predominately by the endothelial form of nitric oxide synthase (eNOS) and under normal circumstances is constitutively expressed and activated by calcium entry into cells. 2 ET-1 is a 21 amino acid peptide formed from a precursor, big ET-1, through the action of an endothelin-converting enzyme and is produced in a number of cell types in addition to endothelial cells, including hepatic stellate cells and biliary epithelium. 3-6 ET-1 is classically recognized as a potent paracrine vasoconstrictor, and its action is mediated by two G protein coupled receptors. 7,8 The endothelin A (ET A ) receptor mainly exists in vascular smooth muscle cells and mediates contraction and vasoconstriction. 9 Two endothelin B (ET B ) receptor types have been found: one in endothelial cells that upregulates eNOS and NO and the other in smooth muscle cells that functions similar to the ET A receptor. 10,11 Increased circulating ET-1, in part derived from increased hepatic production and

Haploid a and alpha cells of yeast respond to the pheromones alpha- and a-factor, respectively, by increasing the transcription of many genes whose products are essential for mating. The STE12 protein acts in this process by binding to the DNA sequence that mediates the increased transcription of pheromone-responsive genes. We show here that a hybrid protein containing STE12 fused to the DNA-binding domain of GAL4 can activate transcription of a reporter gene containing GAL4-binding sites but only after treatment of cells with pheromone. Thus, STE12 alone, when bound to DNA, is sufficient to mediate pheromone-induced transcription. By constructing hybrids of different STE12 regions with the GAL4 domain, we map the domain of STE12 necessary for this activation to the central third of the protein. Upon alpha-factor treatment, the hybrid of GAL4 with the complete STE12 sequence is rapidly phosphorylated, with kinetics consistent with the observed transcriptional induction of pheromone-responsive genes. The domain of STE12 necessary for this phosphorylation correlates with that involved in transcriptional activation. We propose that induction of pheromone-responsive genes is mediated by phosphorylation of STE12 to alter its activation function but not its DNA-binding ability.

Background: PHD2 is the central enzyme that controls hypoxia-inducible factor-␣ (HIF-␣) protein levels. Results: PHD2 binds a Pro-Xaa-Leu-Glu motif in two HSP90 co-chaperones, and knockdown of one of these, p23, augments hypoxia-induced HIF-1␣ protein levels. Conclusion: PHD2 is linked to the HSP90 pathway, facilitating its hydroxylation of HIF-1␣. Significance: This uncovers a new model by which PHD2 controls HIF-1␣.

The biogenesis of cytosolic iron-sulfur (Fe-S) proteins in mammalian cells is poorly understood. In Saccharomyces cerevisiae, there is a pathway dedicated to cytosolic Fe-S protein maturation that involves several essential proteins. One of these is Nar1, which intriguingly is homologous to iron-only hydrogenases, ancient enzymes that catalyze the formation of hydrogen gas in anaerobic bacteria. There are two orthologues of Nar1 in mammalian cells, iron-only hydrogenase-like protein 1 (IOP1) and IOP2 (also known as nuclear prelamin A recognition factor). We examined IOP1 for a potential role in mammalian cytosolic Fe-S protein biogenesis. We found that knockdown of IOP1 in both HeLa and Hep3B cells decreases the activity of cytosolic aconitase, an Fe-S protein, but not that of mitochondrial aconitase. Knockdown of IOP2, in contrast, had no effect on either. The decrease in aconitase activity upon IOP1 knockdown is rescued by expression of a small interference RNA-resistant version of IOP1. Upon loss of its Fe-S cluster, cytosolic aconitase is known to be converted to iron regulatory protein 1, and consistent with this, we found that IOP1 knockdown increases transferrin receptor 1 mRNA levels and decreases ferritin heavy chain protein levels. IOP1 knockdown also leads to a decrease in activity of xanthine oxidase, a distinct cytosolic Fe-S protein. Taken together, these results provide evidence that IOP1 is involved in mammalian cytosolic Fe-S protein maturation. Iron-sulfur (Fe-S)2 proteins play an essential role in multiple physiologic processes, including electron transport, enzyme catalysis, and regulation of gene expression (1). Among the most studied of these proteins are those involved in mitochondrial oxidative phosphorylation, but it is clear that these proteins are present in other cellular compartments, including the cytoplasm and nucleus. The synthesis and maturation of Fe-S proteins in eukaryotes is complex, and much of our current understanding of this pathway derives from studies in yeast (2). The central elements of this pathway are as follows. The cysteine desulfurase Nfs1 (also known as IscS) removes sulfur from the amino acid cysteine. This sulfur is then combined with iron to form Fe-S clusters. These clusters, which can take on many forms, including [2Fe-2S] and [4Fe-4S] clusters, are then combined with apoproteins, resulting in Fe-S proteins. The initial steps of the pathway, including the desulfuration of cysteine and assembly of Fe-S clusters, occur within the mitochondrion. Thus, defects in mitochondrial Fe-S assembly impact Fe-S proteins not only in the mitochondrion but also in the cytosol. The Fe-S clusters are then transported to the appropriate compartments for subsequent assembly of the mature Fe-S proteins. A substantial amount is known about the initial steps. Less is known of the compartment-specific assembly. In yeast, a cytoplasmic iron-sulfur protein assembly pathway has been characterized (3). This pathway comprises at least four distinct proteins: cytosolic Fe-S cluster-defi...