Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element. The ␣-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions. The von Hippel-Lindau protein (pVHL) mediates the ubiquitination and rapid degradation of HIF-␣ (including HIF-1␣ and HIF-2␣). Post-translational hydroxylation of a proline residue in the oxygen-dependent degradation (ODD) domain of HIF-␣ is required for the interaction between HIF and VHL. It has previously been established that cobalt mimics hypoxia and causes accumulation of HIF-1␣ and HIF-2␣. However, little is known about the mechanism by which this occurs. In an earlier study, we demonstrated that cobalt binds directly to the ODD domain of HIF-2␣. Here we provide the first evidence that cobalt inhibits pVHL binding to HIF-␣ even when HIF-␣ is hydroxylated. Deletion of 17 amino acids within the ODD domain of HIF-2␣ that are required for pVHL binding prevented the binding of cobalt and stabilized HIF-2␣ during normoxia. These findings show that cobalt mimics hypoxia, at least in part, by occupying the VHL-binding domain of HIF-␣ and thereby preventing the degradation of HIF-␣.Hypoxia is a critical stimulus in many physiological and disease states (1). Cells respond to hypoxia by regulating the expression of a number of genes, including erythropoietin, vascular endothelial growth factor, and various glycolytic enzymes (2-5). This regulation is mediated in part by transcription factors of the hypoxia-inducible factor (HIF) 1 family (6). HIF-1␣ and HIF-2␣ are basic helix-loop-helix Per-Arnt-Sim (PAS) domain proteins (7) that form a heterodimer with the aryl hydrocarbon nuclear receptor translocator protein. Previous studies have shown that HIF-1␣ protein accumulates rapidly during hypoxia without a significant increase in HIF-1␣ mRNA levels (8). HIF-2␣, which is also known as endothelial PAS domain protein-1, shares close sequence and structural homology with HIF-1␣ (9). Like HIF-1␣, the levels of HIF-2␣ protein are low during normoxia and accumulate when cells are exposed to hypoxia, proteasomal inhibitors, transition metals (e.g. cobalt), iron chelators, or reducing agents (10). During normoxia, the HIF-␣ (HIF-1␣ and HIF-2␣ are referred to here simply as HIF-␣, except where noted otherwise) proteins are continuously degraded by ubiquitin-and proteasome-dependent pathway. Detailed studies of HIF-␣ proteins revealed a 200-amino acid sequence, called the oxygen-dependent degradation domain (ODD) that is responsible for its degradation in the presence of oxygen (11,12). The von Hippel-Lindau (pVHL) protein, a tumor suppressor protein, mediates the ubiquitination and degradation of HIF-␣ by binding to the ODD domain under conditions of normoxia (13,14). Recent findings revealed that pVHL-mediated degradation requires hydroxylation of specific proline residues within the ODD (15-18). The hydroxylation of these proline residues may be critical for regulating the HI...

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Discoidal forms of high density lipoproteins (HDL) are critical intermediates between lipid-poor apolipoprotein A-I (apo A-I), the major protein constituent of HDL, and the mature spherical forms that comprise the bulk of circulating particles. Thus, many studies have focused on understanding apoA-I structure in discs reconstituted in vitro. Recent theoretical and experimental work supports a "belt" model for apoA-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation. To distinguish among these, we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I. After delipidation and tryptic digestion, mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The cross-linking pattern strongly suggests a "double-belt" molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also suggests the presence of an additional double-belt orientation with a shifted helical registry (LL5/2 orientation). Furthermore, a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5. A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.