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Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIF␣ and ARNT. Although this complex involves protein-protein interactions mediated by basic helixloop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2␣ by NMR spectroscopy. We demonstrate that HIF-2␣ PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2␣ central ␤-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this ␤-sheet interface is widely used for both intra-and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins. C ellular responses to oxygen availability are essential for the development and homeostasis of mammalian cells, demonstrated most critically by the link between the cellular adaptation to reduced tissue oxygenation and disease progression (1, 2). In mammalian cells, these responses are mediated in part by the hypoxia-inducible factor (HIF), a heterodimeric transcription factor composed of HIF␣ and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF␤) (3). HIF activity is tightly controlled under normoxic conditions by multiple O 2 -dependent hydroxylation events of the HIF␣ subunit, which coordinately promote the ubiquitin-mediated destruction of this protein (4) and impair its ability to interact with transcriptional coactivators (5, 6) (Fig. 1a). These controls are relieved during hypoxia, allowing HIF to activate the transcription of genes that facilitate metabolic adaptation to low oxygen levels and increase local oxygen supply by angiogenesis (7).All three isoforms of HIF␣ [HIF-1␣, -2␣ (EPAS1), and -3␣] (8, 9) and ARNT belong to the basic helix-loop-helix (bHLH)-Per-ARNT-Sim (PAS) family of eukaryotic transcription factors, which contain bHLH and PAS domains (Fig. 1). The bHLH domains of these proteins serve as dimerization elements, helping determine the specificity of complex formation while providing a DNA-binding interface composed of the basic regions from each monomer (10). PAS domains are widespread components of signal transduction proteins, currently identified in Ͼ2,000 proteins from organisms in all three kingdoms of life. These domains, shown to be ...

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The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

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The heterodimeric transcription factor hypoxia-inducible factor (HIF) plays an important role in the progression of a number of processes in which O 2 availability is compromised and, as such, has become an increasingly attractive therapeutic target. Although tremendous progress has been made in recent years in unraveling the mechanisms underlying O 2 -dependent regulation of HIF through its O 2 -dependent degradation domain and C-terminal transactivation domain, our understanding of the contributions of other structural elements, particularly the Per/ARNT/Sim (PAS)-A and PAS-B domains, to the activity of HIF is incomplete. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point, we have explored the function(s) of the HIF-2␣ PAS domains via mutational analysis. In contrast to recent models, our data reveal that both PAS domains of the HIF-␣ subunit are necessary for heterodimer formation but are not required to mediate other HIF functions in which PAS domains have been implicated. Because disruption of individual PAS domains compromise HIF function independent of the mechanism of HIF induction, these data demonstrate the potential utility of targeting these domains for therapeutic applications.A pathway to sense and respond to changes in O 2 availability is expressed in virtually every mammalian cell, contributing to a host of developmental, physiological, and pathophysiological processes (reviewed in Ref. 1). This ubiquitous hypoxic response pathway involves changes in gene expression mediated through the induction of hypoxiainducible transcription factors (HIFs).2 HIFs are obligate heterodimers composed of single copies of ␣-and ␤-subunits, the latter of which is also called the aryl hydrocarbon receptor nuclear translocator (ARNT) (2). The mammalian genome contains three HIF-␣ genes, HIF-1␣, HIF-2␣ (also called endothelial Per/ARNT/Sim (PAS) domain protein 1 or HIF-like factor), and HIF-3␣ (3-5), that share similar domain structures but likely serve nonoverlapping physiological roles (6, 7).Although the HIF-␤ subunit is essentially insensitive to O 2 availability, both the accumulation and activity of the HIF-␣ subunit are acutely induced in response to low O 2 levels (reviewed in Ref. 8). Briefly, multiple proline residues within the oxygen-dependent degradation domain (9) of the ␣-subunit are selectively hydroxylated under normoxic conditions (10, 11) and subsequently recognized by the product of the von Hippel-Lindau tumor suppressor gene (pVHL). pVHL is a component of a ubiquitin-protein ligase complex that tags the ␣-subunit for rapid proteasomal degradation (12-16). A second independent O 2 -dependent hydroxylation activity directed toward an asparagine residue within the C-terminal transactivation domain of the ␣-subunit blocks coactivator recruitment under normoxic conditions (17). By virtue of their utilization of O 2 as a substrate, the prolyl (18 -20) and asparaginyl hydroxylases (21, 22) that regulate HIF are potential "oxygen sensors" ...