Despite clear epidemiological and genetic evidence for X-linked prostate cancer risk, all prostate cancer genes identified are autosomal. Here we report somatic inactivating mutations and deletion of the X-linked FOXP3 gene residing at Xp11.23 in human prostate cancer. Lineage-specific ablation of FoxP3 in the mouse prostate epithelial cells leads to prostate hyperplasia and prostate intraepithelial neoplasia. In both normal and malignant prostate tissues, FOXP3 is both necessary and sufficient to transcriptionally repress cMYC, the most commonly over-expressed oncogene in prostate cancer as well as among the aggregates of other cancers. FOXP3 is an X-linked prostate tumor suppressor in the male. Since the male has only one X chromosome, our data represents a paradigm of “single-genetic-hit” inactivation-mediated carcinogenesis.

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states typically without changes in ion selectivity 1. In store-operated Ca2+ release-activated Ca2+ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, STIM1 (stromal interaction molecule 1) remains unknown. CRAC channels are distinguished by an extraordinarily high Ca2+ selectivity and are instrumental in generating sustained [Ca2+]i elevations necessary for gene expression and effector function in many eukaryotic cells 2. Here, we probed the central features of the STIM1 gating mechanism in the CRAC channel protein, Orai1, and identified V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produced constitutively active CRAC channels that were open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels were not Ca2+-selective, their Ca2+ selectivity was dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca2+ selectivity also occurred in wild-type (WT) Orai1 channels by increasing the number of STIM1 activation domains directly tethered to Orai1 channels. Thus, exquisite Ca2+ selectivity is not an intrinsic property of CRAC channels, but rather a tunable feature bestowed on otherwise non-selective Orai1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism wherein permeation and gating are closely coupled.

Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca2+ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I–II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca2+ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca2+ block, and channel regulation by Ca2+. We find that alteration of the acidic residues lowers Ca2+ selectivity and results in striking increases in Cs+ permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs+ permeation. Ca2+ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca2+ binding. Unexpectedly, the mutations diminish Ca2+-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca2+ selectivity, increase in Cs+ permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.

Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

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CRAC channels generate Ca 2؉ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca 2؉ selectivity, low Cs ؉ permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiolreactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca 2؉ selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd 2؉ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.Orai1 ͉ STIM1 ͉ store-operated channels C a 2ϩ release-activated Ca 2ϩ (CRAC) channels are the principal route of Ca 2ϩ entry in immune cells and orchestrate functions such as gene expression, motility, and the release of inflammatory mediators (1). Mutations in CRAC channels give rise to devastating immunodeficiencies and abnormalities in muscle, skin, and teeth, highlighting their importance for various organ systems (1). The recent discoveries of STIM1 (the ER Ca 2ϩ sensor), and Orai1 (the CRAC channel pore subunit) have provided major breakthroughs to illuminate the molecular basis of CRAC channel function (2). However, while the identification of these proteins has produced rapid progress in our understanding of the cellular events underlying channel activation, the molecular mechanisms of ion selectivity and permeation remain unclear.CRAC channels are distinguished by an extraordinarily high selectivity for Ca 2ϩ over monovalent ions (P Ca /P Na Ͼ 1,000), a very low unitary conductance (Ͻ1 pS), and low permeability to Cs ϩ and larger monovalent cations (3). The structural underpinnings of these characteristics have been the focus of much debate but are largely unknown. As with most ion channels, the pore properties of CRAC channels are likely shaped by the arrangement and chemistry of pore-lining residues. Thus, to understand the basis of the unique permeation properties of CRAC channels, the residues lining the ion transport pathway need to be elucidated.Orai1 bears little sequence homology to other ion channel proteins, and consequently, there are few clues regarding the contribution of the different parts of the molecule for pore formation. Electrophysiological studies indicate that the exquisite Ca 2ϩ selectivity of CRAC...

E pithelial cells lining the airway represent the first barrier to the entry of respiratory viruses and are their main replication target. In addition to its function as a mechanical barrier and in gas exchange, the airway epithelium plays an important role in pathogen detection and is a source of cytokines and other inflammatory mediators that modulate immunity in the respiratory tract (1-7). Airway epithelial cells (AECs) express Toll-like receptor 1 (TLR1) to , and their activation with TLR agonists has been shown to induce the production of several cytokines, chemokines, and antimicrobial peptides. It is worth noting that the majority of these studies have been done at the mRNA level and using continuous cell lines or nonpolarized primary cells as responders to stimulation. Morphology and differentiation are critical in determining infection and immunity of the airway epithelium. First, AECs cultured under air-liquid interface (ALI) differentiate into ciliated cells that are more resistant to virus infection and mount less exacerbated inflammatory responses (12). Second, mucin is a negative regulator of TLR signaling exclusively expressed on the apical surfaces of differentiated AECs (13). Third, multiple receptors and adhesion molecules have a polarized distribution in AECs, i.e., the alpha/beta interferon (IFN-␣/␤) receptor (IFNAR) is exclusively expressed on the basolateral surface (14). Thus, primary polarized AEC cultures provide a valuable system that is a better representation of the airway epithelial microenvironment in vivo than cell lines (15-17).One of the major downstream products of TLR signaling is the IFN family (18). IFNs are a diverse group of cytokines characterized for inducing antiviral resistance, and there are three types (type I, type II, and type III) based on their biological effects, receptor usage, and structure. Only type I and type III IFNs are directly produced in response to virus infection. Type I IFNs are key immune regulators essential for mounting a robust immune response to many viral infections (19,20). All subtypes of type I IFNs engage the ubiquitously expressed IFNAR and initiate a signaling cascade that leads to the induction of Ͼ300 IFN-stimulated genes (21). Type III IFNs include interleukin-28A (IL-28A), IL-28B, and IL-29 (also known as IFN-1, IFN-2, and IFN-3) (22, 23) and signal through the IFN-receptor (IFNLR) that is composed of an exclusive IFN-R1 chain and a shared IL-10R2 chain (23). Despite the low amino acid homology between type I and type III IFNs, they trigger common signaling pathways and biological activities (24,25). This functional redundancy is contested by the different receptor distributions and by the differential regulation of type I and type III IFN production during infection. Although IFNAR is present in all cells, the expression of IFNLR is limited to epithelial cells (26,27). Type III IFNs are produced at higher levels and during longer times in the lung than type I IFNs during influenza virus infection (28). These differences are likely t...