Species-Specific Inhibition of Necroptosis by HCMV UL36

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.     

Human Cytomegalovirus Inhibits the PARsylation Activity of Tankyrase—A Potential Strategy for Suppression of the Wnt Pathway

Human cytomegalovirus (HCMV) was reported to downregulate the Wnt/β-catenin pathway. Induction of Axin1, the negative regulator of the Wnt pathway, has been reported as an important mechanism for inhibition of β-catenin. Since Tankyrase (TNKS) negatively regulates Axin1, we investigated the effect of HCMV on TNKS expression and poly-ADP ribose polymerase (PARsylation) activity, during virus replication. Starting at 24 h post infection, HCMV stabilized the expression of TNKS and reduced its PARsylation activity, resulting in accumulation of Axin1 and reduction in its PARsylation as well. General PARsylation was not changed in HCMV-infected cells, suggesting specific inhibition of TNKS PARsylation. Similarly, treatment with XAV939, a chemical inhibitor of TNKS’ activity, resulted in the accumulation of TNKS in both non-infected and HCMV-infected cell lines. Reduction of TNKS activity or knockdown of TNKS was beneficial for HCMV, evidenced by its improved growth in fibroblasts. Our results suggest that HCMV modulates the activity of TNKS to induce Axin1, resulting in inhibition of the β-catenin pathway. Since HCMV replication is facilitated by TNKS knockdown or inhibition of its activity, TNKS may serve as an important virus target for control of a variety of cellular processes.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Association of Interleukin-1β-31C/T, -511T/C and -3954C/T Single Nucleotide Polymorphism and Their Blood Plasma Level in Acquired Aplastic Anemia

Aplastic anemia (AA) is an immune-mediated disorder in which hematopoietic stem and progenitor cells are targeted by a number of cellular and molecular pathways. This case control study aims to investigate the association of interleukin-1beta (IL-1β) gene polymorphisms, (IL-1β-31, IL-1β-511 and IL-1β-3954) and their plasma levels with acquired AA. Genotyping was done by Restricted Fragment Length Polymorphism (PCR–RFLP) method and IL-1β plasma levels were evaluated in peripheral blood using ELISA. Increased level of IL-1β was reported to be significant in cases as compared to controls. The susceptibility of developing AA was higher in the cases for IL-1β-3954 genotype. IL-1β-511 genotype showed significant association with the severity groups of AA. No significant association was noticed in responder versus non-responder group. Plasma level of IL-1β gene was found to be significantly higher in severe and very-severe group of AA versus control group. Our findings suggest that IL-1β gene and its genotypes might be involved in the pathophysiology of AA and play a central role in the etiopathogenesis of AA.     

Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome

Dying cells are capable of activating the innate immune system and inducing a sterile inflammatory response. Here, we show that necrotic cells are sensed by the Nlrp3 inflammasome resulting in the subsequent release of the proinflammatory cytokine IL-1β. Necrotic cells produced by pressure disruption, hypoxic injury, or complement-mediated damage were capable of activating the Nlrp3 inflammasome. Nlrp3 inflammasome activation was triggered in part through ATP produced by mitochondria released from damaged cells. Neutrophilic influx into the peritoneum in response to necrotic cells in vivo was also markedly diminished in the absence of Nlrp3. Nlrp3-deficiency moreover protected animals against mortality, renal dysfunction, and neutrophil influx in an in vivo renal ischemic acute tubular necrosis model. These findings suggest that the inhibition of Nlrp3 inflammasome activity can diminish the acute inflammation and damage associated with tissue injury.     

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.     

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β₂-adrenergic receptors (β₂AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β₂AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β₂AR actions favorable for treating obstructive lung disease.

Most G protein–coupled receptors (GPCRs) are now recognized as multisignal transducers (1, 2). Early concepts of agonist–receptor interactions were based on the idea that there was a single “active” receptor conformation induced by the binding of any agonist, resulting in an interaction with the heterotrimeric G protein and a universal, singular signal. Generally, the α-subunit of the G protein, upon its dissociation, was considered the primary activator (or inhibitor) of the effector, resulting in the intracellular signal. Subsequently, it became clear that multiple signaling outcomes from activation of a given GPCR can occur from a single agonist due to specific molecular determinants of the receptor triggering independent mechanisms (3–5). As these multiple functions were being identified, it was apparent that agonists with different structures could act at a given receptor to preferentially activate one signal with minimal engagement of others, a property later termed signal biasing (6–8). Biased agonists, then, could represent important advantages over nonbiased agonists due to this signal selectivity, activating a specified therapeutic pathway while minimally evoking unnecessary or deleterious signaling. The pathway selectivity of biased agonists is thought to be established by the stabilization of specific conformation(s) of the agonist–receptor complex via a set of interactions that differ from those of unbiased (also called balanced) agonists (9–12). While it is conceivable that small modifications of established cognate agonists might yield such specialized signaling, significant deviation from common agonist structures may be necessary to meet this goal (13).The signals/functions of a given GPCR that might be sought for selective activation are defined by the cell type, disease, and desired final physiologic function. In asthma and chronic obstructive pulmonary disease (COPD), active human airway smooth muscle (HASM) cellular contraction limits airflow, representing a major cause of morbidity and mortality. β₂-adrenergic receptors (β₂ARs) expressed on HASM cells are the targets for binding of therapeutically administered β-agonists, which relax the cells via a cyclic adenosine monophosphate–mediated mechanism (14). β-agonists are used for treating acute bronchospasm as well as for long-term prevention. However, the HASM bronchodilator response to acute β-agonist is attenuated by receptor desensitization (15), with typical treatments of humans, or isolated HASM cells, leading to a loss of receptor function over time (16–18), clinically termed tachyphylaxis.Agonist-promoted desensitization of β₂AR (and other GPCRs) is due to partial uncoupling of the receptor to the G protein, which is initiated by phosphorylation of intracellular Ser/Thr residues of the receptor by G protein–coupled receptor kinases (GRKs) (19, 20). The GRK-phosphorylated β₂AR recruits β-arrestin1 or β-arrestin2 to these receptors, with subsequent interactions that appear to compete with the receptor for its binding to the Gα subunit, thus attenuating the intracellular response (11, 21). Such competition has been strongly inferred for the β₂AR (22, 23) and is compelling for rhodopsin–arrestin interactions (24). In addition, β-arrestin binding to GPCRs can initiate receptor internalization and other events such as receptor activation of ERK1/2 (25) through its multiprotein adapter functions. Thus β-arrestin engagement can be considered an early “second signal” of the β₂AR as well as a desensitization initiator for attenuating the Gs signal. An agonist that is biased toward Gαs coupling (cAMP production and airway smooth muscle [ASM] relaxation) and away from β-arrestin binding (desensitization) would be desirable in treating obstructive lung diseases, since efficacy would not be attenuated acutely, nor would tachyphylaxis be experienced from extended treatment. While biased agonists favoring either G protein or β-arrestin (6) signaling have been described for some GPCRs (such as μ-opioid and type 1 angiotensin II receptors), Gαs biasing has not been apparent from most studies with catecholamine-like compounds for the β₂AR. Thus, we have little information as to whether the two β₂AR pathways can be differentially activated in a selective manner by an efficacious agonist, nor is it apparent from a structural standpoint what strategy might be employed to design agonists biased in this manner for this receptor.In order to find this type of biasing for the β₂AR, we screened a 40-million-compound scaffold ranking (SR) library that was agnostic to known β₂AR agonist structures. We found a scaffold in which substitutions of certain R groups led to individual compounds that are apparently Gαs-biased agonists for β₂AR with no apparent engagement of β-arrestin in model systems. Additional studies in HASM cells revealed a lack of tachyphylaxis of the relaxation effect by the lead compound compared with the most widely utilized β₂AR agonist, albuterol. The structure of this biased agonist is very different from that of catecholamine-like agonists. To ascertain the mechanism that may underlie this biased activity, we used structural modeling and molecular simulations and studied homologous compounds with different R groups and receptor mutagenesis to predict the interaction sites with the activated β₂AR. Such studies uncovered distinct structural characteristics that may be responsible for the biasing effect.     

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

Retroviral Gag polyproteins coopt host factors to traffic from cytosolic ribosomes to the plasma membrane, where virions are released. Before membrane transport, the multidomain Gag protein of Rous sarcoma virus (RSV) undergoes importin-mediated nuclear import and CRM1-dependent nuclear export, an intrinsic step in the assembly pathway. Transient nuclear trafficking of Gag is required for efficient viral RNA (vRNA) encapsidation, suggesting that Gag:vRNA binding might occur in the nucleus. Here, we show that Gag is imported into the nucleus through direct interactions of the Gag NC domain with importin-α (imp-α) and the MA domain with importin-11 (imp-11). The vRNA packaging signal, known as ψ, inhibited imp-α binding to Gag, indicating that the NC domain does not bind to imp-α and vRNA simultaneously. Unexpectedly, vRNA binding also prevented the association of imp-11 with both the MA domain alone and with Gag, suggesting that the MA domain may bind to the vRNA genome. In contrast, direct binding of Gag to the nuclear export factor CRM1, via the CRM1-RanGTP heterodimer, was stimulated by ψRNA. These findings suggest a model whereby the genomic vRNA serves as a switch to regulate the ordered association of host import/export factors that mediate Gag nucleocytoplasmic trafficking for virion assembly. The Gag:vRNA interaction appears to serve multiple critical roles in assembly: specific selection of the vRNA genome for packaging, stimulating the formation of Gag dimers, and triggering export of viral ribonucleoprotein complexes from the nucleus.     

Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure

The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1β1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2β3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2β3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO₄ oxidation of the third digitoxose and reductive amination with various R-NH₂ substituents and identified derivatives with significant selectivity for human α2β1 over α1β1 (up to 7.5-fold). When applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits. A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose approaching one residue in the β1 subunit, Gln84, suggesting a role for β in digoxin binding. Gln84 in β1 is replaced by Val88 in β3. Assuming that alkyl substituents might interact with β3Val88, we synthesized perhydro-1,4-oxazepine derivatives of digoxin with diverse alkyl substituents. The methylcyclopropyl and cyclobutyl derivatives are strongly selective for α2β3 over α1β1 (22–33-fold respectively), as determined either with purified human isoform proteins or intact bovine nonpigmented epithelium cells. When applied topically on rabbit eyes, these derivatives potently reduce both pharmacologically raised and basal intraocular pressure. The cyclobutyl derivative is more efficient than Latanoprost, the most widely used glaucoma drug. Thus, the conclusion is that α2β3-selective digoxin derivatives effectively penetrate the cornea and inhibit the Na,K-ATPase, hence reducing aqueous humor production. The new digoxin derivatives may have potential for glaucoma drug therapy.Glaucoma is a disease leading to irreversible blindness. Control of intraocular pressure (IOP) is the mainstay of glaucoma therapy and is achieved by various drugs, such as β-blockers, prostaglandin analogs, α2 adrenergic receptor agonists, cholinergic agonists, and carbonic anhydrase inhibitors given topically or systemically (1). The topical route minimizes systemic side effects and is preferable, provided the drug effectively permeates the cornea. Despite the selection of drugs available, uncontrolled IOP in many patients eventually makes surgical intervention necessary. Thus, fresh approaches to drug treatments are required.The Na,K-ATPase provides the motive power for aqueous humor production in the ciliary epithelium. The sodium, potassium–adenosine triphosphatase (Na,K-ATPase) is a heterodimer of two subunits, α the catalytic subunit and the β subunit that stabilizes the protein and affects functional properties, together with a regulatory FXYD subunit (FXYD1–7) (2). There are four isoforms of α (α1−α4) and three isoforms of β (β1−β3). The α1β1 complex is the ubiquitous complex. α2 is expressed strongly in skeletal and cardiac muscle, brain astrocytes, and also ciliary epithelium. α3 is the neuronal form and α4 is restricted to testes (3). The ciliary epithelium is a syncitium consisting of an inner layer of pigmented epithelium (PE) facing the stroma and the outer nonpigmented epithelium (NPE) layer facing the aqueous humor. Na,K-ATPase is localized to the basolateral surface of both layers (4). Importantly, it is known that the primary isoform of Na,K-ATPase in PE is α1β1, whereas that in NPE is α2β3 in rodents (5).Cardiac glycosides (CGs) consist of a steroid core with a five- or six-membered unsaturated lactone ring and a variable number of sugars bound to the C3 position of the steroid. Each of these structural features has specific effects on the binding properties (6). Crystal structures of the Na,K-ATPase are available at moderate resolution in high-affinity complexes with ouabain (7) or bufalin and digoxin (8) and a low-affinity complex with bound K and ouabain (9). The high-affinity binding site is formed by a deep pocket comprised of transmembrane helices αTM1, 2, 4, 5, and 6, with the lactone-steroid moiety pointing inwards and sugars outwards. Binding of K⁺ occurs via backbone carbonyl oxygens in TM4 that also ligand the lactone moiety in the absence of K⁺, explaining the well-known antagonistic effect of K⁺ on CG binding (7–9).This paper focuses on α2β3-selective digoxin derivatives. The underlying hypothesis is that CGs with selectivity for the principal isoform complex in NPE cells, and sufficiently permeable to enter the eye when applied topically, could effectively inhibit aqueous humor inflow and reduce IOP. As background, digoxin shows moderate selectivity for the α2β1 isoform, and the selectivity is attributable to the tridigitoxose glycan moiety, as inferred from the fact that aglycones such as digoxigenin show no isoform selectivity (10). Following this work, we synthesized a series of perhydro-1,4-oxazepine derivatives of digoxin modified in the third sugar and showed that certain derivatives have enhanced selectivity for α2β1 (up to 7.5-fold) compared with digoxin itself (about fourfold) (11). The two most α2β1-selective derivatives effectively prevented or reversed a pharmacologically induced rise in IOP in rabbits.The current work is based on further insight regarding the β subunit, derived from a recent molecular structure of the Na,K-ATPase with bound digoxin (8). The glycan moiety of digoxin points outwards, and most interestingly, the third digitoxose approaches the β1 subunit near a single residue, Gln84 (Fig. 1). Gln84 in β1 is not conserved in β3 but is replaced by Val88 (Fig. 1). We hypothesized that diverse alkyl substitutions in the perhydro-1,4-oxazepine series of digoxin derivatives might interact with the alkyl side chain of Val88 and so raise selectivity for α2β3:α1β1. Several new alkyl derivatives do show strong selectivity for α2β3 over α1β1 and also potently reduce IOP in rabbits when applied topically.Open in a separate windowFig. 1.Model of human Na,K-ATPase α1β1 and α2β3 isoforms. The model depicts the human α1β1 complex and human α2β3 complex on the pig α1β1 structure with bound digoxin (4RET). α1 and α2 subunits, red; β1 subunit, gray; β3 subunit, blue; digoxin, green.     

Enforcement of γδ-lineage commitment by the pre–T-cell receptor in precursors with weak γδ-TCR signals

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2^−/− DN cells via retroviral transduction. Our results showed that Rag2^−/− DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.T cells can differentiate along distinct αβ- or γδ-cell lineages, but bifurcate from a common bipotent precursor (1, 2). In mice, the earliest subset of T cells contains CD4⁻ CD8⁻ or double-negative (DN) thymocytes, and this can further be divided into four subgroups (DN1–4) based on the expression of CD25 and CD44 (3, 4). Single-cell progenitor analyses have identified the DN3 stage as the point of T-lineage commitment, and also the final stage at which a DN cell specifies its lineage fate as αβ or γδ (1, 5). The αβ- or γδ-lineage choice decision is governed by several factors. Two competing models have been proposed for this process: the stochastic and instructional models (2). Although evidence exists to support either model, a version of the instructional model posits that the strength of signal transduced by the T-cell receptor (TCR) expressed by the DN3 cell dictates its lineage specification (6, 7).The apparent connection between lineage choice and the TCR expressed by the cell can be severed by manipulations of TCR signal strength. We previously noted that stimulating stronger signals via expression of the ERK/MAPK-induced Inhibitor of DNA-binding 3 (Id3) appears to promote the γδ-lineage fate in developing DN3 cells in the absence of TCR expression (8), suggesting a critical role for Id3 in mediating αβ- versus γδ-lineage decisions at this developmental checkpoint. Nevertheless, absence of Id3 also appears to favor the emergence of innate-like Vγ1.1/Vδ6.3 γδ-TCR–bearing T cells from the thymus over other γδ-TCR subsets (9, 10).Several studies have shown that ligand engagement highly influences the αβ- versus γδ-lineage decision because of its effects on γδ-TCR signal strength (6, 7, 9, 11, 12). γδ-TCR–expressing DN3 cells develop along the αβ-lineage and become CD4⁺ CD8⁺ (double-positive, DP) cells in the absence of ligand engagement (7), whereas provision of the ligand, or the use of antibodies to mimic ligand engagement (11), allows these cells to adopt the γδ-lineage fate, remain DN, and down-regulate expression of CD24. Additional signals, such as those mediated by Notch, can also influence αβ- versus γδ-lineage fate outcomes (1, 13–16). We showed that γδ-TCR–bearing thymocytes adopting the γδ-lineage do not require concurrent signals from Notch to mature past the DN3 stage, whereas their pre-TCR–expressing counterparts are completely dependent upon Notch signaling to facilitate their pre-TCR–dependent differentiation to the DP stage (1, 17).Considering the common origin of αβ- and γδ-lineage cells, it is possible for a bipotent DN3 cell to simultaneously express and transmit signals through a functional pre-TCR and a functional γδ-TCR, especially considering that TCR-β, -γ, and -δ genes complete their rearrangements at the DN3 stage. Additionally, γδ-T cells have been shown to contain TCR-β rearrangements (18) and αβ-lineage cells show evidence of both TCR-γ and -δ rearrangements (19–21). In a previous study looking to address the consequences of simultaneously expressing a TCR-β and γδ-TCR in vivo using transgenic (Tg) mice, the numbers of αβ- and γδ-lineage cells in TCR-β/γδ–expressing cells were both high, and comparable to TCR-β- and γδ-TCR-Tg mice, respectively (22). In this case, however, the TCR chains were expressed earlier than physiological for T-cell development, and premature expression of αβ-TCR transgene can lead to aberrant developmental progression (23, 24).Here, we attempt to definitively answer the question of lineage choice by simultaneously expressing TCR-β and γδ-TCR in Rag2^−/− DN3 cells via retroviral transduction followed by in vitro coculture, including limiting dilution and clonal analyses. We now find that Rag2^−/− DN3 cells expressing both pre-TCR and γδ-TCR mature along the γδ-lineage into functionally competent cells that produce IFN-γ in response to stimulation. However, in the absence of Id3 expression and strong γδ-TCR ligand, γδ-expressing cells show a lower propensity to differentiate along the γδ-lineage, but when expressing both pre- and γδ-TCRs, these cells showed increased γδ-lineage differentiation and recover functional competence to produce IFN-γ, indicating that the pre-TCR can serve to enforce to a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells dictates its lineage choice.     

β-Glucans from Trametes versicolor (L.) Lloyd Is Effective for Prevention of Influenza Virus Infection

Coriolus versicolor (C. versicolor) is a higher fungi or mushroom which is now known by its accepted scientific names as Trametes versicolor (L.) Lloyd. Many studies have shown that β-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether β-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether β-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that β-glucans induced the expression of Dectin-1, costimulatory molecules (CD80/86) and cytokines IL-6, IL-1β, IFN-β and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered β-glucans reduced weight loss, mortality and viral titers in the lungs of mice infected with influenza virus and attenuated pathological lung damage caused by the virus in the mice. Orally administered β-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that β-glucans have a significant antiviral effect. Therefore, β-glucans could become a potential immunomodulator against influenza virus.     

Multiple Autonomous Cell Death Suppression Strategies Ensure Cytomegalovirus Fitness

Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease enhances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and -independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host.     

Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.The β-lactams are the most widely used and among the most valuable classes of antibiotics (1). These agents contain a β-lactam ring in their structures and inhibit the activity of transpeptidases or penicillin binding proteins (PBPs), the essential enzymes for the biosynthesis of peptidoglycan (PG) (2, 3), thereby damaging the integrity of bacterial cell wall. One of the principal mechanisms by which bacteria develop resistance to β-lactam antibiotics is the production of intrinsic or horizontally acquired β-lactamases that can degrade and inactivate β-lactams (4).In many bacterial species, β-lactamase expression is inducible in response to β-lactam antibiotic treatment. For example, in Gram-positive bacteria, β-lactamase can be induced directly by β-lactam antibiotics or indirectly by muropeptides that are released from PG after β-lactam treatment (5). In Gram-negative bacteria (e.g., Enterobacteriaceae), muropeptide produced after β-lactam treatment can also induce the expression β-lactamase (6, 7). In addition to β-lactam treatment, β-lactamase expression in Gram-negative bacteria can also be induced by the changes of growth rate (8, 9). The direct role of β-lactam in inducing the expression of β-lactamase in Gram-negative bacteria has not been reported.Two-component systems (TCSs), which are typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are widely present in many bacterial species (10, 11). Typically, environmental signals are sensed by histidine kinase, leading to its autophosphorylation and subsequent phosphoryl transfer to its cognate response regulator (12, 13). Upon phosphorylation, a response regulator usually controls the expression of genes for adaptation to changing environment. Certain TCSs have been shown to contribute to antibiotic (e.g., glycopeptide) resistance. For example, VanS, the histidine kinase of VanSR TCS, directly binds to vancomycin, leading to the expression of genes that are required for the synthesis of alternate peptidoglycan precursors that have low affinity for vancomycin (14–16). Histidine kinases also contribute to the resistance to the antimicrobial peptides, e.g., LL-37, which activates the histidine kinase PhoQ by directly binding to the acidic surface of the PhoQ periplasmic domain (17). TCSs (e.g., BlrAB in Aeromonas or CreBC in Escherichia coli and Pseudomonas) have also been shown to be involved in the expression of β-lactamase and thus are important for β-lactam resistance (3, 9, 18). The response regulator of BlrAB or CreBC triggers the expression of β-lactamase by recognizing the signature sequences (cre/blr-tag: TTCACnnnnnnTTCAC) located in the promoter of β-lactamase gene (3, 9, 18). Despite the evidence that TCS plays an important role in the induction of β-lactamase expression, the identity of the cues that are recognized and transmitted by TCS to control β-lactamase expression remain completely unknown (19).Here, we reported a previously unidentified mechanism that governs β-lactamase production in Gram-negative bacterium Vibrio parahaemolyticus, the leading cause of seafood-borne diarrheal disease worldwide. Most isolates of V. parahaemolyticus from both clinical and environmental settings exhibit resistance to β-lactam antibiotics, thereby limiting treatment options. At the time we initiated these studies, there was minimal knowledge of the mechanisms underlying V. parahaemolyticus resistance to β-lactam antibiotics. However, a recent report revealed that essentially all V. parahaemolyticus isolates encode a class A chromosomal carbenicillin-hydrolyzing (CARB) β-lactamase (bla_V110) (20). We independently identified this chromosome II-encoded enzyme as part of the regulon of a novel TCS (VbrK/VbrR) that controls V. parahaemolyticus resistance to β-lactam antibiotics. Notably, we show that the sensor histidine kinase, VbrK, in this system detects β-lactam antibiotics via direct binding and transmits the signal to the response regulator, VbrR, to control the expression of this CARB β-lactamase gene.     

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974

Wnt signaling is one of the key oncogenic pathways in multiple cancers, and targeting this pathway is an attractive therapeutic approach. However, therapeutic success has been limited because of the lack of therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. We developed a screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific small-molecule Porcupine (PORCN) inhibitor. PORCN is a membrane-bound O-acyltransferase that is required for and dedicated to palmitoylation of Wnt ligands, a necessary step in the processing of Wnt ligand secretion. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo, including reduction of the Wnt-dependent LRP6 phosphorylation and the expression of Wnt target genes, such as AXIN2. LGK974 is potent and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV–Wnt1 and a human head and neck squamous cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wnt-driven cancers through the inhibition of PORCN.Wnt signaling is a key oncogenic pathway in multiple cancers (1, 2). On binding to its receptors LRP5/6 and Frizzled (FZD) at the plasma membrane, Wnt ligand triggers the disruption of the β-catenin degradation machinery (consisting of AXIN2, GSK3β, APC, and others), leading to the accumulation of β-catenin in the cytoplasm (3). Elevated levels of β-catenin ultimately lead to its translocation into the nucleus to form a complex with LEF/TCF and drive downstream gene expression (3). Dysregulation of Wnt signaling (1) can occur through mutations of downstream components, such as APC and β-catenin, which are well-documented in colon cancer (1). In addition, overexpression of Wnt ligands or costimulants, such as R-Spondin 2/3 (RSPO2/3), or silencing of Wnt inhibitor genes has been reported in various cancers (1, 4). Mutations of pan-Wnt pathway components, such as AXIN1/2 or the RSPO coreceptors RNF43/ZNFR3, may potentially play key roles in pancreatic, colon, and hepatocellular carcinoma (4–6).The Wnt gene was originally identified as an oncogene in murine mammary tumors 30 y ago (2) and confirmed to be a key oncogenic pathway in many studies, including an unbiased insertional mutagenesis screen, with Wnt1, Wnt3, and Wnt3A comprising 38% of all unbiased hits (7). In human breast cancer, overexpression of Wnt or silencing of Wnt inhibitor genes has been observed in up to one-half of breast cancer samples (8). Cytoplasmic and nuclear β-catenin have also been correlated with triple-negative and basal-like breast cancer subtypes (9, 10), and Wnt signaling has also been implicated in cancer-initiating cells in multiple cancer types (11–14). Wnt pathway signaling activity is dependent on Wnt ligand. During the biosynthesis of Wnt ligands, Wnt undergoes posttranslational acylation (palmitoylation) that is mediated by Porcupine (PORCN), a membrane bound O-acyltransferase (3, 15). PORCN is specific and dedicated to Wnt posttranslational acylation, which is required for subsequent Wnt secretion (16). Loss of PORCN leads to inhibition of Wnt ligand-driven signaling activities in KO mouse models (17, 18). In humans, loss-of-function (LoF) mutations in the PORCN gene cause focal dermal hypoplasia, an X-linked dominant disorder associated with a variety of congenital abnormalities in both heterozygotes and those individuals with mosaicism for the PORCN gene. This phenotype is consistent with the role of the Wnt signaling pathway during embryogenesis and development (15).Given the key role of Wnt signaling in cancer, targeting this pathway has been an attractive therapeutic approach. However, success has been limited because of the lack of effective therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. Herein, we describe a cellular high-throughput screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific PORCN inhibitor. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo and has strong efficacy in tumor models in vivo. We also show that head and neck squamous cell carcinoma cell lines with LoF mutations in the Notch signaling pathway have a high response rate to LGK974. These findings provide a path forward to target Wnt-driven cancer through the inhibition of PORCN.