FoxO4 controls sGCß transcription in vascular smooth muscle.

Nitric oxide (NO) binds soluble guanylyl cyclase ß (sGCß) to produce cGMP and relax vascular smooth muscle cells (SMCs) needed for vasodilation. Although the regulation of NO-stimulated sGC activity has been well characterized at the posttranslational level, the mechanisms that govern sGC transcription remain incompletely understood. Recently, we identified Forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC; however, the specific FoxO family member responsible for the expression of sGCß in SMC remains unknown. Using FoxO shRNA knockdown adenovirus treatment in rat aortic SMCs, we show that FoxO1 or FoxO3 knockdown causes greater than twofold increases in Gucy1a3 and Gucy1b3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in Gucy1a3 and Gucy1b3 mRNA with 70% loss of sGCα and 50% loss of sGCß protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of GUCY1B3 in human aortic SMCs. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCß expression in SMC.NEW & NOTEWORTHY One of the key mechanisms of vascular smooth muscle cell (SMC) dilation occurs through nitric oxide (NO)-dependent induction of soluble guanylyl cyclase (sGC) by means of its ß-subunit. Herein, we are the first to identify Forkhead box subclass O protein 4 (FoxO4) as a key transcriptional regulator of GUCY1B3 expression, which codes for sGCß protein in human and animal SMCs. This discovery will likely have important implications for the future usage of antihypertensive and vasodilatory therapies which target NO production, sGC, or FoxO transcription factors.

SOD2 V16A amplifies vascular dysfunction in sickle cell patients by curtailing mitochondria complex IV activity.

Superoxide dismutase 2 (SOD2) catalyzes the dismutation of superoxide to hydrogen peroxide in mitochondria, limiting mitochondrial damage. The SOD2 amino acid valine-to-alanine substitution at position 16 (V16A) in the mitochondrial leader sequence is a common genetic variant among patients with sickle cell disease (SCD). However, little is known about the cardiovascular consequences of SOD2V16A in SCD patients or its impact on endothelial cell function. Here, we show SOD2V16A associates with increased tricuspid regurgitant velocity (TRV), systolic blood pressure, right ventricle area at systole, and declined 6-minute walk distance in 410 SCD patients. Plasma lactate dehydrogenase, a marker of oxidative stress and hemolysis, significantly associated with higher TRV. To define the impact of SOD2V16A in the endothelium, we introduced the SOD2V16A variant into endothelial cells. SOD2V16A increases hydrogen peroxide and mitochondrial reactive oxygen species (ROS) production compared with controls. Unexpectedly, the increased ROS was not due to SOD2V16A mislocalization but was associated with mitochondrial complex IV and a concomitant decrease in basal respiration and complex IV activity. In sum, SOD2V16A is a novel clinical biomarker of cardiovascular dysfunction in SCD patients through its ability to decrease mitochondrial complex IV activity and amplify ROS production in the endothelium.

Precise druggability of the PTH type 1 receptor.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

A predicted Francisella tularensis DXD-motif glycosyltransferase blocks immune activation.

Pathogens enhance their survival during infections by manipulating host defenses. Francisella tularensis evades innate immune responses, which we have found to be dependent on an understudied gene ybeX (FTL_0883/FTT_0615c). To understand the function of YbeX, we sought protein interactors in F. tularensis subsp. holarctica live vaccine strain (LVS). An unstudied Francisella protein co-immunoprecipitated with recombinant YbeX, which is a predicted glycosyltransferase with a DXD-motif. There are up to four genomic copies of this gene with identical sequence in strains of F. tularensis pathogenic to humans, despite ongoing genome decay. Disruption mutations were generated by intron insertion into all three copies of this glycosyltransferase domain containing gene in LVS, gdcA1-3. The resulting strains stimulated more cytokines from macrophages in vitro than wild-type LVS and were attenuated in two in vivo infection models. GdcA was released from LVS during culture and was sufficient to block NF-κB activation when expressed in eukaryotic cells. When co-expressed in zebrafish, GdcA and YbeX were synergistically lethal to embryo development. Glycosyltransferases with DXD-motifs are found in a variety of pathogens including NleB, an Escherichia coli type-III secretion system effector that inhibits NF-κB by antagonizing death receptor signaling. To our knowledge, GdcA is the first DXD-motif glycosyltransferase that inhibits NF-κB in immune cells. Together, these findings suggest DXD-motif glycosyltransferases may be a conserved virulence mechanism used by pathogenic bacteria to remodel host defenses.

Redox regulation of soluble guanylyl cyclase.

The nitric oxide/soluble guanylyl cyclase (NO-sGC) signaling pathway regulates the cardiovascular, neuronal, and gastrointestinal systems. Impaired sGC signaling can result in disease and system-wide organ failure. This review seeks to examine the redox control of sGC through heme and cysteine regulation while discussing therapeutic drugs that target various conditions. Heme regulation involves mechanisms of insertion of the heme moiety into the sGC protein, the molecules and proteins that control switching between the oxidized (Fe3+) and reduced states (Fe2+), and the activity of heme degradation. Modifications to cysteine residues by S-nitrosation on the α1 and ß1 subunits of sGC have been shown to be important in sGC signaling. Moreover, redox balance and localization of sGC is thought to control downstream effects. In response to altered sGC activity due to changes in the redox state, many therapeutic drugs have been developed to target decreased NO-sGC signaling. The importance and relevance of sGC continues to grow as sGC dysregulation leads to numerous disease conditions.

Exploiting Analysis of Heterogeneity to Increase the Information Content Extracted from Fluorescence Micrographs of Transgenic Zebrafish Embryos.

Zebrafish embryos are a near-ideal animal model for drug discovery because of their high genetic and physiological similarity to mammals, small size, high fecundity, and optical transparency. The latter properties make zebrafish at larval stages especially suited for high-content analysis and high throughput screening (HTS). However, inherent biological complexity and the inability to screen multiple specimens in a single well present a challenge for HTS because limiting replicates and high variability often prevent assays from reaching the stringent performance criteria demanded of large-scale screening assays. In this report, we present methodology that overcomes these obstacles. We used our previously developed Tg(lhx1a:EGFP)pt303 line, which expresses a fluorescent transgene that enables live real-time measurements of kidney progenitor cell expansion. Since transgenes are expressed in specific cell populations, whose localization is precisely controlled, both spatially and temporally, we considered the developing embryo to be a "host" for a cell population, analogous to a well of a cell culture microplate, rather than a single specimen. By adopting this view, parameters routinely used to analyze cultured cells became applicable to characterize and quantify zebrafish transgene appearance beyond the overall intensity or area measurements, which are analogous to calculating well average data. Using the pixel-level distribution of transgene intensity as a proxy to cell-level data, we applied population-based intensity and heterogeneity measurements to quantitatively describe and characterize transgene expression in each embryo. Subsequent linear discriminant analysis on eight such parameters captured and condensed this information into a single assay parameter that maximizes the difference between positive and negative responses. The improvements in assay performance resulted in the Tg(lhx1a:EGFP)pt303 assay achieving HTS compatible assay performance in multi-day variability studies, documenting readiness for HTS of compounds that expand kidney progenitor cell populations.

Targeted inhibition of pancreatic acinar cell calcineurin is a novel strategy to prevent post-ERCP pancreatitis.

BACKGROUND AND AIMS: There is a pressing need to develop effective preventative therapies for post-ERCP pancreatitis (PEP). We demonstrated that early PEP events are induced through the calcium-activated phosphatase calcineurin and that global calcineurin deletion abolishes PEP in mice. A crucial question is whether acinar cell calcineurin controls the initiation of PEP in vivo. METHODS: We used a mouse model of PEP and examined the effects of in vivo acinar cell-specific calcineurin deletion by either generating a conditional knockout line or infusing a novel AAV-Ela-iCre into the pancreatic duct of a calcineurin floxed line. RESULTS: We found that PEP is dependent on acinar cell calcineurin in vivo, and this led us to determine that calcineurin inhibitors, infused within the radiocontrast, can largely prevent PEP. CONCLUSIONS: These results provide impetus for launching clinical trials to test the efficacy of intraductal calcineurin inhibitors to prevent PEP.

Delayed treatment with PTBA analogs reduces postinjury renal fibrosis after kidney injury.

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.

Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.

Development of high-content assays for kidney progenitor cell expansion in transgenic zebrafish.

Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

Requirement for a uroplakin 3a-like protein in the development of zebrafish pronephric tubule epithelial cell function, morphogenesis, and polarity.

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na(+)/K(+)-ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression.

Distinct and separable activities of the endocytic clathrin-coat components Fcho1/2 and AP-2 in developmental patterning.

Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show that the µ-homology domain of FCHO1/2 represents an endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 µ-homology domain interacts with the Bmp receptor Alk8, uncovering an endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus challenge the primacy of FCHO1/2 in coat initiation.

Structural requirements for PACSIN/Syndapin operation during zebrafish embryonic notochord development.

PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.

Synthesis and structure-activity relationships of 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine analogues as potent, noncompetitive metabotropic glutamate receptor subtype 5 antagonists; search for cocaine medications.

Recent genetic and pharmacological studies have suggested that the metabotropic glutamate receptor subtype 5 (mGluR5) may represent a druggable target in identifying new therapeutics for the treatment of various central nervous system disorders including drug abuse. In particular, considerable attention in the mGluR5 field has been devoted to identifying ligands that bind to the allosteric modulatory site, distinct from the site for the primary agonist glutamate. Both 2-methyl-6-(phenylethynyl)pyridine (MPEP) and its analogue 3-[(2-methyl-4-thiazolyl)ethynyl]pyridine (MTEP) have been shown to be selective and potent noncompetitive antagonists of mGluR5. Because of results presented in this study showing that MTEP prevents the reinstatement of cocaine self-administration caused by the presentation of environmental cues previously associated with cocaine availability, we have prepared a series of analogues of MTEP with the aim of gaining a better understanding of the structural features relevant to its antagonist potency and with the ultimate aim of investigating the effects of such compounds in blunting the self-administration of cocaine. These efforts have led to the identification of compounds showing higher potency as mGluR5 antagonists than either MPEP or MTEP. Two compounds 19 and 59 exhibited functional activity as mGluR5 antagonists that are 490 and 230 times, respectively, better than that of MTEP.

Glycerol-3-phosphate cytidylyltransferase. Structural changes induced by binding of CDP-glycerol and the role of lysine residues in catalysis.

The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates. Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate. Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix. Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B. This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol. Binding of CDP-glycerol also induces smaller movements of residues 92-100. Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate.