Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.

Functional role of Notch signaling in the developing and postnatal heart

In the developing heart, Notch signaling plays an essential role in several key developmental processes, such as epithelial-to-mesenchymal transition and myocyte proliferation and differentiation. The importance of Notch in cardiac development has been demonstrated in knockout mice carrying null mutations in genes encoding components of the Notch pathway. Furthermore, humans with inactivating mutations in the Notch ligand Jagged1 suffer from Alagille syndrome, a condition characterized by several cardiac defects. Notch1 receptor haploinsufficiency has also been involved in aortic valve disease in humans. In addition, accumulating evidence indicates that Notch may also regulate homeostasis in the adult heart. Notch may protect the heart from an excessive and detrimental hypertrophic response and increase cardiomyocyte survival. Emerging evidence also suggests that Notch could be important for cardiac tissue renewal by controlling the maintenance and commitment of a cardiac stem cell compartment.

Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens.

The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the -10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice.

Tissue transglutaminase (TG2) is a protein cross-linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-out mice within 20 hours. Although TG2-/- thymocytes exposed to anti-Fas antibodies die at the same rate as wild-type mice, TG2-/- hepatocytes show increased sensitivity toward anti-Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIP(L) (FLICE-inhibitory protein), or the rate of I-kappaBalpha degradation, but a decrease in the Bcl-xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl-xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand-induced hepatocyte proliferation with a simultaneous inhibition of the Fas-death pathway plays a determinant role.

Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling.

We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses alpha(V)beta(3)- (but not alpha(5)beta(1)-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE(2) and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE(2) accelerated alpha(V)beta(3)-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE(2) effects. alpha(V)beta(3)-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked alpha(V)beta(3)-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued alpha(V)beta(3)-dependent spreading in the presence of NS398 or, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. alpha(5)beta(1)-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE(2), 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE(2) accelerates alpha(V)beta(3)-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces alpha(V)beta(3)-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins alpha(V)beta(3) by COX-2/PGE(2) during tumor angiogenesis and inflammation.

Lack of Smad3 signaling leads to impaired skeletal muscle regeneration.

Smad3 is a key intracellular signaling mediator for both transforming growth factor-β and myostatin, two major regulators of skeletal muscle growth. Previous published work has revealed pronounced muscle atrophy together with impaired satellite cell functionality in Smad3-null muscles. In the present study, we have further validated a role for Smad3 signaling in skeletal muscle regeneration. Here, we show that Smad3-null mice had incomplete recovery of muscle weight and myofiber size after muscle injury. Histological/immunohistochemical analysis suggested impaired inflammatory response and reduced number of activated myoblasts during the early stages of muscle regeneration in the tibialis anterior muscle of Smad3-null mice. Nascent myofibers formed after muscle injury were also reduced in number. Moreover, Smad3-null regenerated muscle had decreased oxidative enzyme activity and impaired mitochondrial biogenesis, evident by the downregulation of the gene encoding mitochondrial transcription factor A, a master regulator of mitochondrial biogenesis. Consistent with known Smad3 function, reduced fibrotic tissue formation was also seen in regenerated Smad3-null muscle. In conclusion, Smad3 deficiency leads to impaired muscle regeneration, which underscores an essential role of Smad3 in postnatal myogenesis. Given the negative role of myostatin during muscle regeneration, the increased expression of myostatin observed in Smad3-null muscle may contribute to the regeneration defects.

A user's guide to the encyclopedia of DNA elements (ENCODE).

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome.

Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

Innate immunity: cytoplasmic DNA sensing by the AIM2 inflammasome.

Cytoplasmic double-stranded DNA triggers cell death and secretion of the pro-inflammatory cytokine IL-1beta in macrophages. Recent reports now describe the mechanism underlying this observation. Upon sensing of DNA, the HIN-200 family member AIM2 triggers the assembly of the inflammasome, culminating in caspase-1 activation, IL-1beta maturation and pyroptotic cell death.

SwissParam: a fast force field generation tool for small organic molecules.

The drug discovery process has been deeply transformed recently by the use of computational ligand-based or structure-based methods, helping the lead compounds identification and optimization, and finally the delivery of new drug candidates more quickly and at lower cost. Structure-based computational methods for drug discovery mainly involve ligand-protein docking and rapid binding free energy estimation, both of which require force field parameterization for many drug candidates. Here, we present a fast force field generation tool, called SwissParam, able to generate, for arbitrary small organic molecule, topologies, and parameters based on the Merck molecular force field, but in a functional form that is compatible with the CHARMM force field. Output files can be used with CHARMM or GROMACS. The topologies and parameters generated by SwissParam are used by the docking software EADock2 and EADock DSS to describe the small molecules to be docked, whereas the protein is described by the CHARMM force field, and allow them to reach success rates ranging from 56 to 78%. We have also developed a rapid binding free energy estimation approach, using SwissParam for ligands and CHARMM22/27 for proteins, which requires only a short minimization to reproduce the experimental binding free energy of 214 ligand-protein complexes involving 62 different proteins, with a standard error of 2.0 kcal mol(-1), and a correlation coefficient of 0.74. Together, these results demonstrate the relevance of using SwissParam topologies and parameters to describe small organic molecules in computer-aided drug design applications, together with a CHARMM22/27 description of the target protein. SwissParam is available free of charge for academic users at www.swissparam.ch.