The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog,mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Cyclic Di-AMP Homeostasis in Bacillus subtilis: BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH*

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.     

Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg²⁺ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.     

p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Class I_B phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class I_B PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.     

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.     

TORC2 Is Required to Maintain Genome Stability during S Phase in Fission Yeast

DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development.     

Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N_in-C_out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.