Leu1283.43Leu128^{3.43} (L128) and Val2476.40Val247^{6.40} (V247) of CXCR1 Are Critical Amino Acid Residues for G Protein Coupling and Receptor Activation

CXCR1, a classic GPCR that binds IL-8, plays a key role in neutrophil activation and migration by activating phospholipase C (PLC)β through $G\alpha_{15}$ and $G\alpha_i$ which generates diacylglycerol and inositol phosphates (IPs). In this study, two conserved amino acid residues of CXCR1 on the transmembrane domain (TM) 3 and TM6, $Leu128^{3.43}$ (L128) and $Val247^{6.40}$ (V247), respectively, were selectively substituted with other amino acids to investigate the role of these conserved residues in CXCR1 activation. Although two selective mutants on Leu128, Leu128Ala (L128A) and Leu128Arg (L128R), demonstrated high binding affinity to IL-8, they were not capable of coupling to G proteins and consequently lost the functional response of the receptors. By contrast, among the four mutants at residue Val247 (TM6.40), replacing Val247 with Ala (V247A) and Asn (V247N) led to constitutive activation of mutant receptors when cotransfected with $G\alpha_{15}$. The V247N mutant also constitutively activated the $G\alpha_i$ protein. These results indicate that L128 on TM3.43 is involved in G protein coupling and receptor activation but is unimportant for ligand binding. On the other hand, V247 on TM6.40 plays a critical role in maintaining the receptor in the inactive state, and the substitution of V247 impaired the receptor constraint and stabilized an active conformation. Functionally, there was an increase in chemotaxis in response to IL-8 in cells expressing V247A and V247N. Our findings indicate that $Leu128^{3.43}$ and $Val247^{6.40}$ are critical for G protein coupling and activation of signaling effectors, providing a valuable insight into the mechanism of CXCR1 activation

Cigarette smoke induces nuclear translocation of heme oxygenase 1 (HO-1) in prostate cancer cells: Nuclear HO-1 promotes vascular endothelial growth factor secretion

Prostate cancer is the second leading cause of male-cancer related death in the United States. Despite a number of evidence-based studies which strongly suggest an association between cigarette smoking and prostate cancer, the underlying biological mechanism is largely unknown. Heme oxygenase 1 (HO-1) has been implicated in maintaining cellular homeostasis, but also in tumor angiogenesis. Nuclear HO-1 protein expression has been observed in various types of tumors including prostate cancer. These studies, however, were reported as clinical and pathological observations, and failed to investigate nuclear HO-1 at the molecular level in cancer. The present study explores the relationship between cigarette smoke and nuclear HO-1-modulated promotion of vascular endothelial growth factor (VEGF) secretion. We have demonstrated that cigarette smoke medium (SM)-induced HO-1 mRNA expression and upregulated HO-1 protein levels in the prostate cancer cell lines DU145 and PC3. We also observed that SM significantly induced nuclear expression of HO-1, and enhanced secretion of VEGF in cells. Nuclear-directed expression of HO-1 activated the transcriptional activity of VEGF and promoted VEGF secretion in prostate cancer cells. This study provides new insights into the molecular mechanism by which cigarette smoke-induced nuclear translocation of HO-1 promotes VEGF secretion in prostate cancer cells. Nuclear HO-1 may, therefore, constitute an attractive therapeutic target to inhibit angiogenesis and the progression of prostate cancer

Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway

Purpose To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. Methods: Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA. Results: HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis. Conclusion: Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression

HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation

Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM) with substrate inhibition kinetics (Ki=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy

Non-Opsonic Phagocytosis of Legionella pneumophila by Macrophages Is Mediated by Phosphatidylinositol 3-Kinase

Background: Legionella pneumophila, is an intracellular pathogen that causes Legionnaires ’ disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis. Methodology/Principal Findings: Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32 P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85a subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells. Conclusion/Significance: Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies fo

A Quasi-Exclusive European Ancestry in the Senepol Tropical Cattle Breed Highlights the Importance of the slick Locus in Tropical Adaptation

Background: The Senepol cattle breed (SEN) was created in the early XXth century from a presumed cross between a European (EUT) breed (Red Poll) and a West African taurine (AFT) breed (N'Dama). Well adapted to tropical conditions, it is also believed trypanotolerant according to its putative AFT ancestry. However, such origins needed to be verified to define relevant husbandry practices and the genetic background underlying such adaptation needed to be characterized. Methodology/Principal Findings: We genotyped 153 SEN individuals on 47,365 SNPs and combined the resulting data with those available on 18 other populations representative of EUT, AFT and Zebu (ZEB) cattle. We found on average 89% EUT, 10.4% ZEB and 0.6% AFT ancestries in the SEN genome. We further looked for footprints of recent selection using standard tests based on the extent of haplotype homozygosity. We underlined i) three footprints on chromosome (BTA) 01, two of which are within or close to the polled locus underlying the absence of horns and ii) one footprint on BTA20 within the slick hair coat locus, involved in thermotolerance. Annotation of these regions allowed us to propose three candidate genes to explain the observed signals (TIAM1, GRIK1 and RAI14). Conclusions/Significance: Our results do not support the accepted concept about the AFT origin of SEN breed. Initial AFT ancestry (if any) might have been counter-selected in early generations due to breeding objectives oriented in particular toward meat production and hornless phenotype. Therefore, SEN animals are likely susceptible to African trypanosomes which questions the importation of SEN within the West African tsetse belt, as promoted by some breeding societies. Besides, our results revealed that SEN breed is predominantly a EUT breed well adapted to tropical conditions and confirmed the importance in thermotolerance of the slick locus. (Résumé d'auteur