Guilin Wang

Epigenetic control of human immunodeficiency virus-1 (HIV-1) genes is critical for viral integration and latency. However, epigenetic changes in the HIV-1-infected host genome have not been well characterized. Here, we report the first large-scale epigenome-wide association study of DNA methylation for HIV-1 infection. We recruited HIV-infected (n = 261) and uninfected (n = 117) patients from the Veteran Aging Cohort Study (VACS) and all samples were profiled for 485,521 CpG sites in DNA extracted from the blood. After adjusting for cell type and clinical confounders, we identified 20 epigenome-wide significant CpGs for HIV-1 infection. Importantly, 2 CpGs in the promoter of the NLR family, CARD domain containing gene 5 (NLRC5), a key regulator of major histocompatibility complex class I gene expression, showed significantly lower methylation in HIV-infected subjects than in uninfected subjects (cg07839457: t = −6.03, Pnominal = 4.96 × 10−9; cg16411857: t = −7.63, Pnominal = 3.07 × 10−13). Hypomethylation of these 2 CpGs was replicated in an independent sample (GSE67705: cg07839457: t = −4.44, Pnominal = 1.61 × 10−5; cg16411857: t = −5.90; P = 1.99 × 10−8). Methylation of these 2 CpGs in NLRC5 was negatively correlated with viral load in the 2 HIV-infected samples (cg07839457: P = 1.8 × 10−4; cg16411857: P = 0.03 in the VACS; and cg07839457: P = 0.04; cg164111857: P = 0.01 in GSE53840). Our findings demonstrate that differential DNA methylation is associated with HIV infection and suggest the involvement of a novel host gene, NLRC5, in HIV pathogenesis.

Treatment of HEK293 cells expressing the δ-opioid receptor with agonist [d-Pen2,5]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e.Thr358 and Ser363, were phosphorylated, with Ser363 being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser363 as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr358 and Ser363, with Thr358being involved in the receptor internalization. Treatment of HEK293 cells expressing the δ-opioid receptor with agonist [d-Pen2,5]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e.Thr358 and Ser363, were phosphorylated, with Ser363 being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser363 as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr358 and Ser363, with Thr358being involved in the receptor internalization. G protein-coupled receptor δ-opioid receptor G protein-coupled receptor kinase [d-Pen2,5]enkephalin hemagglutinin polyacrylamide gel electrophoresis ponasterone A Chinese hamster ovary Prolonged exposure to opioid drugs often produces tolerance, dependence, and addiction, and the molecular mechanisms underlying these phenomena are still poorly understood. Opioid receptors belong to the G protein-coupled receptor (GPCR)1 superfamily (1Evans C.J. Keith D.E. Morrison H. Magendzo K. Edwards R.H. Science. 1992; 258: 1952-1955Crossref PubMed Scopus (1126) Google Scholar, 2Kieffer B.L. Befort K. Gaveriaux-Ruff C. Hirth C.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12048-12052Crossref PubMed Scopus (977) Google Scholar). Therefore, one component of opioid tolerance is likely to be mediated by a phosphorylation-dependent desensitization of the receptor. Phosphorylation of the prototypic β2-adrenergic receptor by protein kinases, including the G protein-coupled receptor kinases (GRKs), promotes the association with the receptor of inhibitory proteins called arrestins (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar, 4Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (850) Google Scholar). This association uncouples the receptor from the G proteins and promotes targeting of activated receptors to clathrin-coated pits for subsequent internalization, thus blunting receptor signaling. Agonist-induced phosphorylation of opioid receptors has been demonstrated in different systems (5Arden J.R. Segredo V. Wang Z. Lameh J. Sadee W. J. Neurochem. 1995; 65: 1636-1645Crossref PubMed Scopus (283) Google Scholar, 6Pei G. Kieffer B.L. Lefkowitz R.J. Freedman N.J. Mol. Pharmacol. 1995; 48: 173-177PubMed Google Scholar, 7Zhang L., Yu, Y. Mackin S. Weight F.F. Uhl G.R. Wang J.B. J. Biol. Chem. 1996; 271: 11449-11454Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 8Yu Y. Zhang L. Yin X. Sun H. Uhl G.R. Wang J.B. J. Biol. Chem. 1997; 272: 28869-28874Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 9Appleyard S.M. Patterson T.A. Jin W. Chavkin C. J. Neurochem. 1997; 69: 2405-2412Crossref PubMed Scopus (50) Google Scholar, 10El Kouhen R. Maestri-El Kouhen O. Law P.-Y. Loh H.H. J. Biol. Chem. 1999; 274: 9207-9215Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Concrete demonstration of the phosphorylation of δ-opioid receptor (DOR) was first reported by Pei and colleagues (6Pei G. Kieffer B.L. Lefkowitz R.J. Freedman N.J. Mol. Pharmacol. 1995; 48: 173-177PubMed Google Scholar). Although the sites of agonist-dependent phosphorylation have not yet been identified, truncation of the carboxyl tail (C-tail) of DOR showed that major phosphorylation sites are localized within this domain (11Zhao J. Pei G. Huang Y.L. Zhong F.M. Ma L. Biochem. Biophys. Res. Commun. 1997; 238: 71-76Crossref PubMed Scopus (36) Google Scholar, 12Murray S.R. Evans C.J. von Zastrow M. J. Biol. Chem. 1998; 273: 24987-24991Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Phosphorylation of DOR appears to be the mechanism for agonist-induced receptor desensitization (6Pei G. Kieffer B.L. Lefkowitz R.J. Freedman N.J. Mol. Pharmacol. 1995; 48: 173-177PubMed Google Scholar, 13Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 14Zhang J. Ferguson S.S.G. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.-Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (451) Google Scholar). [d-Pen2,5]enkephalin (DPDPE)-induced phosphorylation of DOR seems to involve one or more GRKs (6Pei G. Kieffer B.L. Lefkowitz R.J. Freedman N.J. Mol. Pharmacol. 1995; 48: 173-177PubMed Google Scholar), and Ala substitution of the last four carboxyl-terminal Ser and Thr of the receptor impaired the GRK- and arrestin-mediated receptor desensitization (13Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In Xenopus oocytes, co-expression of GRK3 and β-arrestin2 resulted in an increased rate of agonist-induced homologous desensitization of DOR (13Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). However, truncation of the COOH-terminal 31 amino acids of DOR did not affect the agonist-induced desensitization of the receptor in CHO cells (15Wang C. Zhou D. Cheng Z. Wei Q. Chen J. Li G. Pei G. Chi Z. Biochem. Biophys. Res. Commun. 1998; 249: 321-324Crossref PubMed Scopus (22) Google Scholar), unlike what Zhao and colleagues (11Zhao J. Pei G. Huang Y.L. Zhong F.M. Ma L. Biochem. Biophys. Res. Commun. 1997; 238: 71-76Crossref PubMed Scopus (36) Google Scholar) reported in NG108-15 cells. Opioid receptors are endocytosed in a dynamin-dependent manner by clathrin-coated pits (14Zhang J. Ferguson S.S.G. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.-Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (451) Google Scholar, 16Keith D.E. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Kang L. Evans C.J. von Zastrow M. J. Biol. Cell. 1996; 271: 19021-19024Scopus (479) Google Scholar, 17Trapaidze N. Keith D.E. Cvejic S. Evans C.J. Devi L.A. J. Biol. Chem. 1996; 271: 29279-29285Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 18Chu P. Murray S.R. Lissin D. von Zastrow M. J. Biol. Cell. 1997; 272: 27124-27130Scopus (128) Google Scholar). However, the precise role of phosphorylation in the mechanism of opioid receptor endocytosis is still not fully understood. A truncated mutant δ-opioid receptor undergoes rapid agonist-induced internalization in HEK293 cells, but is not phosphorylated in the presence of agonist, whereas the same mutant remained predominantly in the plasma membrane of CHO cells, suggesting that cell type-specific differences may exist in the biochemical requirements for the agonist-induced endocytosis (12Murray S.R. Evans C.J. von Zastrow M. J. Biol. Chem. 1998; 273: 24987-24991Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In light of these discrepancies, it is imperative to identify the agonist-induced phosphorylation sites and to investigate the role of the phosphorylation of these sites in the regulation of the δ-opioid receptor. We used series of receptor mutants to identify the phosphorylation sites at the C-tail of DOR. In this study, we reported that two sites (Thr358 and Ser363) are phosphorylated in the presence of DPDPE and, furthermore, that the phosphorylation of the receptor is hierarchical. Additionally, we investigated the role played by these two phosphorylation sites in internalization of the receptor. Here, we also demonstrated that internalization of the activated receptor plays a role in the loss of δ-opioid receptor-mediated inhibition of adenylyl cyclase activity. Dulbecco's modified Eagle's medium and Geneticin (G418) were purchased from Life Technologies, Inc. [3H]Diprenorphine (58 Ci/mmol) was supplied by Amersham Pharmacia Biotech and [32P]orthophosphate (>400 Ci/ml) by ICN (Costa Mesa, CA). 125I-Acetylated cAMP (2200 Ci/mmol) was purchased from Linco Research Inc. (St. Charles, MO). Polyclonal antibodies that recognize the acetylated cAMP were purchased from Calbiochem (La Jolla, CA). NIDA (National Institutes on Health, Bethesda, MD) supplied the [d-Pen2,5]enkephalin ligand. All other chemicals were purchased from Sigma. The human influenza virus hemagglutinin (HA) epitope-tagged mouse δ-opioid receptor (described in Ref. 19Ko J.L. Arvidson U. Williams F.G. Law P.-Y. Elde R. Loh H.H. Mol. Brain Res. 1999; 69: 171-185Crossref PubMed Scopus (60) Google Scholar) (Dortag) subcloned into the expression vector pcDNA3 (Invitrogen, Carlsbad, CA), was used to generate most of the point mutations. Ser and/or Thr present at the C-tail of the receptor (from Thr335 to Ser363) were point-mutated to Ala or Asp by oligonucleotide-directed mutagenesis using a QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer's directions, except for the following mutants. The T358A, S363A, and S363D mutants were constructed using the Altered SitesTM in vitro mutagenesis system provided by Promega Corp. (Madison, WI), using DOR-1 cDNA subcloned into the phagmid pAlter-1 as template. The nucleotide of mutants were by using of the different were and to the in with the same HEK293 cells were in medium with at were transiently by the and the were of stably cells expressing the or mutant receptors were in the presence of of to to the of cDNA into the were by cell using in is as the the to the cells in the and presence of HEK293 cells stably expressing the receptor in the vector were purchased from and stably the or mutant receptors were in Ref. Maestri-El Kouhen O. J. Wang W. Loh H.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google or ponasterone A used to of protein for the of protein for the or of protein for the of protein for the receptor were HEK293 cells in were transiently with of or mutant receptor as The cells were and in were the following The cells were with DPDPE for to and were with the same of The cAMP was by cAMP and antibodies that recognize the acetylated as in Ref. 10El Kouhen R. Maestri-El Kouhen O. Law P.-Y. Loh H.H. J. Biol. Chem. 1999; 274: 9207-9215Abstract Full Text Full Text PDF PubMed Scopus (49) Google The of cAMP in was by the of the to for cAMP to the antibodies with that of of acetylated or stably HEK293 cells were in and proteins were as in Ref. 10El Kouhen R. Maestri-El Kouhen O. Law P.-Y. Loh H.H. J. Biol. Chem. 1999; 274: 9207-9215Abstract Full Text Full Text PDF PubMed Scopus (49) Google DOR and mutant receptors were using the and a of protein at proteins were from the by of and The were at for and on a proteins were and by using the system and were with the antibodies to the was using the system Pharmacia were by of in the HEK293 cells expressing or mutant epitope-tagged receptors were with DPDPE for the were on to membrane and receptors were by using a of the mouse CA) and a of the antibodies with were at receptor of cells was using CA). were using the from were by a of with subsequent from by investigate the in phosphorylation of HEK293 cells expressing the receptor were with [32P]orthophosphate with a of and receptors were and by in these was In presence of phosphorylation of the receptor was with the phosphorylation in of agonist and a at corresponding to that of the epitope-tagged DOR Phosphorylation of this protein was and not and the phosphorylation were in HEK293 cells transiently or stably expressing the receptor and were in with an molecular corresponding to the receptor with of DOR J.L. Arvidson U. Williams F.G. Law P.-Y. Elde R. Loh H.H. Mol. Brain Res. 1999; 69: 171-185Crossref PubMed Scopus (60) Google Scholar, Devi L.A. 1999; PubMed Scopus Google of mutant δ-opioid receptors transiently with of cDNA were with and with DPDPE for from and mutant is the of a of at receptor proteins by of the corresponding phosphorylated receptor phosphorylated were with and were expressed as of phosphorylation for DOR in presence of the into the receptors was expressed as a of the of receptor present in by of the are phosphorylation of DOR. HEK293 cells stably expressing the or mutant receptors were and the of DPDPE to phosphorylation was as amino acid of the C-tail of DOR. The different Ala or Asp are and the from the receptors were and on a by and of the phosphorylated were expressed as a of DPDPE-induced phosphorylation of the receptor. of at with the phosphorylation of the that agonist-induced phosphorylation of DOR at the carboxyl tail domain (11Zhao J. Pei G. Huang Y.L. Zhong F.M. Ma L. Biochem. Biophys. Res. Commun. 1997; 238: 71-76Crossref PubMed Scopus (36) Google Scholar, 12Murray S.R. Evans C.J. von Zastrow M. J. Biol. Chem. 1998; 273: 24987-24991Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The C-tail of the mouse DOR Ser and Thr phosphorylation sites identify Ser and/or Thr are phosphorylated in to or were substituted into All these mutant receptors were stably expressed in HEK293 cells, and differences were in to the or the agonist with that of the receptor Ala substitution of Ser and Thr at the C-tail of the receptor the DPDPE-induced phosphorylation that the phosphorylation are likely within the of the receptor. We substituted these carboxyl to with the the or mutants did not differences in agonist-induced receptor that of these four are involved in the DPDPE-induced phosphorylation of the receptor In of or Ser363 to Ala the phosphorylation to or as with the that these are involved in the agonist-induced phosphorylation of the receptor or agonist-induced phosphorylation be with the that critical for the phosphorylation of DOR. The of the DPDPE-induced phosphorylation of the receptor by the and that this is a phosphorylation or is a kinase site. The phosphorylation of this was demonstrated by the mutant receptor in the at the except Ser363, were substituted to Ala This mutant showed a agonist-induced phosphorylation and of that of the receptor. This showed that Ser363 is the primary phosphorylation and that phosphorylation of DOR be with Ser363 being the first to be of the δ-opioid receptor and mutant receptors phosphorylation were on membrane as in Ref. R. Loh H.H. Law J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google of were by and for the of receptor in the for the were by from and were by using the are of at two and mutant receptors have for and and of the at the C-tail did not affect the of receptors in the in a were on membrane as in Ref. R. Loh H.H. Law J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google of were by and for the of receptor in the for the were by from and were by using the are of at two and mutant receptors have for and and of the at the C-tail did not affect the of receptors in the In order to identify the other phosphorylation Ser363 was to aspartic acid a phosphorylated of this and a of the receptor the mechanism is hierarchical. in the S363D mutant the DPDPE-induced phosphorylation with the to of that of the receptor. This that Ser363 has to be phosphorylated and that Thr358 and/or is the other phosphorylation be with a mutant only as a potential phosphorylation with the Ser363 that is not phosphorylated Ser363, but Thr358 the of phosphorylation in the mutant is not different from the of phosphorylation in the mutant Ser363 as the only potential phosphorylation the of phosphorylation in the S363D mutant only of that of the that only one is phosphorylated with Ser363 and that this other is the the agonist-induced phosphorylation of the receptor. in the phosphorylation of the receptor as a kinase but not as a phosphorylation site. The was constructed to the phosphorylation mechanism of but not of the phosphorylation as with that of the affect to a the to the receptor. This Ser363 was into Ala with the substitution with the critical role of Ser363 in the phosphorylation of the receptor. we the role of phosphorylation in the agonist-induced desensitization of DOR in HEK293 cells. The of the loss of DPDPE inhibition of the adenylyl cyclase were and the rate of desensitization was with at of not with Kouhen R. Maestri-El Kouhen O. Law P.-Y. Loh H.H. J. Biol. Chem. 1999; 274: 9207-9215Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In DPDPE-induced phosphorylation of the receptor a within of DPDPE exposure not The in these two that receptor phosphorylation not to DOR The to phosphorylation to desensitization of DOR be to the of receptor expression of in HEK293 cells. In an study, by the expression of the receptor in HEK293 cells with an expression system Maestri-El Kouhen O. J. Wang W. Loh H.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we were to that the rate of DOR desensitization is on the receptor expressed at the cell the same system to the expression of the and mutant in HEK293 cells we that receptor desensitization in the presence of DPDPE were receptor of receptor expression of the desensitization of receptors were and suggesting that the of receptor phosphorylation did not the rate of were the receptor and a mutant receptor of being phosphorylated were transiently expressed in HEK293 cells not of expression of the receptor of receptor was the DPDPE-induced loss of was and the receptor following DPDPE The of receptor phosphorylation the of desensitization, expressed at as the receptor. following agonist of the the phosphorylation of DOR at Ser363 is an but not in the DPDPE-induced internalization is involved in the desensitization of several J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (850) Google Scholar), and in Maestri-El Kouhen O. J. Wang W. Loh H.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) we demonstrated that receptor internalization also in the rapid desensitization of we the DPDPE-induced phosphorylation of Thr358 and Ser363 in the agonist-induced receptor internalization. The of internalization of the and mutant receptors from the cell were using that DOR in presence of DPDPE and that of cell receptor were in to agonist with at This rate with (12Murray S.R. Evans C.J. von Zastrow M. J. Biol. Chem. 1998; 273: 24987-24991Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 17Trapaidze N. Keith D.E. Cvejic S. Evans C.J. Devi L.A. J. Biol. Chem. 1996; 271: 29279-29285Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, Maestri-El Kouhen O. J. Wang W. Loh H.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the four first Ser or Thr at the C-tail into Ala to did not the rate or the of internalization of the corresponding activated that of these four are involved in the DPDPE-induced internalization of the receptor in HEK293 cells In the mutant as as the the DPDPE-induced phosphorylation of the a rate and of internalization, suggesting that phosphorylation of Ser363 and/or to the receptor internalization and Phosphorylation of Ser363 the receptor from as as the suggesting that the phosphorylation of this Ser only is to receptor internalization. Ala substitution of Thr358 also the agonist-induced internalization of the receptor. that phosphorylation of Thr358 is critical for receptor internalization. the S363D mutant the DPDPE-induced phosphorylation of a of receptor internalization to the receptor. Therefore, internalization of DOR the phosphorylation of subsequent to the phosphorylation of of the and mutant receptors in HEK293 of internalization of the and mutant receptors were by one using the internalization the using the receptors be into two internalization to that of the receptor. The first receptors as of and the receptors a rate and/or of internalization. as to the receptor. in a The of internalization of the and mutant receptors were by one using the internalization the using the receptors be into two internalization to that of the receptor. The first receptors as of and the receptors a rate and/or of internalization. as to the receptor. mutagenesis it is that Ser363 is the primary phosphorylation that agonist-induced phosphorylation of DOR in a and that Thr358 and Ser363 are the only two being in receptor substitution into Ala the of phosphorylation with the receptor. the phosphorylation with the mutant from Ser363, Ser363 is phosphorylated of present at the C-tail of the receptor of the of a kinase to the receptor phosphorylation phosphorylation has been demonstrated for only a including the and the receptor H. K. PubMed Scopus Google Scholar, Benovic J.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). this phosphorylation mechanism is of the or to of to be is that phosphorylation of Ser363 a for the subsequent phosphorylation of Thr358 by the same or by have that of the DOR (6Pei G. Kieffer B.L. Lefkowitz R.J. Freedman N.J. Mol. Pharmacol. 1995; 48: 173-177PubMed Google Scholar, A. J. S. L. D. P. J. Neurochem. 1998; PubMed Scopus Google Scholar). Although the of has not been to an or amino acid Lefkowitz R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Ser363 and Thr358 are of and residues, Ser363 is from an aspartic as a However, Thr358 is from an Ala phosphorylation of Ser363 and Thr358 involved a to be that agonist-induced phosphorylation of the opioid receptors be mediated by protein kinase P. V. J. Neurochem. 1997; 69: PubMed Scopus Google Scholar), or by protein kinase S.M. V. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), but the amino acid Thr358 or Ser363 not to that of it is to that a yet kinase is for the DPDPE-induced phosphorylation of DOR. The of this kinase be one of the of a mutant of not the agonist-induced phosphorylation of the receptor (6Pei G. Kieffer B.L. Lefkowitz R.J. Freedman N.J. Mol. Pharmacol. 1995; 48: 173-177PubMed Google Scholar, 10El Kouhen R. Maestri-El Kouhen O. Law P.-Y. Loh H.H. J. Biol. Chem. 1999; 274: 9207-9215Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The rapid and desensitization of DOR is on the of expression of the receptor. The rate of a loss of DPDPE inhibition of adenylyl cyclase in cells expressing a of receptors not with the rapid DPDPE-induced phosphorylation of the receptor not The of receptor expressed at the cell that is not phosphorylated and to the activity, with the DOR and the adenylyl cyclase R. D. Mol. Pharmacol. Google Scholar), the phosphorylation of the receptor did not with the loss of The that the mutants agonist-induced as and mutants not and a rate and of desensitization as the receptor at receptor that other receptor be involved in the desensitization of the receptor. of receptor to the of expression in expressing cells, the of the agonist-induced receptor phosphorylation the of rapid desensitization, that the receptor phosphorylation at Ser363 in receptor the of the receptor to still that the agonist-induced internalization also to the loss of as it was for other for for the receptor J. Williams Mol. Pharmacol. 1995; 48: Google Scholar) or the receptor Y. A. J. S.R. Mol. Pharmacol. 1996; Google Scholar, R. L. J. Wang W. Loh H.H. Mol. Pharmacol. PubMed Scopus Google Scholar). We reported at expression of and δ-opioid a of receptor internalization to a of the agonist-induced rapid desensitization of these receptors Maestri-El Kouhen O. J. Wang W. Loh H.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Y. A. J. S.R. Mol. Pharmacol. 1996; Google Scholar). of Thr358 the agonist-induced internalization of that phosphorylation of this is for receptor internalization and to the DPDPE-induced rapid of DOR von Zastrow M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. L. D. G. J. P. J. Pharmacol. Google Scholar), receptor and likely to the loss of receptor activity, of or not the receptor is phosphorylated has been reported to be for the agonist-induced of DOR in CHO cells S. N. C. Devi L.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the DPDPE-induced phosphorylation of DOR in HEK293 cells not involve the of the receptor to not be by the of the receptor to be for the of Ser363 to Ala agonist-induced being an agonist-induced phosphorylation R. Loh H.H. Law J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), that phosphorylation of a is not a for the of receptor at the or not is involved in DPDPE-induced of the receptor in HEK293 cells to be a truncated δ-opioid receptor predominantly in the plasma membrane of CHO cells, still in HEK293 cells (12Murray S.R. Evans C.J. von Zastrow M. J. Biol. Chem. 1998; 273: 24987-24991Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). a to including the opioid receptors W. H. Z. J. 1996; PubMed Scopus Google Scholar), present at the the and the C-tail of the or a J. C. V. L. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar) present within the domain of be in the of the receptor. We that phosphorylation of Ser363 the of the activated receptor from G phosphorylation of Thr358 internalization of the receptor-mediated signaling. Therefore, of receptor and G by the to opioid or drugs are to in the Therefore, opioid receptors may this regulation mechanism to the same mechanism in in vitro cell or in to be We Kouhen for

Suberin acts as stress-induced antipathogen barrier in the root cell wall. CYP86A1 encodes cytochrome P450 fatty acid ω-hydroxylase, which has been reported to be a key enzyme for suberin biosynthesis; however, its role in resistance to fungi and the mechanisms related to immune responses remain unknown. Here, we identified a disease resistance-related gene, GbCYP86A1-1, from Gossypium barbadense cv. Hai7124. There were three homologs of GbCYP86A1 in cotton, which are specifically expressed in roots and induced by Verticillium dahliae. Among them, GbCYP86A1-1 contributed the most significantly to resistance. Silencing of GbCYP86A1-1 in Hai7124 resulted in severely compromised resistance to V. dahliae, while heterologous overexpression of GbCYP86A1-1 in Arabidopsis improved tolerance. Tissue sections showed that the roots of GbCYP86A1-1 transgenic Arabidopsis had more suberin accumulation and significantly higher C16-C18 fatty acid content than control. Transcriptome analysis revealed that overexpression of GbCYP86A1-1 not only affected lipid biosynthesis in roots, but also activated the disease-resistant immune pathway; genes encoding the receptor-like kinases (RLKs), receptor-like proteins (RLPs), hormone-related transcription factors, and pathogenesis-related protein genes (PRs) were more highly expressed in the GbCYP86A1-1 transgenic line than control. Furthermore, we found that when comparing V. dahliae -inoculated and noninoculated plants, few differential genes related to disease immunity were detected in the GbCYP86A1-1 transgenic line; however, a large number of resistance genes were activated in the control. This study highlights the role of GbCYP86A1-1 in the defence against fungi and its underlying molecular immune mechanisms in this process.

We present an integrative machine learning method, incRNA, for whole-genome identification of noncoding RNAs (ncRNAs). It combines a large amount of expression data, RNA secondary-structure stability, and evolutionary conservation at the protein and nucleic-acid level. Using the incRNA model and data from the modENCODE consortium, we are able to separate known C. elegans ncRNAs from coding sequences and other genomic elements with a high level of accuracy (97% AUC on an independent validation set), and find more than 7000 novel ncRNA candidates, among which more than 1000 are located in the intergenic regions of C. elegans genome. Based on the validation set, we estimate that 91% of the approximately 7000 novel ncRNA candidates are true positives. We then analyze 15 novel ncRNA candidates by RT-PCR, detecting the expression for 14. In addition, we characterize the properties of all the novel ncRNA candidates and find that they have distinct expression patterns across developmental stages and tend to use novel RNA structural families. We also find that they are often targeted by specific transcription factors (∼59% of intergenic novel ncRNA candidates). Overall, our study identifies many new potential ncRNAs in C. elegans and provides a method that can be adapted to other organisms.