RESUMO
With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.
Assuntos
Encéfalo/metabolismo , Mapas de Interação de Proteínas/fisiologia , Receptores Opioides delta/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Técnicas de Introdução de Genes , Genes Reporter/genética , Masculino , Camundongos , Camundongos Transgênicos , Dobramento de Proteína , Mapeamento de Interação de Proteínas/métodos , Proteômica , Receptores Opioides delta/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas em TandemRESUMO
Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1-/- mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity.
Assuntos
Encefalomielite Autoimune Experimental/etiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Adulto , Animais , Astrócitos/fisiologia , Estudos de Casos e Controles , Sistema Nervoso Central/patologia , Códon sem Sentido , Doenças Desmielinizantes , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Imunidade Inata , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Adulto JovemRESUMO
OBJECTIVE: To evaluate the effect of intrauterine administration of activated peripheral blood mononuclear cells (PBMC) on intrauterine insemination (IUI) success rates. METHODS: This prospective double-blind randomized parallel clinical trial included 213 patients undergoing IUI at the Fertilys clinic. PBMC were isolated on the day of ovulation (day 0; D0) and stimulated with phytohemagglutinin (PHA) and human chorionic gonadotropin (hCG) for 48 hours (day 2; D2). Patients in the PBMC group (n = 108) underwent in utero administration of 1.106 cells on D2, while patients in the control group (n = 105) were administered sperm-washing medium. Distribution of CD4 T lymphocyte populations (n = 61) was assessed on D0 and D2. Pregnancy and live birth rates were also evaluated. RESULTS: Demographic and clinical characteristics, pregnancy rates, and live birth rates were not significantly different between the PBMC and control groups. Significantly higher levels of T helper (Th) 2, Th22, and T regulatory cells (P < 0.0001) and lower levels of Th17 cells were observed in hCG-activated PBMC at D2 than at D0. CONCLUSION: Intrauterine administration of PBMC was not beneficial in IUI patients. New clinical approaches to better identify patients requiring endometrium immunomodulation needs to be addressed.
Assuntos
Fertilização in vitro , Leucócitos Mononucleares , Gonadotropina Coriônica , Feminino , Humanos , Inseminação , Masculino , Indução da Ovulação , Gravidez , Taxa de Gravidez , Estudos ProspectivosRESUMO
The use of opioids for the relief of pain and headache disorders has been studied for years. Nowadays, particularly because of its ability to produce analgesia in various pain models, delta opioid receptor (DOPr) emerges as a promising target for the development of new pain therapies. Indeed, their potential to avoid the unwanted effects commonly observed with clinically used opioids acting at the mu opioid receptor (MOPr) suggests that DOPr agonists could be a therapeutic option. In this review, we discuss the use of opioids in the management of pain in addition to describing the evidence of the analgesic potency of DOPr agonists in animal models.
Assuntos
Dor Aguda , Analgésicos Opioides/farmacologia , Dor do Câncer , Dor Crônica , Transtornos de Enxaqueca , Neuralgia , Receptores Opioides delta , Dor Aguda/tratamento farmacológico , Dor Aguda/metabolismo , Animais , Dor do Câncer/tratamento farmacológico , Dor do Câncer/metabolismo , Dor Crônica/tratamento farmacológico , Dor Crônica/metabolismo , Humanos , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismoRESUMO
In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1-4] (i.e. Dmt-d-Ala-Phe-GlyNH2, Dmtâ¯=â¯2',6'-dimethyl-(S)-tyrosine) for the µ opioid receptor (MOP) and δ opioid receptor (DOP) were evaluated using radioligand binding studies, functional cell-based assays and isolated organ bath experiments. By means of solid-phase or solution-phase Suzuki-Miyaura cross-couplings, various substituted regioisomers of the phenylalanine moiety in position 3 of the sequence were prepared. An 18-membered library of opioid tetrapeptides was generated via screening of the chemical space around the Phe3 side chain. These substitutions modulated bioactivity, receptor subtype selectivity and highly effective ligands with subnanomolar binding affinities, contributed to higher functional activities and potent analgesic actions. In search of selective peptidic ligands, we show here that the Suzuki-Miyaura reaction is a versatile and robust tool which could also be deployed elsewhere.
Assuntos
Analgésicos Opioides/uso terapêutico , Oligopeptídeos/uso terapêutico , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Analgésicos Opioides/síntese química , Analgésicos Opioides/química , Analgésicos Opioides/farmacologia , Animais , Cobaias , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ratos Sprague-DawleyRESUMO
The delta opioid receptor (DOPr) is known to be mainly expressed in intracellular compartments. It remains unknown why DOPr is barely exported to the cell surface, but it seems that a substantial proportion of the immature receptor is trapped within the endoplasmic reticulum (ER) and the Golgi network. In the present study, we performed LC-MS/MS analysis to identify putative protein partners involved in the retention of DOPr. Analysis of the proteins co-immunoprecipitating with Flag-DOPr in transfected HEK293 cells revealed the presence of numerous subunits of the coatomer protein complex I (COPI), a vesicle-coating complex involved in recycling resident proteins from the Golgi back to the ER. Further analysis of the amino acid sequence of DOPr identified multiple consensus di-lysine and di-arginine motifs within the intracellular segments of DOPr. Using cell-surface ELISA and GST pulldown assays, we showed that DOPr interacts with COPI through its intracellular loops 2 and 3 (ICL2 and ICL3, respectively) and that the mutation of the K164AK166 (ICL2) or K250EK252 (ICL3) putative COPI binding sites increased the cell-surface expression of DOPr in transfected cells. Altogether, our results indicate that COPI is a binding partner of DOPr and provide a putative mechanism to explain why DOPr is highly retained inside the cells.
Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Sinais Direcionadores de Proteínas , Receptores Opioides delta/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico , Receptores Opioides delta/químicaRESUMO
During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K(38)KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7's N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by â¼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.
Assuntos
Caspase 7/química , Caspase 7/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteólise , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoptose , Caspase 3/metabolismo , Linhagem Celular , Humanos , Oxirredutases Intramoleculares/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Prostaglandina-E Sintases , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.
Assuntos
Caspases/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Endopeptidases/metabolismo , Células HeLa , Humanos , RatosRESUMO
Caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.
Assuntos
Caspase 8/metabolismo , Apoptose , Sítios de Ligação , Caspases/metabolismo , Domínio Catalítico , Linhagem Celular , Dimerização , Endopeptidases/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Células Jurkat , Modelos Biológicos , Mutação , Estrutura Terciária de ProteínaRESUMO
We have cloned and sequenced a cDNA from the surf clam (Spisula solidissima, a pelecypod mollusc) that encodes an octopamine receptor which we have named Spi-OAR. The sequence of Spi-OAR shares many similarities with two Aplysia and three Drosophila octopamine receptors belonging to a sub-group of beta-adrenergic-like octopamine receptors. Using an expression vector and transient transfections of Spi-OAR into HEK 293 cells, we observed an increase of cAMP upon addition of octopamine and, to a lesser extent, of tyramine, but not after addition of dopamine, serotonin, or histamine. Using a battery of known agonists and antagonists for octopamine receptors, we observed a rather unique pharmacological profile for Spi-OAR through measurements of cAMP. Spi-OAR exhibited some constitutive activity in HEK 293 cells and no Ca(2+) responses could be detected following addition of octopamine to Spi-OAR-transfected cells. RT-PCR analysis revealed ubiquitous expression of Spi-OAR mRNA in all adult tissues, oocytes and early embryos examined. While addition of serotonin to isolated clam oocytes resulted in meiotic activation, similar additions of octopamine had no effect, suggesting that its potential role in clam reproductive physiology differs significantly from that of serotonin. This work identifies Spi-OAR as a novel mollusc octopamine receptor closely related to other invertebrate beta-adrenergic-like octopamine receptors, with possible reproductive and other physiological functions. This initial characterization of Spi-OAR makes possible further investigations and comparisons with more studied and familiar insect or gastropod mollusc octopamine receptors.
Assuntos
Bivalves/genética , Receptores de Amina Biogênica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Aminas Biogênicas/farmacologia , Bivalves/metabolismo , Linhagem Celular , Clonagem Molecular , AMP Cíclico/metabolismo , DNA Complementar/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Octopamina/farmacologia , Filogenia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Amina Biogênica/agonistas , Receptores de Amina Biogênica/antagonistas & inibidores , Receptores de Amina Biogênica/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , TransfecçãoRESUMO
Nowadays, pain represents one of the most important societal burdens. Current treatments are, however, too often ineffective and/or accompanied by debilitating unwanted effects for patients dealing with chronic pain. Indeed, the prototypical opioid morphine, as many other strong analgesics, shows harmful unwanted effects including respiratory depression and constipation, and also produces tolerance, physical dependence, and addiction. The urgency to develop novel treatments against pain while minimizing adverse effects is therefore crucial. Over the years, the delta-opioid receptor (DOP) has emerged as a promising target for the development of new pain therapies. Indeed, targeting DOP to treat chronic pain represents a timely alternative to existing drugs, given the weak unwanted effects spectrum of DOP agonists. Here, we review the current knowledge supporting a role for DOP and its agonists for the treatment of pain. More specifically, we will focus on the cellular and subcellular localization of DOP in the nervous system. We will also discuss in further detail the molecular and cellular mechanisms involved in controlling the cellular trafficking of DOP, known to differ significantly from most G protein-coupled receptors. This review article will allow a better understanding of how DOP represents a promising target to develop new treatments for pain management as well as where we stand as of our ability to control its cellular trafficking and cell surface expression.
RESUMO
Opioid and neurotensin (NT) receptors are expressed in both central and peripheral nervous systems where they modulate nociceptive responses. Nowadays, opioid analgesics like morphine remain the most prescribed drugs for the treatment of moderate to severe pain. However, despite their daily used, opioids can produce life-threatening side effects, such as constipation or respiratory depression. Besides, NT analogs exert strong opioid-independent analgesia. Here, we thus hypothesized that the combined use of opioid and NT agonists would require lower doses to produce significant analgesic effects, hence decreasing opioid-induced adverse effects. We used isobologram analyses to determine if the combination of a NT brain-penetrant analog, An2-NT(8-13) with morphine results in an inhibitory, synergistic or additive analgesic response. We found that intravenous administration of An2-NT(8-13) reduced by 90% the nocifensive behaviors induced by formalin injection, at the dose of 0.018â¯mg/kg. Likewise, subcutaneous morphine reduced pain by 90% at 1.8â¯mg/kg. Importantly, isobologram analyses revealed that the co-injection of An2-NT(8-13) with morphine induced an additive analgesic response. We finally assessed the effects of morphine and An2-NT(8-13) on the gastrointestinal tract motility using the charcoal meal test. As opposed to morphine which significantly reduced the intestinal motility at the analgesic effective dose of 1.8â¯mg/kg, An2-NT(8-13) did not affect the charcoal meal intestinal transit at 0.018â¯mg/kg. Interestingly, at the dose providing 90% pain relief, the co-administration of morphine with An2-NT(8-13) had a reduced effect on constipation. Altogether, these results suggest that combining NT agonists with morphine may improve its analgesic/adverse effect ratio.
Assuntos
Analgésicos Opioides/administração & dosagem , Neurotensina/administração & dosagem , Medição da Dor/efeitos dos fármacos , Fragmentos de Peptídeos/administração & dosagem , Peptídeos/administração & dosagem , Receptores de Neurotensina/agonistas , Receptores Opioides mu/agonistas , Animais , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Masculino , Morfina/administração & dosagem , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Receptores de Neurotensina/metabolismo , Receptores Opioides mu/metabolismoRESUMO
Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of ß-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.
Assuntos
Encefalina Leucina/análogos & derivados , Oligopeptídeos/farmacologia , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Animais , Células HEK293 , Humanos , Peptídeos Opioides/efeitos dos fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismoRESUMO
Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to µ-opioid receptor (MOP) agonists that are in the spotlight of the current "opioid crisis." Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects.
Assuntos
Simulação de Acoplamento Molecular , Peptídeos/química , Receptores Opioides delta/agonistas , Receptores Opioides delta/química , Animais , Cristalografia por Raios X , Humanos , Domínios Proteicos , Receptores Opioides mu/agonistas , Receptores Opioides mu/química , Células Sf9 , SpodopteraRESUMO
Genetically-modified animal models have significantly increased our understanding of the complex central nervous system circuits. Among these models, inducible transgenic mice whose specific gene expression can be modulated through a Cre recombinase/LoxP system are useful to study the role of specific peptides and proteins in a given population of cells. In the present study, we describe an efficient approach to selectively deliver a Cre-GFP to dorsal root ganglia (DRG) neurons. First, mice of different ages were injected in both hindpaws with a recombinant adeno-associated virus (rAAV2/9-CBA-Cre-GFP). Using this route of injection in mice at 5 days of age, we report that approximately 20% of all DRG neurons express GFP, 6 to 8 weeks after the infection. The level of infection was reduced by 50% when the virus was administered at 2 weeks of age. Additionally, the virus-mediated delivery of the Cre-GFP was also investigated via the intrathecal route. When injected intrathecally, the rAAV2/9-CBA-Cre-GFP virus infected a much higher proportion of DRG neurons than the intraplantar injection, with up to 51.6% of infected lumbar DRG neurons. Noteworthy, both routes of injection predominantly transduced DRG neurons over spinal and brain neurons.
Assuntos
Dependovirus/fisiologia , Gânglios Espinais/citologia , Integrases/metabolismo , Transdução Genética/métodos , Animais , DNA Recombinante/genética , Dependovirus/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Camundongos , Neurônios/metabolismoRESUMO
Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex. In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function. Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo. We show that recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro. By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1. Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo. Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells. The recombination defect is rescued by expression of a bacterial Holliday junction resolvase. These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Mitose/genética , Interferência de RNA , Recombinação Genética/genética , Proteínas de Schizosaccharomyces pombe/genética , Sequência de Aminoácidos , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Sobrevivência Celular/fisiologia , Clonagem Molecular , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/genética , Células HeLa , Resolvases de Junção Holliday/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Bloom syndrome is a rare, autosomal recessive inherited disorder in humans. The product of the Bloom syndrome mutated gene, designated BLM, is a member of the RecQ helicase family. BLM has been proposed to function at the interface of replication and recombination, and to facilitate the repair of DNA damage. Here, we report in vivo physical interaction and colocalization of BLM and a DNA structure-specific endonuclease, Mus81, at sites of stalled replication forks outside the promyelocytic leukemia nuclear bodies during the S-phase arrest of the cell cycle. Amino acids 125 to 244 of Mus81 interact with the C-terminal region (amino acids 1,007-1,417) of BLM. Whereas Mus81 does not have any effect on the helicase activity of BLM, BLM can stimulate Mus81 endonuclease activity on the nicked Holliday junctions and 3' flap. This stimulation is due to enhanced binding of Mus81 to the DNA substrates. These data suggest a new function of BLM in cooperating with Mus81 during processing and restoration of stalled replication forks.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Adenosina Trifosfatases/genética , Sítios de Ligação , Linhagem Celular , DNA/biossíntese , DNA/metabolismo , DNA Helicases/genética , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Fibroblastos/enzimologia , Células HCT116 , Humanos , Mapeamento de Peptídeos , RecQ Helicases , TransfecçãoRESUMO
A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CFâCH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [125I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cells. Together with our previous observations, our findings strongly suggest that the third amide bond of Leu-enkephalin primarily acts as a hydrogen bond acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide, or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding analogs. The lipophilicity (LogD7.4) of the active analog was also measured. It appears that fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.
Assuntos
Ligação Competitiva/efeitos dos fármacos , Encefalina Leucina/química , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/farmacologia , Peptidomiméticos/síntese química , Receptores Opioides delta/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Dipeptídeos/química , Encefalina Leucina/análogos & derivados , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos , Peptidomiméticos/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Receptores Opioides delta/genética , Transfecção , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismoRESUMO
Given the putative selectivity of the antagonist TIPP (Tyr-Tic-Phe-Phe) for δ-opioid receptors (DOP), this compound was selected for the design of a novel 64Cu-radiolabeled potent and selective DOP positron emission tomography (PET) imaging agent. Ex vivo autoradiography of TIPPD-PEG-K(NOTA/64Cu)-NH2 on rat brain sections produced a distribution pattern consistent with the known expression of DOP. Taken together, the in vitro and ex vivo data indicate that this 64Cu-tracer holds promise for studying the DOP by means of PET.