RESUMO
Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.
Assuntos
Adenosina Trifosfatases , Montagem de Vírus , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/química , Montagem de Vírus/genética , Proteínas Virais/genética , Proteínas Virais/química , Empacotamento do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/química , DNA Viral/genética , DNA Viral/químicaRESUMO
The voltage-gated Na+ channel ß1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. ß1 is cleaved by α/ß and γ-secretases, releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on ß1 function in breast cancer cells. Using a series of GFP-tagged ß1 constructs, we show that ß1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal ß1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report ß1ICD-GFP accumulation in the nucleus. Furthermore, ß1-GFP and ß1ICD-GFP both increased Na+ current, whereas ß1STOP-GFP, which lacks the ICD, did not, thus highlighting that the ß1-ICD is necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Nav1.5), the Na+ current increased by ß1-GFP or ß1ICD-GFP was TTX-sensitive. Finally, we found ß1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Nav1.1 and SCN9A/Nav1.7. Taken together, this work suggests that the ß1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating ß1 function in breast cancer.
Assuntos
Neoplasias da Mama , Canais de Sódio , Secretases da Proteína Precursora do Amiloide/metabolismo , Feminino , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7 , Sódio/metabolismo , Canais de Sódio/metabolismo , Tetrodotoxina/farmacologia , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genéticaRESUMO
Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The "α-oxo-Mannich" and "catalyst-free aldol" ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the "Mannich" pathway at acidic pH to afford bifunctionalized bioconjugates, and the "catalyst-free aldol" pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH's.
Assuntos
Aldeídos/química , Bases de Mannich/química , Proteínas/química , Compostos de Anilina/química , Catálise , Concentração de Íons de Hidrogênio , Peptídeos/químicaRESUMO
Cancer therapies based on in vivo stimulation, or on adoptive T cell transfer of Vγ9Vδ2 T cells, have been tested in the past decades but have failed to provide consistent clinical efficacy. New, promising concepts such as γδ Chimeric Antigen Receptor (CAR) -T cells and γδ T-cell engagers are currently under preclinical evaluation. Since the impact of factors, such as the relatively low abundance of γδ T cells within tumor tissue is still under investigation, it remains to be shown whether these effector T cells can provide significant efficacy against solid tumors. Here, we highlight key learnings from the natural role of Vγ9Vδ2 T cells in the elimination of host cells bearing intracellular bacterial agents and we translate these into the setting of tumor therapy. We discuss the availability and relevance of preclinical models as well as currently available tools and knowledge from a drug development perspective. Finally, we compare advantages and disadvantages of existing therapeutic concepts and propose a role for Vγ9Vδ2 T cells in immune-oncology next to Cluster of Differentiation (CD) 3 activating therapies.
Assuntos
Infecções/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Animais , Plasticidade Celular , Humanos , Neoplasias/patologia , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Psoriasis is a chronic inflammatory disease appearing as scaly erythematous cutaneous lesions, which are characterized by parakeratosis and acanthosis as well as the infiltration of immune cells, such as T helper-1 and T helper-17 cells. Here, we demonstrated that KdPT, a tripeptide structurally related to the C-terminal amino acids of alpha-melanocyte-stimulating hormone, which was previously shown to exhibit anti-inflammatory effects in intestinal inflammation, ameliorated ongoing disease in the mouse model of imiquimod-induced psoriasis-like skin inflammation and in the small xenotransplant mouse model of psoriasis. We could show that systemic KdPT treatment significantly reduced hyperkeratosis and acanthosis in murine as well as human skin. Moreover, KdPT upregulated Foxp3 in CD4+ T cells from mice and from peripheral blood of individuals with psoriasis and decreased the expression of type 1 inflammatory cytokines, indicating that the beneficial effect of KdPT was, at least in part, mediated by the induction of functional regulatory T cells that suppressed the activation of pathogenic CD4+ IFN-γ+ and CD4+ IL-17+ T cells. Thus, these data might suggest KdPT as a potential novel therapeutic alternative for the treatment of psoriasis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Oligopeptídeos/uso terapêutico , Psoríase/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Ceratose/tratamento farmacológico , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/farmacologia , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/patologia , Células Th1/efeitos dos fármacos , Células Th1/patologia , Células Th17/efeitos dos fármacos , Células Th17/patologia , Transplante HeterólogoRESUMO
A nickel-catalyzed alkylation of polycyclic aromatic methyl ethers as well as methyl enol ethers with B-alkyl 9-BBN and trialkylborane reagents that involves the cleavage of stable C(sp2 )-OMe bonds is described. The transformation has a wide substrate scope and good chemoselectivity profile while proceeding under mild reaction conditions; it provides a versatile way to form C(sp2 )-C(sp3 ) bonds that does not suffer from ß-hydride elimination. Furthermore, a selective and sequential alkylation process by cleavage of inert C-O bonds is presented to demonstrate the advantage of this method.
RESUMO
Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of cell-cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-competitive dual inhibitor of MEK and Aurora kinases. Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic and cellular assays. Equipotent effects were observed in BRAF-mutant cells, whereas in KRAS-mutant cells, MEK inhibition required higher concentrations than Aurora kinase inhibition. Daily oral administration of BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models. Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF-mutant models but of Aurora kinase in KRAS-mutant models. Inhibition of both MEK and Aurora kinase in KRAS-mutant tumors was observed when BI 847325 was administered once weekly at 70 mg/kg. Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF and KRAS mutation. These preclinical data are discussed in the light of the results of a recently completed clinical phase I trial assessing safety, tolerability, pharmacokinetics, and efficacy of BI 847325 in patients with cancer. Mol Cancer Ther; 15(10); 2388-98. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Aurora Quinases/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Aurora Quinases/química , Aurora Quinases/metabolismo , Ligação Competitiva , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems.
Assuntos
Imunidade Inata , Lipídeos/imunologia , Animais , Membrana Celular/química , Fibroblastos/metabolismo , Doença de Gaucher/imunologia , Humanos , Interleucina-6/imunologia , Leucodistrofia de Células Globoides/imunologia , Redes e Vias Metabólicas , Camundongos , Esfingolipídeos/metabolismo , Receptores Toll-Like/imunologiaRESUMO
Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Imunidade Inata/genética , Inflamação/genética , Peritonite/genética , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/imunologia , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Inflamação/patologia , Lipídeos/imunologia , Macrófagos/imunologia , Camundongos , Peritonite/imunologia , Peritonite/patologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.
Assuntos
Proteínas de Bactérias/metabolismo , Endocitose , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Interações Hospedeiro-Patógeno , Microtúbulos/metabolismo , Pseudomonas aeruginosa/fisiologia , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Sistemas de Secreção Tipo VI , Fatores de Virulência/metabolismoRESUMO
The application of cyclic and acyclic enol ethers as electrophiles in cross coupling reactions offers new possibilities for the preparation of functional compounds. A novel nickel catalyzed dealkoxylative cross coupling reaction allows access to structurally diverse allylsilanes and alcohol derivatives with high stereospecificity and in good yields under mild reaction conditions directly from the corresponding enol ethers.
RESUMO
C-modified 7-deazaadenosines containing a diphenylacetylene moiety have been synthesised using cross-coupling approaches. The C-modified nucleosides exhibit remarkable fluorescence properties, including high quantum yields. Solvatochromic studies show a near linear correlation between the Stokes shift and solvent polarity which is indicative of intramolecular charge transfer. DFT calculations have allowed us to correlate the experimentally observed photophysical properties with the calculated HOMO-LUMO energy gaps within a series of real and model compounds.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Tubercidina/química , Tubercidina/síntese química , Técnicas de Química Sintética , Transporte de Elétrons , Modelos Moleculares , Conformação Molecular , Teoria QuânticaRESUMO
Recognition of pathogens by the innate immune system requires proteins that detect conserved molecular patterns. Nucleic acids are recognized by cytoplasmic sensors as well as by endosomal Toll-like receptors (TLRs). It has become evident that TLRs require additional proteins to be activated by their respective ligands. In this study, we show that CD14 (cluster of differentiation 14) constitutively interacts with the MyD88-dependent TLR7 and TLR9. CD14 was necessary for TLR7- and TLR9-dependent induction of proinflammatory cytokines in vitro and for TLR9-dependent innate immune responses in mice. CD14 associated with TLR9 stimulatory DNA in precipitation experiments and confocal imaging. The absence of CD14 led to reduced nucleic acid uptake in macrophages. Additionally, CD14 played a role in the stimulation of TLRs by viruses. Using various types of vesicular stomatitis virus, we showed that CD14 is dispensable for viral uptake but is required for the triggering of TLR-dependent cytokine responses. These data show that CD14 has a dual role in nucleic acid-mediated TLR activation: it promotes the selective uptake of nucleic acids, and it acts as a coreceptor for endosomal TLR activation.
Assuntos
Receptores de Lipopolissacarídeos/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptor 7 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia , Aminoquinolinas/farmacologia , Animais , Sequência de Bases , Células Cultivadas , Endossomos/metabolismo , Feminino , Humanos , Imiquimode , Vírus da Influenza A/imunologia , Interleucina-6/genética , Receptores de Lipopolissacarídeos/análise , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/farmacologia , Proteômica , Receptor 7 Toll-Like/análise , Receptor Toll-Like 9/análiseRESUMO
Pd/Cu-mediated direct arylation of 2'-deoxyadenosine with various aryl iodides provides 8-arylated 2'-deoxyadenosine derivatives in good yields. Following significant reaction optimization, it has been determined that a substoichiometric quantity of piperidine (secondary amine) in combination with cesium carbonate is necessary for effective direct arylation. The general synthetic protocol allows lower temperature direct arylations, which minimizes deglycosylation. The origin of the piperidine effect primarily derives from the in situ generation of Pd(OAc)(2)[(CH(2))(5)NH](2). Various copper(I) salts have been evaluated; only CuI provides good yields of the 8-arylated-2'-deoxyadenosines. Copper(I) appears to have a high binding affinity for 2'-deoxyadenosine, which explains the mandatory requirement for stoichiometric amounts of this key component. The conditions are compared with more general direct arylation protocols, e.g., catalytic Pd, ligand, acid additives, which do not employ copper(I). In each case, no detectable arylation of 2'-deoxyadenosine was noted. The conformational preferences of the 8-aryl-2'-deoxyadenosine products have been determined by detailed spectroscopic (NMR) and single crystal X-ray diffraction studies. Almost exclusively, the preferred solution-state conformation was determined to be syn-C2'-endo (ca. 80%). The presence of a 2-pyridyl group at the 8-position further biases the solution-state equilibrium toward this conformer (ca. 88%), due to an additional H-bond between H1' and the pyridyl nitrogen atom. The Pd/Cu catalyst system has been found to be unique for adenosine type substrates, the reactivity of which has been placed into context with the reported direct arylations of related 1H-imidazoles. The reactivity of other purine nucleosides has been assessed, which has revealed that both 2'-deoxyguanosine and guanosine are incompatible with the Pd/Cu-direct arylation conditions. Both substrates appear to hinder catalysis, akin to the established inhibitory effects in Suzuki cross-couplings with arylboronic acids.
Assuntos
Cobre/química , Desoxiadenosinas/química , Paládio/química , Aminas/química , Catálise , Cristalografia por Raios X , Iodobenzenos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação MolecularRESUMO
This review examines the 'Quiet Embryo Hypothesis' which proposes that viable preimplantation embryos operate at metabolite or nutrient turnover rates distributed within lower ranges than those of their less viable counterparts. The 'quieter' metabolism consistent with this hypothesis is considered in terms of (i) 'functional' quietness; the contrasting levels of intrinsic metabolic activity in different cell types as a consequence of their specialized functions, (ii) inter-individual embryo/cell differences in metabolism and (iii) loss of quietness in response to environmental stress. Data are reviewed which indicate that gametes and early embryos function in vivo at a lower temperature than core body temperature, which could encourage the expression of a quiet metabolism. We call for research to determine the optimum temperature for mammalian gamete/embryo culture. The review concludes by examining the key role of reactive oxygen species, which can induce molecular damage, trigger a cellular stress response and lead to a loss of quietness.
Assuntos
Blastocisto/metabolismo , Sobrevivência Celular , Técnicas de Cultura Embrionária , Metabolismo Energético , Trifosfato de Adenosina/metabolismo , Animais , Temperatura Corporal , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
We identify PICH (Plk1-interacting checkpoint "helicase"), a member of the SNF2 ATPase family, as an interaction partner and substrate of Plk1. Following phosphorylation of PICH on the Cdk1 site T1063, Plk1 is recruited to PICH and controls its localization. Starting in prometaphase, PICH accumulates at kinetochores and inner centromeres. Moreover, it decorates threads that form during metaphase before increasing in length and progressively diminishing during anaphase. PICH-positive threads connect sister kinetochores and are dependent on tension, sensitive to DNase, and exacerbated in response to premature loss of cohesins or inhibition of topoisomerase II, suggesting that they represent stretched centromeric chromatin. Depletion of PICH causes the selective loss of Mad2 from kinetochores and completely abrogates the spindle checkpoint, resulting in massive chromosome missegregation. These data identify PICH as a novel essential component of checkpoint signaling. We propose that PICH binds to catenated centromere-related DNA to monitor tension developing between sister kinetochores.