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1.
Cell ; 135(1): 49-60, 2008 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-18854154

RESUMEN

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.


Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas/metabolismo , Replicación Viral , Línea Celular , Humanos , Interferencia de ARN , Técnicas del Sistema de Dos Híbridos
2.
Int J Mol Sci ; 20(2)2019 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-30669362

RESUMEN

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.


Asunto(s)
Alarminas/metabolismo , Calreticulina/metabolismo , Lipopolisacáridos/metabolismo , Alarminas/química , Animales , Calreticulina/química , Cromatografía en Gel , Endotoxinas/metabolismo , Humanos , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
3.
PLoS Pathog ; 12(3): e1005466, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27031835

RESUMEN

Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.


Asunto(s)
Infecciones por Alphavirus/genética , Alphavirus/genética , Movimiento Celular/genética , ARN Interferente Pequeño/genética , Actinas/metabolismo , Alphavirus/metabolismo , Infecciones por Alphavirus/metabolismo , Movimiento Celular/fisiología , Replicación del ADN/genética , Humanos , Transporte de Proteínas/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismo
4.
BMC Microbiol ; 15: 259, 2015 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-26545875

RESUMEN

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.


Asunto(s)
Burkholderia mallei/inmunología , Burkholderia pseudomallei/inmunología , Quimiocinas/genética , Macrófagos/inmunología , Macrófagos/microbiología , Actinas/metabolismo , Animales , Burkholderia mallei/aislamiento & purificación , Burkholderia mallei/patogenicidad , Burkholderia pseudomallei/aislamiento & purificación , Burkholderia pseudomallei/patogenicidad , Regulación de la Expresión Génica , Células Gigantes/metabolismo , Inmunidad Innata , Macrófagos/citología , Ratones , Células RAW 264.7
5.
Cancer Res ; 84(6): 887-904, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38241710

RESUMEN

PARP inhibitor (PARPi)-resistant BRCA-mutant (BRCAm) high-grade serous ovarian cancer (HGSOC) represents a new clinical challenge with unmet therapeutic needs. Here, we performed a quantitative high-throughput drug combination screen that identified the combination of an ATR inhibitor (ATRi) and an AKT inhibitor (AKTi) as an effective treatment strategy for both PARPi-sensitive and PARPi-resistant BRCAm HGSOC. The ATRi and AKTi combination induced DNA damage and R loop-mediated replication stress (RS). Mechanistically, the kinase domain of AKT1 directly interacted with DHX9 and facilitated recruitment of DHX9 to R loops. AKTi increased ATRi-induced R loop-mediated RS by mitigating recruitment of DHX9 to R loops. Moreover, DHX9 was upregulated in tumors from patients with PARPi-resistant BRCAm HGSOC, and high coexpression of DHX9 and AKT1 correlated with worse survival. Together, this study reveals an interaction between AKT1 and DHX9 that facilitates R loop resolution and identifies combining ATRi and AKTi as a rational treatment strategy for BRCAm HGSOC irrespective of PARPi resistance status. SIGNIFICANCE: Inhibition of the AKT and ATR pathways cooperatively induces R loop-associated replication stress in high-grade serous ovarian cancer, providing rationale to support the clinical development of AKT and ATR inhibitor combinations. See related commentary by Ramanarayanan and Oberdoerffer, p. 793.


Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Humanos , Femenino , Estructuras R-Loop , Proteínas Proto-Oncogénicas c-akt/metabolismo , Resistencia a Antineoplásicos/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Antineoplásicos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Neoplasias/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo
6.
J Biol Chem ; 287(6): 3704-9, 2012 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-22158869

RESUMEN

Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP(3)) production and subsequent calcium (Ca(2+)) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP(3)-Ca(2+) signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP(3)-Ca(2+) cascade in the LPS-induced innate immune response pathway where release of intracellular Ca(2+) mediates TLR4 trafficking and subsequent activation of IRF3.


Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Endocitosis/efectos de los fármacos , Factor 3 Regulador del Interferón/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Fosfolipasa C gamma/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Calcio/inmunología , Señalización del Calcio/fisiología , Línea Celular , Endocitosis/fisiología , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/fisiología , Inositol 1,4,5-Trifosfato/genética , Inositol 1,4,5-Trifosfato/inmunología , Inositol 1,4,5-Trifosfato/metabolismo , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/inmunología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología
7.
Nat Methods ; 7(10): 801-5, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20936779

RESUMEN

Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.


Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteómica/métodos , Secuencia de Bases , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Línea Celular , Membrana Celular/metabolismo , Clonación Molecular , ADN Complementario/genética , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Luciferasas/genética , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/fisiología , Datos de Secuencia Molecular , Mucinas/genética , Mucinas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Interferente Pequeño/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Técnicas del Sistema de Dos Híbridos
8.
Sci Transl Med ; 15(701): eadd7872, 2023 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-37343085

RESUMEN

Poly(ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in breast cancer gene (BRCA)-mutant high-grade serous ovarian carcinoma (HGSC). However, most patients eventually develop resistance to PARPis, highlighting an unmet need for improved therapeutic strategies. Using high-throughput drug screens, we identified ataxia telangiectasia and rad3-related protein/checkpoint kinase 1 (CHK1) pathway inhibitors as cytotoxic and further validated the activity of the CHK1 inhibitor (CHK1i) prexasertib in PARPi-sensitive and -resistant BRCA-mutant HGSC cells and xenograft mouse models. CHK1i monotherapy induced DNA damage, apoptosis, and tumor size reduction. We then conducted a phase 2 study (NCT02203513) of prexasertib in patients with BRCA-mutant HGSC. The treatment was well tolerated but yielded an objective response rate of 6% (1 of 17; one partial response) in patients with previous PARPi treatment. Exploratory biomarker analyses revealed that replication stress and fork stabilization were associated with clinical benefit to CHK1i. In particular, overexpression of Bloom syndrome RecQ helicase (BLM) and cyclin E1 (CCNE1) overexpression or copy number gain/amplification were seen in patients who derived durable benefit from CHK1i. BRCA reversion mutation in previously PARPi-treated BRCA-mutant patients was not associated with resistance to CHK1i. Our findings suggest that replication fork-related genes should be further evaluated as biomarkers for CHK1i sensitivity in patients with BRCA-mutant HGSC.


Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Neoplasias Ováricas , Animales , Femenino , Humanos , Ratones , Antineoplásicos/uso terapéutico , Biomarcadores , Proteína BRCA1/genética , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico
9.
Front Microbiol ; 13: 803041, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35369443

RESUMEN

Current therapies for anthrax include the use of antibiotics (i.e., doxycycline, and ciprofloxacin), an anthrax vaccine (BioThrax) and Bacillus anthracis-specific, monoclonal antibody (mAb) (i.e., Raxibacumab and obiltoxaximab). In this study, we investigated the activity of immunomodulators, which potentiate inflammatory responses through innate immune receptors. The rationale for the use of innate immune receptor agonists as adjunctive immunomodulators for infectious diseases is based on the concept that augmentation of host defense should promote the antimicrobial mechanism of the host. Our aim was to explore the anti-B. anthracis effector function of Toll-like receptor (TLR) agonists using a mouse model. Amongst the six TLR ligands tested, Pam3CSK4 (TLR1/2 ligand) was the best at protecting mice from lethal challenge of B. anthracis. We then evaluated the activity of a novel TLR2 ligand, DA-98-WW07. DA-98-WW07 demonstrated enhanced protection in B. anthracis infected mice. The surviving mice that received DA-98-WW07 when re-challenged with B. anthracis 20 days post the first infection showed increased survival rate. Moreover, ciprofloxacin, when treated in adjunct with a suboptimal concentration of DA-98-WW07 demonstrated augmented activity in protecting mice from B. anthracis infection. Taken together, we report the prophylactic treatment potential of DA-98-WW07 for anthrax and the utility of immunomodulators in combination with an antibiotic to treat infections caused by the B. anthracis bacterium.

10.
Front Microbiol ; 12: 625211, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33967974

RESUMEN

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1ß (IL-1ß). On the contrary, the expression of IL-1ß receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1ß production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model.

11.
Lancet Infect Dis ; 20(9): e231-e237, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32563280

RESUMEN

The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Vacunas contra el Virus del Ébola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/terapia , Humanos , Profilaxis Posexposición
12.
Neurobiol Learn Mem ; 90(1): 164-70, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18442936

RESUMEN

Drug-associated cues are critical in reinstating the drug taking behavior even during prolonged abstinence and thus are thought to be a key factor to induce drug craving and to cause relapse. Amygdaloid complex has been known for its physiological function in mediating emotional experience storage and emotional cues-regulated memory retrieval. This study was undertaken to examine the role of basolateral nuclei of amygdala and the intracellular signaling molecule in drug cues-elicited cocaine memory retrieval. Systemic anisomycin treatment prior to the retrieval test abolished the cues-provoked cocaine conditioned place preference (CPP) memory. Likewise, a similar blockade of cues-provoked cocaine CPP performance was achieved by infusion of anisomycin and cycloheximide into the basolateral nuclei of amygdala before the test. Intra-amygdaloid infusion of H89, a protein kinase A inhibitor, or U0126, a MEK inhibitor, did not affect retrieval of the cues-elicited cocaine CPP memory. In contrast, intra-amygdaloid infusion of NPC 15437, a PKC inhibitor, abolished the cues-elicited cocaine CPP expression, while left the memory per se intact. Intra-amygdaloid infusion of NPC 15437 did not seem to affect locomotor activity or exert observable aversive effect. Taken together, our results suggest that activation of PKC signaling pathway and probably downstream de novo protein synthesis in the basolateral nuclei of amygdala is required for the cues-elicited cocaine memory performance. However, temporary inhibition of this signaling pathway does not seem to affect cocaine CPP memory per se.


Asunto(s)
Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/enzimología , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Memoria/efectos de los fármacos , Proteína Quinasa C/metabolismo , Animales , Anisomicina/farmacología , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Cicloheximida/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Isoquinolinas/farmacología , Masculino , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Piperidinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Percepción Espacial/efectos de los fármacos , Percepción Espacial/fisiología , Sulfonamidas/farmacología
13.
Chin J Physiol ; 51(6): 408-13, 2008 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-19280886

RESUMEN

The purpose of the current study was to evaluate the effects of chronic administration of alcohol on the olfactory bulbectomy (OBX)-induced mouse-killing behavior (MKB), an animal model for screening antidepressants. The rats were divided into three groups, which were given alcohol (0, 0.5, or 1 g/kg/day) orally for 28 days. MKB was analyzed before and at the end of each week of the alcohol treatment. The results showed that chronic alcohol treatment produced a significant increase in the latency of MKB, implying that alcohol may have an antidepressant-like activity. This suggests that alcohol dependence or abuse in depressed patients may result from "self-medication". Since it has been reported that OBX causes a decrease in the density of N-methyl-D-aspartate (NMDA) receptors in the brain and that alcohol is a potent and selective inhibitor of NMDA receptors, the possible role of NMDA receptors in this effect is discussed.


Asunto(s)
Alcoholes/farmacología , Bulbo Olfatorio/cirugía , Conducta Predatoria/efectos de los fármacos , Alcoholismo , Animales , Masculino , Ratones , Modelos Animales , Ratas
14.
mBio ; 9(1)2018 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-29382729

RESUMEN

Increasing incidences of multidrug resistance in pathogenic bacteria threaten our ability to treat and manage bacterial infection. The development and FDA approval of novel antibiotics have slowed over the past decade; therefore, the adoption and improvement of alternative therapeutic strategies are critical for addressing the threat posed by multidrug-resistant bacteria. Host-directed therapies utilize small-molecule drugs and proteins to alter the host response to pathogen infection. Here, we highlight strategies for modulating the host inflammatory response to enhance bacterial clearance, small-molecule potentiation of innate immunity, and targeting of host factors that are exploited by pathogen virulence factors. Application of state-of-the-art "omic" technologies, including proteomics, transcriptomics, and image-omics (image-based high-throughput phenotypic screening), combined with powerful bioinformatics tools will enable the modeling of key signaling pathways in the host-pathogen interplay and aid in the identification of host proteins for therapeutic targeting and the discovery of host-directed small molecules that will regulate bacterial infection. We conclude with an outlook on research needed to overcome the challenges associated with transitioning host-directed therapies into a clinical setting.


Asunto(s)
Infecciones Bacterianas/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple , Factores Inmunológicos/uso terapéutico , Biología Computacional , Descubrimiento de Drogas/tendencias , Interacciones Huésped-Patógeno , Humanos
15.
Emerg Microbes Infect ; 4(10): e61, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26576339

RESUMEN

Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness. The recent global spread of the virus and lack of approved therapeutic options makes it imperative to gain greater insight into the molecular mechanisms underlying CHIKV pathogenesis, in particular host factors recruited by the virus. In the current study, we identify sphingosine kinase 2 (SK2) as a CHIKV host factor co-localized with the viral replication complex (VRC) during infection. SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins. Targeted impairment of SK2 expression or function significantly inhibited CHIKV infection. Furthermore, affinity purification-mass spectrometry studies revealed that SK2 associates with a number of proteins involved in cellular gene expression specifically during viral infection, suggesting a role in replication. Collectively these results identify SK2 as a novel CHIKV host factor.


Asunto(s)
Infecciones por Alphavirus/virología , Virus Chikungunya/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Infecciones por Alphavirus/prevención & control , Animales , Línea Celular , Células Cultivadas , Virus Chikungunya/genética , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Músculo Esquelético/citología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Interferencia de ARN , ARN Viral/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología
16.
Front Microbiol ; 6: 683, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26284031

RESUMEN

Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3ß suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments.

17.
PLoS One ; 9(2): e87201, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24516547

RESUMEN

Alveolar macrophages (AMs) phagocytose Bacillus anthracis following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of B. anthracis, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of B. anthracis, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the B. anthracis. Meta-analyses revealed genes differentially expressed in response to B. anthracis infection were also induced upon infections with multiple pathogens such as Francisella Novicida or Staphylococcus aureus. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of B. anthracis. The reported differences may account for the marked difference in pathogenicity between these two strains.


Asunto(s)
Bacillus anthracis , Regulación de la Expresión Génica , Macrófagos Alveolares/microbiología , Animales , Antígenos Bacterianos/inmunología , Inmunidad Innata , Macaca mulatta , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Fagocitosis/inmunología , Esporas Bacterianas/inmunología , Esporas Bacterianas/metabolismo
18.
PLoS One ; 9(2): e89284, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24586659

RESUMEN

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses.


Asunto(s)
Células Dendríticas/inmunología , Regulación de la Expresión Génica , Inmunidad Innata , Macrófagos/inmunología , Proteína Fosfatasa 1/fisiología , Infecciones Estreptocócicas/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Western Blotting , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Ensayo de Inmunoadsorción Enzimática , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoprecipitación , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Mutación/genética , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Infecciones Estreptocócicas/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus/patogenicidad , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
19.
Cell Host Microbe ; 11(3): 306-18, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22423970

RESUMEN

Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.


Asunto(s)
Inmunidad Innata/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Embrión de Pollo , Simulación por Computador , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Endosomas/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Biológicos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Transporte de Proteínas , Interferencia de ARN , Transducción de Señal , Máquina de Vectores de Soporte
20.
Cell Stem Cell ; 6(1): 37-47, 2010 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-20085741

RESUMEN

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/análisis , Neoplasias Encefálicas/química , Diferenciación Celular , Transformación Celular Neoplásica/química , Glioblastoma/química , Células Madre Neoplásicas/química , Proteínas Nucleares/análisis , Interferencia de ARN , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Ratones SCID , Células Madre Neoplásicas/citología , Proteínas Nucleares/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
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