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1.
Cell ; 147(2): 436-46, 2011 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-22000020

RESUMEN

STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser(407) by TBK1 and Tyr(641), independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6(-/-) mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum.


Asunto(s)
Inmunidad Innata , Proteínas de la Membrana/metabolismo , Infecciones por Virus ARN/inmunología , Virus ARN , Factor de Transcripción STAT6/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Secuencia de Bases , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción STAT6/genética
2.
PLoS Pathog ; 16(10): e1009020, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33108406

RESUMEN

Brucellosis, caused by a number of Brucella species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo. However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. STING suppression occurred in MyD88-/- macrophages and was not induced by Toll-like receptor agonists or purified Brucella lipopolysaccharide (LPS). Rather, Brucella induced a STING-targeting microRNA, miR-24-2, in a type IV secretion system-dependent manner. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in Mirn23a locus-deficient macrophages. Failure to suppress STING expression in Mirn23a-/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Mirn23a-/- mice were also more resistant to splenic colonization one week post infection. Anti-miR-24 potently suppressed replication in wild type, but much less in STING-/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages cytosolic surveillance by miR-24-dependent suppression of STING expression; post-STING activation "damage control" via targeted STING destruction may enable establishment of chronic infection.


Asunto(s)
Brucella/metabolismo , Brucelosis/metabolismo , Proteínas de la Membrana/biosíntesis , MicroARNs/metabolismo , Animales , Brucella/genética , Brucelosis/genética , Femenino , Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , ARN Mensajero/genética , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismo
3.
Mol Cell ; 50(1): 5-15, 2013 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-23478444

RESUMEN

How the cell recognizes cytosolic DNA including DNA-based microbes to trigger host-defense-related gene activation remains to be fully resolved. Here, we demonstrate that STING (stimulator of interferon genes), an endoplasmic reticulum translocon-associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self- (apoptotic, necrotic) or pathogen-related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and proinflammatory genes in addition to type I IFN. Our data indicate that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.


Asunto(s)
Citoplasma/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Apoptosis , Sitios de Unión , Citoplasma/inmunología , ADN de Cadena Simple/inmunología , Genes Reporteros , Células HEK293 , Herpesvirus Humano 1/inmunología , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Proteínas de la Membrana/genética , Ratones , Necrosis , Multimerización de Proteína , Interferencia de ARN , Telomerasa/genética , Telomerasa/metabolismo , Transcripción Genética , Transfección
4.
J Immunol ; 200(2): 607-622, 2018 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-29203515

RESUMEN

Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by Brucella abortus DNA involves TLR9, AIM2, and stimulator of IFN genes (STING). In this study, we observed by microarray analysis that several type I IFN-associated genes, such as IFN-ß and guanylate-binding proteins (GBPs), are downregulated in STING knockout (KO) macrophages infected with Brucella or transfected with DNA. Additionally, we determined that STING and cyclic GMP-AMP synthase (cGAS) are important to engage the type I IFN pathway, but only STING is required to induce IL-1ß secretion, caspase-1 activation, and GBP2 and GBP3 expression. Furthermore, we determined that STING but not cGAS is critical for host protection against Brucella infection in macrophages and in vivo. This study provides evidence of a cGAS-independent mechanism of STING-mediated protection against an intracellular bacterial infection. Additionally, infected IFN regulatory factor-1 and IFNAR KO macrophages had reduced GBP2 and GBP3 expression and these cells were more permissive to Brucella replication compared with wild-type control macrophages. Because GBPs are critical to target vacuolar bacteria, we determined whether GBP2 and GBPchr3 affect Brucella control in vivo. GBPchr3 but not GBP2 KO mice were more susceptible to bacterial infection, and small interfering RNA treated-macrophages showed reduction in IL-1ß secretion and caspase-1 activation. Finally, we also demonstrated that Brucella DNA colocalizes with AIM2, and AIM2 KO mice are less resistant to B. abortus infection. In conclusion, these findings suggest that the STING-dependent type I IFN pathway is critical for the GBP-mediated release of Brucella DNA into the cytosol and subsequent activation of AIM2.


Asunto(s)
Brucella abortus/inmunología , Brucelosis/inmunología , Brucelosis/metabolismo , Proteínas de Unión al GTP/metabolismo , Inflamasomas/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Animales , Brucella abortus/genética , Brucelosis/genética , Brucelosis/microbiología , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Citocinas/metabolismo , Proteínas de Unión al GTP/genética , Expresión Génica , Perfilación de la Expresión Génica , Granuloma/metabolismo , Granuloma/microbiología , Granuloma/patología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Mediadores de Inflamación , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Modelos Biológicos , FN-kappa B/metabolismo
5.
Proc Natl Acad Sci U S A ; 109(47): 19386-91, 2012 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-23132945

RESUMEN

Inflammatory autoimmune diseases such as systemic lupus erythematosus (SLE) and polyarthritis are characterized by chronic cytokine overproduction, suggesting that the stimulation of host innate immune responses, speculatively by persistent infection or self nucleic acids, plays a role in the manifestation of these disorders. Mice lacking DNase II die during embryonic development through comparable inflammatory disease because phagocytosed DNA from apoptotic cells cannot be adequately digested and intracellular host DNA sensor pathways are engaged, resulting in the production of a variety of cytokines including type I IFN. The cellular sensor pathway(s) responsible for triggering DNA-mediated inflammation aggravated autoimmune disease remains to be determined. However, we report here that Stimulator of IFN Genes (STING) is responsible for inflammation-related embryonic death in DNase II defective mice initiated by self DNA. DNase II-dependent embryonic lethality was rescued by loss of STING function, and polyarthritis completely prevented because cytosolic DNA failed to robustly trigger cytokine production through STING-controlled signaling pathways. Our data provides significant molecular insight into the causes of DNA-mediated inflammatory disorders and affords a target that could plausibly be therapeutically controlled to help prevent such diseases.


Asunto(s)
ADN/inmunología , Inflamación/inmunología , Inflamación/patología , Proteínas de la Membrana/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Animales , Apoptosis , Artritis/inmunología , Artritis/patología , Citocinas/biosíntesis , Pérdida del Embrión/genética , Pérdida del Embrión/inmunología , Embrión de Mamíferos/metabolismo , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Necrosis
6.
PLoS Pathog ; 8(10): e1002934, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23055924

RESUMEN

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.


Asunto(s)
Células Dendríticas/metabolismo , Virus del Dengue/inmunología , Virus del Dengue/patogenicidad , Interferón Tipo I/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas no Estructurales Virales/metabolismo , Aedes , Animales , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Células Dendríticas/virología , Virus del Dengue/metabolismo , Células HEK293 , Humanos , Evasión Inmune , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Células Vero , Replicación Viral
7.
J Immunol ; 189(9): 4630-9, 2012 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-23028052

RESUMEN

IFN regulatory factor 3 (IRF3) regulates early type I IFNs and other genes involved in innate immunity. We have previously shown that cells undergoing an endoplasmic reticulum (ER) stress response called the unfolded protein response produce synergistically augmented IFN-ß when stimulated with pattern recognition receptor agonists such as LPS. Concomitant ER stress and LPS stimulation resulted in greater recruitment of the IRF3 transcription factor to ifnb1 gene regulatory elements. In this study, we used murine cells to demonstrate that both oxygen-glucose deprivation and pharmacologic unfolded protein response inducers trigger phosphorylation and nuclear translocation of IRF3, even in the absence of exogenous LPS. Different ER stressors used distinct mechanisms to activate IRF3: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin treatment, oxygen-glucose deprivation) critically depended upon stimulator of IFN gene, an ER-resident nucleic acid-responsive molecule. However, calcium mobilization alone by ionomycin was insufficient for IRF3 phosphorylation. In contrast, other forms of ER stress (e.g., tunicamycin treatment) promote IRF3 phosphorylation independently of stimulator of IFN gene and TANK-binding kinase 1. Rather, IRF3 activation by tunicamycin and 2-deoxyglucose was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor that blocks activating transcription factor 6 processing. Interfering with ER stress-induced IRF3 activation abrogated IFN-ß synergy. Together, these data suggest ER stress primes cells to respond to innate immune stimuli by activating the IRF3 transcription factor. Our results also suggest certain types of ER stress accomplish IRF3 phosphorylation by co-opting existing innate immune pathogen response pathways. These data have implications for diseases involving ER stress and type I IFN.


Asunto(s)
Estrés del Retículo Endoplásmico/inmunología , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/inmunología , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Lactonas/farmacología , Lipopolisacáridos/fisiología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Proteínas Serina-Treonina Quinasas/fisiología , Sesquiterpenos/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/inmunología
8.
Blood ; 118(5): 1329-39, 2011 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-21659544

RESUMEN

Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-x(L)/Bcl-2 dependence. Surprisingly, analysis of Mcl-1-dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-x(L) in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Proteínas de la Membrana/fisiología , Mieloma Múltiple/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteína bcl-X/fisiología , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Nitrofenoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/farmacología , Sulfonamidas/farmacología , Distribución Tisular , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
9.
Blood ; 111(10): 5152-62, 2008 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-18354037

RESUMEN

The use of arsenic trioxide (ATO) to treat multiple myeloma (MM) is supported by preclinical studies as well as several phase 2 studies, but the precise mechanism(s) of action of ATO has not been completely elucidated. We used gene expression profiling to determine the regulation of apoptosis-related genes by ATO in 4 MM cell lines and then focused on Bcl-2 family genes. ATO induced up-regulation of 3 proapoptotic BH3-only proteins (Noxa, Bmf, and Puma) and down-regulation of 2 antiapoptotic proteins Mcl-1 and Bcl-X(L). Coimmunoprecipitation demonstrated that Noxa and Puma bind Mcl-1 to release Bak and Bim within 6 hours of ATO addition. Bak and Bim are also released from Bcl-X(L). Silencing of Bmf, Noxa, and Bim significantly protected cells from ATO-induced apoptosis, while Puma silencing had no effect. Consistent with a role for Noxa inhibition of Mcl-1, the Bad-mimetic ABT-737 synergized with ATO in the killing of 2 MM lines. Finally, Noxa expression was enhanced by GSH depletion and inhibited by increasing GSH levels in the cells. Understanding the pattern of BH3-only protein response should aid in the rational design of arsenic-containing regimens.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Mieloma Múltiple/tratamiento farmacológico , Óxidos/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Reguladoras de la Apoptosis/fisiología , Trióxido de Arsénico , Proteína 11 Similar a Bcl2 , Muerte Celular , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Mieloma Múltiple/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología
10.
J Biol Chem ; 284(19): 12886-95, 2009 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-19279006

RESUMEN

Arsenicals are both environmental carcinogens as well as therapeutic agents for the treatment of trypanosomiasis and more recently cancer. Arsenic trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has activity in multiple myeloma (MM). While signaling events associated with carcinogenesis have been well studied, it still remains to be determined which of these events are involved in anti-cancer signaling. To better define this response, gene expression profiling following ATO treatment of four MM cell lines was performed. The pattern was consistent with a strong antioxidative response, particularly of genes activated by Nrf2. While Nrf2 is expressed constitutively at the mRNA level, the protein is not detected in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized and present in the nucleus within 6 h of ATO treatment. Despite the activation of this antioxidative response, ROS may not be important in ATO-induced death. Inhibition of ATO-induced ROS with butylated hydroxyanisole (BHA) does not affect Nrf2 activation or cell death. Moreover, silencing Nrf2 had no effect on ATO-induced apoptosis. Together these data suggest that ROS is not important in the induction of the antioxidative response or cellular death by ATO.


Asunto(s)
Antineoplásicos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Mieloma Múltiple/tratamiento farmacológico , Óxidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Trióxido de Arsénico , Western Blotting , Hidroxianisol Butilado/farmacología , Perfilación de la Expresión Génica , Inhibidores de Crecimiento/farmacología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares
11.
Mol Cancer Ther ; 8(5): 1197-206, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19417148

RESUMEN

Here, we report on the organic arsenical darinaparsin (ZIO-101, S-dimethylarsino-glutathione) and its anti-myeloma activity compared with inorganic arsenic trioxide. Darinaparsin induced apoptosis in multiple myeloma cell lines in a dose-dependent manner, and the addition of N-acetylcysteine, which increases intracellular glutathione (GSH), blocked cytotoxicity of both darinaparsin and arsenic trioxide. In contrast to arsenic trioxide, intracellular GSH does not appear to be important for darinaparsin metabolism, as an inhibitor of GSH synthesis, buthionine sulfoximine, had little effect on drug activity. This discrepancy was resolved when we determined the effects of thiols on drug uptake. The addition of exogenous GSH, L-cysteine, or D-cysteine prevented darinaparsin cellular uptake and cell death but had no effect on the uptake or activity of arsenic trioxide, suggesting a difference in the transport mechanism of these two drugs. In addition, gene expression profiling revealed differences in the signaling of protective responses between darinaparsin and arsenic trioxide. Although both arsenicals induced a transient heat shock response, only arsenic trioxide treatment induced transcription of metal response genes and anti-oxidant genes related to the Nrf2-Keap1 pathway. In contrast to the protective responses, both arsenicals induced up-regulation of BH3-only proteins. Moreover, silencing of BH3-only proteins Noxa, Bim, and Bmf protected myeloma cells from darinaparsin-induced cell death. Finally, treatment of an arsenic trioxide-resistant myeloma cell line with darinaparsin resulted in dose-dependent apoptosis, indicating that cross-resistance does not necessarily develop between these two forms of arsenic in multiple myeloma cell lines. These results suggest darinaparsin may be useful as an alternative treatment in arsenic trioxide-resistant hematologic cancers.


Asunto(s)
Arsenicales/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Glutatión/análogos & derivados , Mieloma Múltiple/metabolismo , Óxidos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Trióxido de Arsénico , Arsenicales/metabolismo , Butionina Sulfoximina/metabolismo , Butionina Sulfoximina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisteína/metabolismo , Cisteína/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Glutatión/farmacología , Humanos , Óxidos/metabolismo
12.
Blood ; 107(12): 4907-16, 2006 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-16507771

RESUMEN

Multiple myeloma (MM) is an incurable plasma cell malignancy. The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in MM cells; however, the nature of its selectivity remains unknown. Here we demonstrate that 5 different MM cell lines display similar patterns of sensitivity to 3 proteasome inhibitors (PIs) but respond differently to specific NF-kappaB inhibition. We further show that PIs initiate the unfolded protein response (UPR), a signaling pathway activated by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). Consistent with reports that prosurvival/physiologic UPR components are required for B-cell differentiation into antibody-secreting cells, we found that MM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. However, bortezomib rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress-specific eIF-2alpha kinase; ATF4, an ER stress-induced transcription factor; and its proapoptotic target, CHOP/GADD153. Consistent with our hypothesis that PIs induce the accumulation of misfolded ER-processed proteins, we found that the amount of immunoglobulin subunits retained within MM cells correlated with their sensitivity to PIs. These findings suggest that MM cells have a lower threshold for PI-induced UPR induction and ER stress-induced apoptosis because they constitutively express ER stress survival factors to function as secretory cells.


Asunto(s)
Ácidos Borónicos/farmacología , Mieloma Múltiple/enzimología , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Pirazinas/farmacología , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ácidos Borónicos/uso terapéutico , Bortezomib , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/enzimología , Chaperón BiP del Retículo Endoplásmico , Humanos , Mieloma Múltiple/tratamiento farmacológico , Proteínas de Neoplasias/biosíntesis , Células Plasmáticas/enzimología , Células Plasmáticas/patología , Inhibidores de Proteasas/uso terapéutico , Inhibidores de Proteasoma , Pirazinas/uso terapéutico
13.
J Biol Chem ; 277(40): 37349-58, 2002 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-12147687

RESUMEN

Identification of the environmental triggers involved in the expression of virulence genes is a fundamental objective in studies of bacterial pathogens. For uropathogens, urea, found in the urinary tract at concentrations of up to 500 mm, functions as an environmental signal. Urea freely diffuses into the bacterium Providencia stuartii and activates UreR, a member of the AraC family of transcriptional activators. Active UreR promotes transcription of virulence-associated urease genes and alerts the organisms of its immediate milieu. Thus, the UreR.urea complex has a dual role, acting as both a transcriptional activator as well as an environmental sensor. Here, we describe the molecular events associated with activation of gene expression by urea-bound UreR. The K(d) of the urea.UreR binding reaction was measured as 0.2 mm by fluorescence quenching assays, and the shape of the binding curve indicated a single specific urea-binding site on UreR. Histidine residues are critical for urea binding in urease, and therefore to identify the urea-binding site in UreR, five mutant UreR forms were generated with histidine to alanine substitutions. Two of the mutants (UreR(c)) exhibited a constitutive phenotype by both activating transcription and binding to DNA with an increased affinity in the absence of urea. The UreR(c) bound urea with an affinity similar to that of wild-type UreR. We concluded, therefore, that the mutations resulting in constitutive activity were not involved in the UreR.urea interaction. UreR was activated, then, either by binding urea or by histidine to alanine substitutions at one of two positions. Circular dichroism indicated little change in the structure of UreR when activated, and size-exclusion chromatography demonstrated that both rUreR and rUreR(c) were dimers in both the presence and absence of urea. Thus, the structural changes associated with activation are subtle.


Asunto(s)
Proteínas Bacterianas , Transducción de Señal/efectos de los fármacos , Transactivadores/genética , Urea/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Codón/genética , Escherichia coli/genética , Secuencias Hélice-Giro-Hélice , Datos de Secuencia Molecular , Plásmidos , Providencia/efectos de los fármacos , Providencia/genética , Providencia/fisiología , Transducción de Señal/fisiología , Espectrometría de Fluorescencia , Espectrofotometría , Transactivadores/efectos de los fármacos , Transactivadores/metabolismo , Transcripción Genética
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