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1.
Cell Rep ; 42(12): 113403, 2023 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-37979174

RESUMO

The inheritance of a functional endoplasmic reticulum (ER) is ensured by the ER stress surveillance (ERSU) pathway. Here, we made the unexpected discovery that reticulon 1 (Rtn1) and Yop1, well-known ER-curvature-generating proteins, each possess two sphingolipid-binding motifs within their transmembrane domains and that these motifs recognize the ER-stress-induced sphingolipid phytosphingosine (PHS), resulting in an ER inheritance block. Upon binding PHS, Rtn1/Yop1 accumulate on the ER tubule, poised to enter the emerging daughter cell, and cause its misdirection to the bud scars (i.e., previous cell division sites). Amino acid changes in the conserved PHS-binding motifs preclude Rtn1 or Yop1 from binding PHS and diminish their enrichment on the tubular ER, ultimately preventing the ER-stress-induced inheritance block. Conservation of these sphingolipid-binding motifs in human reticulons suggests that sphingolipid binding to Rtn1 and Yop1 represents an evolutionarily conserved mechanism that enables cells to respond to ER stress.


Assuntos
Saccharomyces cerevisiae , Esfingolipídeos , Humanos , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Retículo Endoplasmático/metabolismo , Pontos de Checagem do Ciclo Celular , Estresse do Retículo Endoplasmático
2.
Mol Biol Cell ; 33(13): ar117, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36044344

RESUMO

Assembly of the nucleus following mitosis requires rapid and coordinate recruitment of diverse constituents to the inner nuclear membrane. We have identified an unexpected role for the nucleoporin Nup153 in promoting the continued addition of a subset of nuclear envelope (NE) proteins during initial expansion of nascent nuclei. Specifically, disrupting the function of Nup153 interferes with ongoing addition of B-type lamins, lamin B receptor, and SUN1 early in telophase, after the NE has initially enclosed chromatin. In contrast, effects on lamin A and SUN2 were minimal, pointing to differential requirements for the ongoing targeting of NE proteins. Further, distinct mistargeting phenotypes arose among the proteins that require Nup153 for NE targeting. Thus, disrupting the function of Nup153 in nuclear formation reveals several previously undescribed features important for establishing nuclear architecture: 1) a role for a nuclear basket constituent in ongoing recruitment of nuclear envelope components, 2) two functionally separable phases of NE formation in mammalian cells, and 3) distinct requirements of individual NE residents for continued targeting during the expansion phase of NE reformation.


Assuntos
Membrana Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Lamina Tipo A/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Mitose , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
3.
Mol Biol Cell ; 32(2): 109-119, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33448894

RESUMO

The endoplasmic reticulum (ER) is one of the largest cytoplasmic organelles in eukaryotic cells and plays a role in many cellular processes, such as the production and quality control of secretory protein, lipid synthesis, and calcium homeostasis. The ER cannot be generated de novo, and thus its proper inheritance during cell division is paramount to the health and survival of the daughter cells. Although previous work has uncovered the cytoskeletal components involved, we still lack a comprehensive understanding of the intricate steps of and the cytoplasmic and membrane-bound components involved in ER inheritance. To directly address these issues, we utilized microfluidics and genetic analyses to show that before nuclear migration, early ER inheritance can be further divided into three distinctive steps. Moreover, we demonstrated that perturbing each of these steps affects the cell's ability to mitigate ER stress. Thus, proper ER inheritance is essential to ensuring a healthy, functional cell.


Assuntos
Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/genética , Padrões de Herança/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Biochim Biophys Acta Mol Cell Res ; 1867(12): 118825, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32828757

RESUMO

The generation of new cells is one of the most fundamental aspects of cell biology. Proper regulation of the cell cycle is critical for human health, as underscored by many diseases associated with errors in cell cycle regulation, including both cancer and hereditary diseases. A large body of work has identified regulatory mechanisms and checkpoints that ensure accurate and timely replication and segregation of chromosomal DNA. However, few studies have evaluated the extent to which similar checkpoints exist for the division of cytoplasmic components, including organelles. Such checkpoint mechanisms might be crucial for compartments that cannot be generated de novo, such as the endoplasmic reticulum (ER). In this review, we highlight recent work in the model organism Saccharomyces cerevisiae that led to the discovery of such a checkpoint that ensures that cells inherit functional ER into the daughter cell.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Segregação de Cromossomos/genética , Replicação do DNA/genética , Retículo Endoplasmático/genética , Divisão Celular/genética , Estresse do Retículo Endoplasmático/genética , Humanos , Saccharomyces cerevisiae/genética
5.
Cells ; 9(4)2020 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-32290509

RESUMO

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.


Assuntos
Diarreia Infantil/genética , Molécula de Adesão da Célula Epitelial/genética , Síndromes de Malabsorção/genética , Resposta a Proteínas não Dobradas/genética , Animais , Doença Crônica , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Humanos , Recém-Nascido , Camundongos
6.
Dev Cell ; 51(2): 173-191.e5, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31564614

RESUMO

During cell division, the inheritance of a functional endoplasmic reticulum (ER) is ensured by the endoplasmic reticulum stress surveillance (ERSU) pathway. Activation of ERSU causes the septin ring to mislocalize, which blocks ER inheritance and cytokinesis. Here, we uncover that the septin ring in fact translocates to previously utilized cell division sites called cytokinetic remnants (CRMs). This unconventional translocation requires Nba1, a negative polarity regulator that normally prevents repolarization and re-budding at CRMs. Furthermore, septin ring translocation relies on the recruitment and activation of a key ERSU component Slt2 by Bem1, without activating Cdc42. Failure to transfer all septin subunits to CRMs delays the cell's ability to re-enter the cell cycle when ER homeostasis is restored and hinders cell growth after ER stress recovery. Thus, these deliberate but unprecedented rearrangements of cell polarity factors during ER stress safeguard cell survival and the timely cell-cycle re-entry upon ER stress recovery.


Assuntos
Ciclo Celular/fisiologia , Polaridade Celular/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Divisão Celular/fisiologia , Citocinese/fisiologia , Citoesqueleto/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Septinas/metabolismo
7.
Dev Cell ; 46(3): 327-343.e7, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30086303

RESUMO

The unfolded protein response (UPR) is induced by proteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.


Assuntos
Fator 6 Ativador da Transcrição/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Fator 6 Ativador da Transcrição/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fenretinida/farmacologia , Humanos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Transcrição Gênica/efeitos dos fármacos
8.
J Cell Biol ; 217(2): 495-505, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29317528

RESUMO

Proper inheritance of functional organelles is vital to cell survival. In the budding yeast, Saccharomyces cerevisiae, the endoplasmic reticulum (ER) stress surveillance (ERSU) pathway ensures that daughter cells inherit a functional ER. Here, we show that the ERSU pathway is activated by phytosphingosine (PHS), an early biosynthetic sphingolipid. Multiple lines of evidence support this: (1) Reducing PHS levels with myriocin diminishes the ability of cells to induce ERSU phenotypes. (2) Aureobasidin A treatment, which blocks conversion of early intermediates to downstream complex sphingolipids, induces ERSU. (3) orm1Δorm2Δ cells, which up-regulate PHS, show an ERSU response even in the absence of ER stress. (4) Lipid analyses confirm that PHS levels are indeed elevated in ER-stressed cells. (5) Lastly, the addition of exogenous PHS is sufficient to induce all ERSU phenotypes. We propose that ER stress elevates PHS, which in turn activates the ERSU pathway to ensure future daughter-cell viability.


Assuntos
Estresse do Retículo Endoplasmático , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Esfingolipídeos/antagonistas & inibidores , Esfingolipídeos/genética
9.
J Immunol ; 198(8): 3017-3022, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28275141

RESUMO

In this study, we used cre-lox techniques to generate mice selectively deficient in ORMDL3 in airway epithelium (Ormdl3Δ2-3/Δ2-3/CC10) to simulate an inhaled therapy that effectively inhibited ORMDL3 expression in the airway. In contrast to the anticipated reduction in airway hyperresponsiveness (AHR), OVA allergen-challenged Ormdl3Δ2-3/Δ2-3/CC10 mice had a significant increase in AHR compared with wild-type mice. Levels of airway inflammation, mucus, fibrosis, and airway smooth muscle were no different in Ormdl3Δ2-3/Δ2-3/CC10 and wild-type mice. However, levels of sphingosine-1-phosphate (S1P) were significantly increased in Ormdl3Δ2-3/Δ2-3/CC10 mice as well as in airway epithelial cells in which ORMDL3 was inhibited with small interfering RNA. Incubation of S1P with airway smooth muscle cells significantly increased contractility. Overall, Ormdl3Δ2-3/Δ2-3/CC10 mice exhibit increased allergen-induced AHR independent of inflammation and associated with increased S1P generation. These studies raise concerns for inhaled therapies that selectively and effectively inhibit ORMDL3 in airway epithelium in asthma.


Assuntos
Asma/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Hipersensibilidade Respiratória/metabolismo , Animais , Asma/imunologia , Modelos Animais de Doenças , Lisofosfolipídeos/imunologia , Lisofosfolipídeos/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Hipersensibilidade Respiratória/imunologia , Esfingosina/análogos & derivados , Esfingosina/imunologia , Esfingosina/metabolismo
10.
Sci Rep ; 6: 33353, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27634301

RESUMO

Activation of the IRE1α-XBP1 branch of the unfolded protein response (UPR) has been implicated in multiple types of human cancers, including multiple myeloma (MM). Through an in silico drug discovery approach based on protein-compound virtual docking, we identified the anthracycline antibiotic doxorubicin as an in vitro and in vivo inhibitor of XBP1 activation, a previously unknown activity for this widely utilized cancer chemotherapeutic drug. Through a series of mechanistic and phenotypic studies, we showed that this novel activity of doxorubicin was not due to inhibition of topoisomerase II (Topo II). Consistent with its inhibitory activity on the IRE1α-XBP1 branch of the UPR, doxorubicin displayed more potent cytotoxicity against MM cell lines than other cancer cell lines that have lower basal IRE1α-XBP1 activity. In addition, doxorubicin significantly inhibited XBP1 activation in CD138(+) tumor cells isolated from MM patients. Our findings suggest that the UPR-modulating activity of doxorubicin may be utilized clinically to target IRE1α-XBP1-dependent tumors such as MM.


Assuntos
Doxorrubicina/farmacologia , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/química , Etoposídeo/química , Etoposídeo/farmacologia , Humanos , Splicing de RNA/genética , Inibidores da Topoisomerase/farmacologia
11.
Mol Cancer Ther ; 15(9): 2055-65, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27307600

RESUMO

Using a luciferase reporter-based high-throughput chemical library screen and topological data analysis, we identified N-acridine-9-yl-N',N'-dimethylpropane-1,3-diamine (DAPA) as an inhibitor of the inositol requiring kinase 1α (IRE1α)-X-box binding protein-1 (XBP1) pathway of the unfolded protein response. We designed a collection of analogues based on the structure of DAPA to explore structure-activity relationships and identified N(9)-(3-(dimethylamino)propyl)-N(3),N(3),N(6),N(6)-tetramethylacridine-3,6,9-triamine (3,6-DMAD), with 3,6-dimethylamino substitution on the chromophore, as a potent inhibitor. 3,6-DMAD inhibited both IRE1α oligomerization and in vitro endoribonuclease (RNase) activity, whereas the other analogues only blocked IRE1α oligomerization. Consistent with the inhibition of IRE1α-mediated XBP1 splicing, which is critical for multiple myeloma cell survival, these analogues were cytotoxic to multiple myeloma cell lines. Furthermore, 3,6-DMAD inhibited XBP1 splicing in vivo and the growth of multiple myeloma tumor xenografts. Our study not only confirmed the utilization of topological data analysis in drug discovery but also identified a class of compounds with a unique mechanism of action as potent IRE1α-XBP1 inhibitors in the treatment of multiple myeloma. Mol Cancer Ther; 15(9); 2055-65. ©2016 AACR.


Assuntos
Acridinas/farmacologia , Antineoplásicos/farmacologia , Endorribonucleases/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Endorribonucleases/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mieloma Múltiplo/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Dev Cell ; 37(1): 7-8, 2016 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-27046826

RESUMO

In a recent issue of Science, Lei and Spradling (2016) uncover how germ cells differentiate into oocytes in mouse embryos. Mouse germ cells form cysts, in which sister cells nurse the developing oocyte by donating their organelles and cytoplasmic materials.


Assuntos
Células Gigantes/citologia , Oócitos/citologia , Oogênese , Organelas/fisiologia , Animais , Feminino
13.
Dev Cell ; 37(3): 279-88, 2016 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-27117666

RESUMO

Segregation of functional organelles during the cell cycle is crucial to generate healthy daughter cells. In Saccharomyces cerevisiae, ER stress causes an ER inheritance block to ensure cells inherit a functional ER. Here, we report that formation of tubular ER in the mother cell, the first step in ER inheritance, depends on functional symmetry between the cortical ER (cER) and perinuclear ER (pnER). ER stress induces functional asymmetry, blocking tubular ER formation and ER inheritance. Using fluorescence recovery after photobleaching, we show that the ER chaperone Kar2/BiP fused to GFP and an ER membrane reporter, Hmg1-GFP, behave differently in the cER and pnER. The functional asymmetry and tubular ER formation depend on Reticulons/Yop1, which maintain ER structure. LUNAPARK1 deletion in rtn1Δrtn2Δyop1Δ cells restores the pnER/cER functional asymmetry, tubular ER generation, and ER inheritance blocks. Thus, Reticulon/Yop1-dependent changes in ER structure are linked to ER inheritance during the yeast cell cycle.


Assuntos
Núcleo Celular/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Elife ; 42015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26327697

RESUMO

Stress induced by cytoplasmic protein aggregates can have deleterious consequences for the cell, contributing to neurodegeneration and other diseases. Protein aggregates are also formed within the endoplasmic reticulum (ER), although the fate of ER protein aggregates, specifically during cell division, is not well understood. By simultaneous visualization of both the ER itself and ER protein aggregates, we found that ER protein aggregates that induce ER stress are retained in the mother cell by activation of the ER Stress Surveillance (ERSU) pathway, which prevents inheritance of stressed ER. In contrast, under conditions of normal ER inheritance, ER protein aggregates can enter the daughter cell. Thus, whereas cytoplasmic protein aggregates are retained in the mother cell to protect the functional capacity of daughter cells, the fate of ER protein aggregates is determined by whether or not they activate the ERSU pathway to impede transmission of the cortical ER during the cell cycle.


Assuntos
Divisão Celular , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/fisiologia , Agregados Proteicos , Estresse Fisiológico , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/fisiologia
15.
Semin Cancer Biol ; 33: 48-56, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25986851

RESUMO

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress, which is characteristic of cells with high level of secretory activity and implicated in a variety of disease conditions. In response to ER stress, the cell elicits an adaptive process called the unfolded protein response (UPR) to support cellular homeostasis and survival. However, prolonged and unsolvable ER stress also induces apoptosis. As the most conserved signaling branch of the UPR, the IRE1α-XBP1 pathway plays important roles in both physiological and pathological settings and its activity has profound effects on disease progression and prognosis. Recently, modulating this pathway with small molecule compounds has been demonstrated as a promising approach for disease therapy. In this review, we summarize a list of current investigational compounds targeting this pathway and their therapeutic features for treating human diseases.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Aldeídos/química , Aldeídos/metabolismo , Animais , Apoptose , Sobrevivência Celular , Cumarínicos/química , Modelos Animais de Doenças , Progressão da Doença , Desenho de Fármacos , Descoberta de Drogas , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Homeostase , Humanos , Camundongos , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Prognóstico , Dobramento de Proteína , Quercetina/química , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Sulfonamidas , Tiofenos , Toiocamicina/química , Proteína 1 de Ligação a X-Box
16.
Cell Rep ; 9(3): 850-8, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25437541

RESUMO

An evolutionarily conserved unfolded protein response (UPR) component, IRE1, cleaves XBP1/HAC1 introns in order to generate spliced mRNAs that are translated into potent transcription factors. IRE1 also cleaves endoplasmic-reticulum-associated RNAs leading to their decay, an activity termed regulated IRE1-dependent decay (RIDD); however, the mechanism by which IRE1 differentiates intron cleavage from RIDD is not well understood. Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase and mutations at oligomerization interfaces. Using an IRE1 RNase inhibitor, STF-083010, selective inhibition of XBP1 splicing indicates that XBP1 promotes cell survival, whereas RIDD leads to cell death, revealing modulation of IRE1 activities as a drug-development strategy.


Assuntos
Biocatálise , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Estabilidade de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Animais , Morte Celular , Linhagem da Célula , Endorribonucleases , Glicoproteínas de Membrana/química , Camundongos , Modelos Moleculares , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , RNA Fúngico/metabolismo , Fatores de Transcrição de Fator Regulador X , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Relação Estrutura-Atividade , Especificidade por Substrato , Proteína 1 de Ligação a X-Box
17.
J Immunol ; 192(8): 3475-87, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24623133

RESUMO

Orosomucoid-like (ORMDL)3 has been strongly linked with asthma in genetic association studies. Because allergen challenge induces lung ORMDL3 expression in wild-type mice, we have generated human ORMDL3 zona pellucida 3 Cre (hORMDL3(zp3-Cre)) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3(zp3-Cre) mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3(zp3-Cre) mice had spontaneously increased airway responsiveness to methacholine compared to wild-type mice. This increased airway remodeling was associated with selective activation of the unfolded protein response pathway transcription factor ATF6 (but not Ire1 or PERK). The ATF6 target gene SERCA2b, implicated in airway remodeling in asthma, was strongly induced in the lungs of hORMDL3(zp3-Cre) mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-ß1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3(zp3-Cre) mice. hORMDL3(zp3-Cre) mice had increased levels of IgE, with no change in levels of IgG, IgM, and IgA. These studies provide evidence that ORMDL3 plays an important role in vivo in airway remodeling potentially through ATF6 target genes such as SERCA2b and/or through ATF6-independent genes (TGF-ß1, ADAM8).


Assuntos
Remodelação das Vias Aéreas/genética , Remodelação das Vias Aéreas/imunologia , Asma/genética , Asma/imunologia , Proteínas de Membrana/genética , Fator 6 Ativador da Transcrição/metabolismo , Alérgenos/imunologia , Animais , Especificidade de Anticorpos/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/induzido quimicamente , Quimiocinas CC/metabolismo , Quimiocinas CXC/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/metabolismo , Expressão Gênica , Ordem dos Genes , Marcação de Genes , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Cloreto de Metacolina/administração & dosagem , Camundongos , Camundongos Transgênicos , Ovalbumina/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Transgenes , Resposta a Proteínas não Dobradas , eIF-2 Quinase/metabolismo
18.
PLoS One ; 7(10): e45078, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23110043

RESUMO

NF-κB, a transcription factor, becomes activated during the Unfolded Protein Response (UPR), an endoplasmic reticulum (ER) stress response pathway. NF-κB is normally held inactive by its inhibitor, IκBα. Multiple cellular pathways activate IKK (IκBα Kinase) which phosphorylate IκBα leading to its degradation and NF-κB activation. Here, we find that IKK is required for maximum activation of NF-κB in response to ER stress. However, unlike canonical NFκB activation, IKK activity does not increase during ER stress, but rather the level of basal IKK activity is critical for determining the extent of NF-κB activation. Furthermore, a key UPR initiator, IRE1, acts to maintain IKK basal activity through IRE1's kinase, but not RNase, activity. Inputs from IRE1 and IKK, in combination with translation repression by PERK, another UPR initiator, lead to maximal NF-κB activation during the UPR. These interdependencies have a significant impact in cancer cells with elevated IKK/NF-κB activity such as renal cell carcinoma cells (786-0). Inhibition of IKK by an IKK inhibitor, which significantly decreases NF-κB activity, is overridden by UPR induction, arguing for the importance of considering UPR activation in cancer treatment.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Animais , Western Blotting , Células CHO , Linhagem Celular , Imunoprecipitação da Cromatina , Cricetinae , Ensaio de Desvio de Mobilidade Eletroforética , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Quinase I-kappa B/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , NF-kappa B/genética , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/genética , eIF-2 Quinase/genética
19.
Proc Natl Acad Sci U S A ; 109(41): 16648-53, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-23011799

RESUMO

Orosomucoid like 3 (ORMDL3) has been strongly linked with asthma in genetic association studies, but its function in asthma is unknown. We demonstrate that in mice ORMDL3 is an allergen and cytokine (IL-4 or IL-13) inducible endoplasmic reticulum (ER) gene expressed predominantly in airway epithelial cells. Allergen challenge induces a 127-fold increase in ORMDL3 mRNA in bronchial epithelium in WT mice, with lesser 15-fold increases in ORMDL-2 and no changes in ORMDL-1. Studies of STAT-6-deficient mice demonstrated that ORMDL3 mRNA induction highly depends on STAT-6. Transfection of ORMDL3 in human bronchial epithelial cells in vitro induced expression of metalloproteases (MMP-9, ADAM-8), CC chemokines (CCL-20), CXC chemokines (IL-8, CXCL-10, CXCL-11), oligoadenylate synthetases (OAS) genes, and selectively activated activating transcription factor 6 (ATF6), an unfolded protein response (UPR) pathway transcription factor. siRNA knockdown of ATF-6α in lung epithelial cells inhibited expression of SERCA2b, which has been implicated in airway remodeling in asthma. In addition, transfection of ORMDL3 in lung epithelial cells activated ATF6α and induced SERCA2b. These studies provide evidence of the inducible nature of ORMDL3 ER expression in particular in bronchial epithelial cells and suggest an ER UPR pathway through which ORMDL3 may be linked to asthma.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Quimiocinas/metabolismo , Pulmão/metabolismo , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , Fator 6 Ativador da Transcrição/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Quimiocinas/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Pulmão/citologia , Proteínas de Membrana/genética , Metaloproteases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética
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