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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34080635

RESUMO

Despite the recognized significance of reversible protein lipidation (S-acylation) for T cell receptor signal transduction, the enzymatic control of this post-translational modification in T cells remains poorly understood. Here, we demonstrate that DHHC21 (also known as ZDHHC21), a member of the DHHC family of mammalian protein acyltransferases, mediates T cell receptor-induced S-acylation of proximal T cell signaling proteins. Using Zdhhc21dep mice, which express a functionally deficient version of DHHC21, we show that DHHC21 is a Ca2+/calmodulin-dependent enzyme critical for activation of naïve CD4+ T cells in response to T cell receptor stimulation. We find that disruption of the Ca2+/calmodulin-binding domain of DHHC21 does not affect thymic T cell development but prevents differentiation of peripheral CD4+ T cells into Th1, Th2 and Th17 effector T helper lineages. Our findings identify DHHC21 as an essential component of the T cell receptor signaling machinery and define a new role for protein acyltransferases in regulation of T cell-mediated immunity.

2.
Mol Biol Rep ; 47(8): 6471-6478, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32789573

RESUMO

S-acylation reversible-post-translational lipidation of cysteine residues-is emerging as an important regulatory mechanism in T cell signaling. Dynamic S-acylation is critical for protein recruitment into the T cell receptor complex and initiation of the subsequent signaling cascade. However, the enzymatic control of protein S-acylation in T cells remains poorly understood. Here, we report a previously uncharacterized role of DHHC21, a member of the mammalian family of DHHC protein acyltransferases, in regulation of the T cell receptor pathway. We found that loss of DHHC21 prevented S-acylation of key T cell signaling proteins, resulting in disruption of the early signaling events and suppressed expression of T cell activation markers. Furthermore, downregulation of DHHC21 prevented activation and differentiation of naïve T cells into effector subtypes. Together, our study provides the first direct evidence that DHHC protein acyltransferases can play an essential role in regulation of T cell-mediated immunity.

3.
Biophys J ; 118(4): 826-835, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-31547976

RESUMO

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here, we investigated whether ß-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that ß-adrenergic stimulation led to rapidly increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and contractile responses. We identified the plasma membrane-localized palmitoyl acyltransferase DHHC5 as an important mediator of the stimulus-dependent palmitoylation in cardiomyocytes. Knockdown of DHHC5 showed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and functional responses downstream of ß-adrenergic stimulation. A palmitoylation assay with purified components revealed that Gαs and Gαi are direct substrates of DHHC5. Finally, we provided evidence that the C-terminal tail of DHHC5 can be palmitoylated in response to stimulation and such modification is important for its dynamic localization and function in the plasma membrane. Our results reveal that DHHC5 is a central regulator of signaling downstream of ß-adrenergic receptors in cardiomyocytes.

4.
Cell Death Dis ; 10(12): 870, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31740668

RESUMO

Burn patients experiencing hypermetabolism develop hepatic steatosis, which is associated with liver failure and poor outcomes after the injury. These same patients also undergo white adipose tissue (WAT) browning, which has been implicated in mediating post-burn cachexia and sustained hypermetabolism. Despite the clinical presentation of hepatic steatosis and WAT browning in burns, whether or not these two pathological responses are linked remains poorly understood. Here, we show that the burn-induced WAT browning and its associated increased lipolysis leads to the accelerated development of hepatic steatosis in mice. Deletion of interleukin 6 (IL-6) and the uncoupling protein 1 (UCP1), regulators of burn-induced WAT browning completely protected mice from hepatic steatosis after the injury. Treatment of post-burn mice with propranolol or IL-6 receptor blocker attenuated burn-induced WAT browning and its associated hepatic steatosis pathology. Lipidomic profiling in the plasma of post-burn mice and burn patients revealed elevated levels of damage-inducing lipids (palmitic and stearic acids), which induced hepatic endoplasmic reticulum (ER) stress and compromised hepatic fat oxidation. Mechanistically, we show that hepatic ER stress after a burn injury leads to a greater ER-mitochondria interaction, hepatocyte apoptosis, oxidative stress, and impaired fat oxidation. Collectively, our findings uncover an adverse "cross-talk" between the adipose and liver tissue in the context of burn injury, which is critically mediated by WAT browning.


Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Queimaduras/complicações , Fígado Gorduroso/patologia , Animais , Humanos , Camundongos
5.
Front Cell Dev Biol ; 7: 198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31620439

RESUMO

Chronic ER stress occurs when protein misfolding in the Endoplasmic reticulum (ER) lumen remains unresolved despite activation of the unfolded protein response. We have shown that traumatic injury such as a severe burn leads to chronic ER stress in vivo leading to systemic inflammation which can last for more than a year. The mechanisms linking chronic ER stress to systemic inflammatory responses are not clear. Here we show that induction of chronic ER stress leads to the release of known and novel damage-associated molecular patterns (DAMPs). The secreted DAMPs are aggregated and markedly protease resistant. ER stress-derived DAMPs activate dendritic cells (DCs) which are then capable of polarizing naïve T cells. Our findings indicate that induction of chronic ER stress may lead to the release of hyperstable DAMPs into the circulation resulting in persistent systemic inflammation and adverse outcomes.

6.
Biomed Pharmacother ; 106: 411-418, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29990828

RESUMO

Ellagic acid is a botanical polyphenol which has been shown to have numerous effects on cellular function. Ellagic acid can induce apoptosis and inhibit the proliferation of various cancer cell types in vitro and in vivo. As such, ellagic acid has attracted significant interest as a potential chemotherapeutic compound. One mechanism by which ellagic acid has been proposed to affect cellular physiology is by regulating metabolic pathways. Here we show the dose-dependent effects of ellagic acid on cellular energy production and downstream induction of the apoptotic program in HEK293, HeLa, MCF7, and HepG2 cells. At physiologically relevant doses, ellagic acid has pleiotropic and cell-type specific effects on mitochondrial function. At high doses ellagic acid can also influence glycolytic pathways and induce cell death. Our results demonstrate that ellagic acid can influence mitochondrial function at therapeutically relevant concentrations. The observed effects of ellagic acid on cellular respiration are complex and cell type-specific, which may limit the chemotherapeutic utility of this compound.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ácido Elágico/farmacologia , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 6/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Fatores de Tempo
7.
J Mol Cell Cardiol ; 112: 95-103, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28923351

RESUMO

Calcium plays an integral role to many cellular processes including contraction, energy metabolism, gene expression, and cell death. The inositol 1, 4, 5-trisphosphate receptor (IP3R) is a calcium channel expressed in cardiac tissue. There are three IP3R isoforms encoded by separate genes. In the heart, the IP3R-2 isoform is reported to being most predominant with regards to expression levels and functional significance. The functional roles of IP3R-1 and IP3R-3 in the heart are essentially unexplored despite measureable expression levels. Here we show that all three IP3Rs isoforms are expressed in both neonatal and adult rat ventricular cardiomyocytes, and in human heart tissue. The three IP3R proteins are expressed throughout the cardiomyocyte sarcoplasmic reticulum. Using isoform specific siRNA, we found that expression of all three IP3R isoforms are required for hypertrophic signaling downstream of endothelin-1 stimulation. Mechanistically, IP3Rs specifically contribute to activation of the hypertrophic program by mediating the positive inotropic effects of endothelin-1 and leading to downstream activation of nuclear factor of activated T-cells. Our findings highlight previously unidentified functions for IP3R isoforms in the heart with specific implications for hypertrophic signaling in animal models and in human disease.


Assuntos
Cardiomegalia/metabolismo , Hiperglicemia/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/complicações , Cardiomegalia/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/efeitos dos fármacos , Citosol/metabolismo , Endotelina-1/farmacologia , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Hiperglicemia/patologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fatores de Transcrição NFATC/metabolismo , Isoformas de Proteínas/metabolismo , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Front Oncol ; 7: 138, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28706877

RESUMO

Calcium is a critical regulator of cell death pathways. One of the most proximal events leading to cell death is activation of plasma membrane and endoplasmic reticulum-resident calcium channels. A large body of evidence indicates that defects in this pathway contribute to cancer development. Although we have a thorough understanding of how downstream elevations in cytosolic and mitochondrial calcium contribute to cell death, it is much less clear how calcium channels are activated upstream of the apoptotic stimulus. Recently, it has been shown that protein lipidation is a potent regulator of apoptotic signaling. Although classically thought of as a static modification, rapid and reversible protein acylation has emerged as a new signaling paradigm relevant to many pathways, including calcium release and cell death. In this review, we will discuss the role of protein lipidation in regulating apoptotic calcium signaling with direct therapeutic relevance to cancer.

9.
Cell Calcium ; 61: 44-49, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28073595

RESUMO

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.


Assuntos
Apoptose , Cálcio/metabolismo , Calmodulina/metabolismo , Apoptose/efeitos dos fármacos , Calmodulina/genética , Células HeLa , Humanos , Estaurosporina/farmacologia , Fatores de Tempo
10.
Biochim Biophys Acta Mol Cell Res ; 1864(6): 907-914, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27884701

RESUMO

Calcium is a second messenger that regulates almost all cellular functions. In cardiomyocytes, calcium plays an integral role in many functions including muscle contraction, gene expression, and cell death. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of calcium channels that are ubiquitously expressed in all tissues. In the heart, IP3Rs have been associated with regulation of cardiomyocyte function in response to a variety of neurohormonal agonists, including those implicated in cardiac disease. Notably, IP3R activity is thought to be essential for mediating the hypertrophic response to multiple stimuli including endothelin-1 and angiotensin II. In this review, we will explore the functional implications of IP3R activity in the heart in health and disease.


Assuntos
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miocárdio/metabolismo , Animais , Cardiomegalia/metabolismo , Humanos
11.
Am J Obstet Gynecol ; 215(6): 750.e1-750.e8, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27371355

RESUMO

BACKGROUND: Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors primarily used for treatment of hyperlipidemia. Recently, they have been shown to inhibit proliferation of uterine fibroid cells and inhibit tumor growth in fibroid animal models. OBJECTIVE: We sought to examine the association between statin use and the risk of uterine fibroids and fibroid-related symptoms in a nationally representative sample of commercially insured women diagnosed with hyperlipidemia. STUDY DESIGN: We performed a nested case-control study of >190,000 women enrolled in one of the nation's largest commercial health insurance programs. From a cohort of women aged 18-65 years diagnosed with hyperlipidemia from January 2004 through March 2011, we identified 47,713 cases (women diagnosed with uterine fibroids) and 143,139 controls (women without uterine fibroids) matched at a 1:3 ratio on event/index date (month and year) and age (±1 year). We used conditional and unconditional logistic regression to calculate odds ratios and 95% confidence intervals for the risk of uterine fibroids and fibroid-related symptoms associated with prior use of statins. RESULTS: Exposure to statins within 2 years before the event/index date was associated with a decreased risk of uterine fibroids (odds ratio, 0.85; 95% confidence interval, 0.83-0.87). In a separate subanalysis restricted to cases, statin users had a lower likelihood of having menorrhagia (odds ratio, 0.88; 95% confidence interval, 0.84-0.91), anemia (odds ratio, 0.84; 95% confidence interval, 0.79-0.88), or pelvic pain (odds ratio, 0.85; 95% confidence interval, 0.81-0.91) and of undergoing myomectomy (odds ratio, 0.76; 95% confidence interval, 0.66-0.87) compared to nonusers. CONCLUSION: The use of statins was associated with a lower risk of uterine fibroids and fibroid-related symptoms. Further studies, including randomized controlled trials, may be warranted.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperlipidemias/tratamento farmacológico , Histerectomia/estatística & dados numéricos , Leiomioma/epidemiologia , Menorragia/epidemiologia , Dor Pélvica/epidemiologia , Miomectomia Uterina/estatística & dados numéricos , Neoplasias Uterinas/epidemiologia , Adolescente , Adulto , Idoso , Anemia/epidemiologia , Estudos de Casos e Controles , Feminino , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Razão de Chances , Fatores de Proteção , Estados Unidos/epidemiologia , Adulto Jovem
12.
Proc Natl Acad Sci U S A ; 112(38): 11876-80, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26351666

RESUMO

Palmitoylation is the posttranslational modification of proteins with a 16-carbon fatty acid chain through a labile thioester bond. The reversibility of protein palmitoylation and its profound effect on protein function suggest that this modification could play an important role as an intracellular signaling mechanism. Evidence that palmitoylation of proteins occurs with the kinetics required for signal transduction is not clear, however. Here we show that engagement of the Fas receptor by its ligand leads to an extremely rapid and transient increase in palmitoylation levels of the tyrosine kinase Lck. Lck palmitoylation kinetics are consistent with the activation of downstream signaling proteins, such as Zap70 and PLC-γ1. Inhibiting Lck palmitoylation not only disrupts proximal Fas signaling events, but also renders cells resistant to Fas-mediated apoptosis. Knockdown of the palmitoyl acyl transferase DHHC21 eliminates activation of Lck and downstream signaling after Fas receptor stimulation. Our findings demonstrate highly dynamic Lck palmitoylation kinetics that are essential for signaling downstream of the Fas receptor.


Assuntos
Lipoilação , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Transdução de Sinais , Receptor fas/metabolismo , Aciltransferases/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Células Jurkat , Lipoilação/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Ácido Palmítico/metabolismo , Fosfolipase C gama/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Coloração e Rotulagem , Linfócitos T/metabolismo , Temperatura
13.
Am J Obstet Gynecol ; 213(2): 196.e1-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25840272

RESUMO

OBJECTIVE: Uterine leiomyomas represent a common gynecologic problem with no satisfactory long-term medical treatment. The purpose of this study is to examine the effects of simvastatin on uterine leiomyoma, both in vitro and in vivo. STUDY DESIGN: This is a laboratory-based experimental study. For in vitro studies, we used human and rat leiomyoma cells. For in vivo studies, we used immunodeficient mice supplemented with estrogen/progesterone pellets xenografted with human leiomyoma tissue explant. RESULTS: For in vitro studies, cells were treated with different concentrations of simvastatin for 48 hours. Simvastatin induced dose-dependent apoptosis in leiomyoma cells as measured by a fluorometric caspase-3 activity assay, and inhibited proliferation as demonstrated by an (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (both were significant at 5 and 10 µM). In addition, simvastatin decreased Akt signaling pathway phosphorylation as examined using Western blot analysis. For in vivo studies, animals were treated for 28 days with simvastatin (20 µg/gm body weight/day) vs vehicle control. The treatment inhibited tumor growth as measured weekly using calipers and/ or ultrasound (P < .01). Finally, simvastatin decreased expression of the proliferation marker Ki67 in xenograft tumor tissue as examined by immunohistochemistry (P = .02). CONCLUSION: Simvastatin can be a promising treatment for uterine leiomyoma. Further studies, including pharmacokinetic and drug delivery studies, are required.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Leiomioma/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Sinvastatina/farmacologia , Neoplasias Uterinas/metabolismo , Animais , Linhagem Celular Tumoral , Estrogênios/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Antígeno Ki-67/efeitos dos fármacos , Antígeno Ki-67/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Progesterona/farmacologia , Progestinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Mol Med ; 21: 242-56, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25879625

RESUMO

Uterine leiomyomas are the most common tumors of the female genital tract, affecting 50% to 70% of females by the age of 50. Despite their prevalence and enormous medical and economic impact, no effective medical treatment is currently available. This is, in part, due to the poor understanding of their underlying pathobiology. Although they are thought to start as a clonal proliferation of a single myometrial smooth muscle cell, these early cytogenetic alterations are considered insufficient for tumor development and additional complex signaling pathway alterations are crucial. These include steroids, growth factors, transforming growth factor-beta (TGF-ß)/Smad; wingless-type (Wnt)/ß-catenin, retinoic acid, vitamin D, and peroxisome proliferator-activated receptor γ (PPARγ). An important finding is that several of these pathways converge in a summative way. For example, mitogen-activated protein kinase (MAPK) and Akt pathways seem to act as signal integrators, incorporating input from several signaling pathways, including growth factors, estrogen and vitamin D. This underlines the multifactorial origin and complex nature of these tumors. In this review, we aim to dissect these pathways and discuss their interconnections, aberrations and role in leiomyoma pathobiology. We also aim to identify potential targets for development of novel therapeutics.


Assuntos
Leiomioma/metabolismo , Transdução de Sinais , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leiomioma/etiologia , Leiomioma/terapia , Esteroides/metabolismo
15.
J Biol Chem ; 290(11): 7304-13, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25645916

RESUMO

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.


Assuntos
Apoptose , Proteína BRCA1/metabolismo , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Modelos Moleculares , Neoplasias/metabolismo
16.
J Biol Chem ; 289(51): 35075-86, 2014 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-25359773

RESUMO

Statins are drugs commonly used for the treatment of high plasma cholesterol levels. Beyond these well known lipid-lowering properties, they possess broad-reaching effects in vivo, including antitumor effects. Statins inhibit the growth of multiple tumors. However, the mechanisms remain incompletely understood. Here we show that simvastatin inhibits the proliferation of human leiomyoma cells. This was associated with decreased mitogen-activated protein kinase signaling and multiple changes in cell cycle progression. Simvastatin potently stimulated leiomyoma cell apoptosis in a manner mechanistically dependent upon apoptotic calcium release from voltage-gated calcium channels. Therefore, simvastatin possesses antitumor effects that are dependent upon the apoptotic calcium release machinery.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Sinvastatina/farmacologia , Western Blotting , Canais de Cálcio Tipo L/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Leiomioma/metabolismo , Leiomioma/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos
17.
Sci Signal ; 7(345): ra93, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25270259

RESUMO

The guanine nucleotide exchange factor SLAT (SWAP-70-like adaptor of T cells) regulates T cell activation and differentiation by enabling Ca(2+) release from intracellular stores in response to stimulation of the T cell receptor (TCR). We found a TCR-induced association between SLAT and inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1). The N-terminal region of SLAT, which contains two EF-hand motifs that we determined bound Ca(2+), and the SLAT pleckstrin homology (PH) domain independently bound to IP3R1 by associating with a conserved motif within the IP3R1 ligand-binding domain. Disruption of the SLAT-IP3R1 interaction with cell-permeable, IP3R1-based fusion peptides inhibited TCR-stimulated Ca(2+) signaling, activation of the transcription factor NFAT (nuclear factor of activated T cells), and production of cytokines, suggesting that this interaction is required for optimal T cell activation. The finding that SLAT is an IP3R1-interacting protein required for T cell activation suggests that this interaction could be a potential target for a selective immunosuppressive drug.


Assuntos
Sinalização do Cálcio/imunologia , Proteínas de Ligação a DNA/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ativação Linfocitária/imunologia , Proteínas Nucleares/metabolismo , Linfócitos T/imunologia , Animais , Western Blotting , Fracionamento Celular , Proteínas de Ligação a DNA/genética , Vetores Genéticos/genética , Fatores de Troca do Nucleotídeo Guanina , Imunoprecipitação , Interferon gama/metabolismo , Luciferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Linfócitos T/metabolismo
18.
FEBS Lett ; 588(21): 3977-81, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-25240199

RESUMO

Molecular chaperones are required for successful folding and assembly of sarcomeric myosin in skeletal and cardiac muscle. Here, we show that the chaperone UNC-45B inhibits the actin translocation function of myosin. Further, we show that Hsp90, another chaperone involved in sarcomere development, allows the myosin to resume actin translocation. These previously unknown activities may play a key role in sarcomere development, preventing untimely myosin powerstrokes from disrupting the precise alignment of the sarcomere until it has formed completely.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miosinas/metabolismo , Sarcômeros/metabolismo , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Chaperonas Moleculares , Movimento , Estrutura Terciária de Proteína , Coelhos
19.
PLoS One ; 9(1): e87513, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24475300

RESUMO

Huntington's Disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in huntingtin (htt) protein. The expansion leads to increased htt aggregation and toxicity. Factors that aid in the clearance of mutant huntingtin proteins should relieve the toxicity. We previously demonstrated that overexpression of ubiqulin-1, which facilitates protein clearance through the proteasome and autophagy pathways, reduces huntingtin aggregates and toxicity in mammalian cell and invertebrate models of HD. Here we tested whether overexpression of ubiquilin-1 delays or prevents neurodegeneration in R6/2 mice, a well-established model of HD. We generated transgenic mice overexpressing human ubiquilin-1 driven by the neuron-specific Thy1.2 promoter. Immunoblotting and immunohistochemistry revealed robust and widespread overexpression of ubiquilin-1 in the brains of the transgenic mice. Similar analysis of R6/2 animals revealed that ubiquilin is localized in huntingtin aggregates and that ubiquilin levels decrease progressively to 30% during the end-stage of disease. We crossed our ubiquilin-1 transgenic line with R6/2 mice to assess whether restoration of ubiquilin levels would delay HD symptoms and pathology. In the double transgenic progeny, ubiquilin levels were fully restored, and this correlated with a 20% increase in lifespan and a reduction in htt inclusions in the hippocampus and cortex. Furthermore, immunoblots indicated that endoplasmic reticulum stress response that is elevated in the hippocampus of R6/2 animals was attenuated by ubiquilin-1 overexpression. However, ubiquilin-1 overexpression neither altered the load of htt aggregates in the striatum nor improved motor impairments in the mice.


Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Doença de Huntington/tratamento farmacológico , Proteínas do Tecido Nervoso/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Análise de Variância , Animais , Proteínas Relacionadas à Autofagia , Encéfalo/patologia , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Cruzamentos Genéticos , Primers do DNA/genética , Humanos , Proteína Huntingtina , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Teste de Desempenho do Rota-Rod
20.
Mol Med ; 19: 72-8, 2013 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-23508570

RESUMO

The first 24 h following burn injury is known as the ebb phase and is characterized by a depressed metabolic rate. While the postburn ebb phase has been well described, the molecular mechanisms underlying this response are poorly understood. The endoplasmic reticulum (ER) regulates metabolic rate by maintaining glucose homeostasis through the hepatic ER stress response. We have shown that burn injury leads to ER stress in the liver during the first 24 h following thermal injury. However, whether ER stress is linked to the metabolic responses during the ebb phase of burn injury is poorly understood. Here, we show in an animal model that burn induces activation of activating transcription factor 6 (ATF6) and inositol requiring enzyme-1 (IRE-1) and this leads to increased expression of spliced X-box binding protein-1 (XBP-1s) messenger ribonucleic acid (mRNA) during the ebb phase. This is associated with increased expression of XBP-1 target genes and downregulation of the key gluconeogenic enzyme glucose-6-phosphatase (G6Pase). We conclude that upregulation of the ER stress response after burn injury is linked to attenuated gluconeogenesis and sustained glucose tolerance in the postburn ebb phase.


Assuntos
Queimaduras/genética , Proteínas de Ligação a DNA/genética , Estresse do Retículo Endoplasmático/genética , Gluconeogênese/genética , Fatores de Transcrição/genética , Animais , Glicemia/análise , Fatores de Transcrição Forkhead/metabolismo , Glucose-6-Fosfatase/metabolismo , Insulina/sangue , Fígado/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição de Fator Regulador X , Proteína 1 de Ligação a X-Box
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