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Shahzina Kanwal Yann Fardini Patrick Pagesy Thierry N’Tumba-Byn Cécile Pierre-Eugène Elodie Masson Cornelia Hampe Tarik Issad 《PloS one》2013,8(7)
O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine
residues) is a post-translational modification that regulates stability,
activity or localization of cytosolic and nuclear proteins. O-linked
N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the
hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc
from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent
evidences suggest that O-GlcNAcylation may affect the growth of cancer cells.
However, the consequences of O-GlcNAcylation on anti-cancer therapy have not
been evaluated. In this work, we studied the effects of O-GlcNAcylation on
tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells.
Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected
MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT
expression by siRNA potentiated the effect of tamoxifen on cell death. Since the
PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to
evaluate the effect of PUGNAc+glucosamine on PIP3 production. We
observed that these treatments stimulated PIP3 production in MCF-7
cells. This effect was associated with an increase in Akt phosphorylation.
However, the PI-3 kinase inhibitor , which abolished the effect of
PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects
of PUGNAc+glucosamine against tamoxifen-induced cell death. These results
suggest that the protective effects of O-GlcNAcylation are independent of the
PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen
receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine
on the expression of this receptor. We observed that O-GlcNAcylation-inducing
treatment significantly reduced the expression of ERα mRNA and protein,
suggesting a potential mechanism for the decreased tamoxifen sensitivity induced
by these treatments. Therefore, our results suggest that inhibition of
O-GlcNAcylation may constitute an interesting approach to improve the
sensitivity of breast cancer to anti-estrogen therapy. LY294002相似文献
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Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
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Virginija Jazbutyte Franz Kehl Theo Pelzer 《Biochemical and biophysical research communications》2009,384(4):450-6
Estrogen receptor alpha (ERα) is present in the nucleus, the cytosol and in mitochondria. The rat ERα ligand binding domain was employed as bait in a bacterial two-hybrid screening of a human heart cDNA library to detect novel protein-protein interaction partners of ERα in the heart. 17β-Hydroxysteroid dehydrogenase type 10 (17β-HSD10), which converts potent (17β-estradiol) to less potent estrogens (estrone), co-localized with 17β-HSD10 in the mitochondria of rat cardiac myocytes. GST pull-down experiments confirmed the interaction of ERα and 17β-HSD10. These findings suggest that the ERα estrogen receptor might be involved in regulating intracellular estrogen levels by modulating 17β-HSD10 activity. 相似文献
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Cheng-Ping Yu Jar-Yi Ho Yi-Ting Huang Tai-Lung Cha Guang-Huan Sun Dah-Shyong Yu Fung-Wei Chang Shu-Pin Chen Ren-Jun Hsu 《PloS one》2013,8(2)
Renal cell carcinoma (RCC) originates in the lining of the proximal convoluted tubule and accounts for approximately 3% of adult malignancies. The RCC incidence rate increases annually and is twofold higher in males than in females. Female hormones such as estrogen may play important roles during RCC carcinogenesis and result in significantly different incidence rates between males and females. In this study, we found that estrogen receptor β (ERβ) was more highly expressed in RCC cell lines (A498, RCC-1, 786-O, ACHN, and Caki-1) than in breast cancer cell lines (MCF-7 and HBL-100); however, no androgen receptor (AR) or estrogen receptor α (ERα) could be detected by western blot. In addition, proliferation of RCC cell lines was significantly decreased after estrogen (17-β-estradiol, E2) treatment. Since ERβ had been documented to be a potential tumor suppressor gene, we hypothesized that estrogen activates ERβ tumor suppressive function, which leads to different RCC incidence rates between males and females. We found that estrogen treatment inhibited cell proliferation, migration, invasion, and increased apoptosis of 786-O (high endogenous ERβ), and ERβ siRNA-induced silencing attenuated the estrogen-induced effects. Otherwise, ectopic ERβ expression in A498 (low endogenous ERβ) increased estrogen sensitivity and thus inhibited cell proliferation, migration, invasion, and increased apoptosis. Analysis of the molecular mechanisms revealed that estrogen-activated ERβ not only remarkably reduced growth hormone downstream signaling activation of the AKT, ERK, and JAK signaling pathways but also increased apoptotic cascade activation. In conclusion, this study found that estrogen-activated ERβ acts as a tumor suppressor. It may explain the different RCC incidence rates between males and females. Furthermore, it implies that ERβ may be a useful prognostic marker for RCC progression and a novel developmental direction for RCC treatment improvement. 相似文献
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Orwah Saleh Bertolt Gust Bj?rn Boll Hans-Peter Fiedler Lutz Heide 《The Journal of biological chemistry》2009,284(21):14439-14447
The bacterium Streptomyces anulatus 9663, isolated from the
intestine of different arthropods, produces prenylated derivatives of
phenazine 1-carboxylic acid. From this organism, we have identified the
prenyltransferase gene ppzP. ppzP resides in a gene cluster
containing orthologs of all genes known to be involved in phenazine
1-carboxylic acid biosynthesis in Pseudomonas strains as well as
genes for the six enzymes required to generate dimethylallyl diphosphate via
the mevalonate pathway. This is the first complete gene cluster of a phenazine
natural compound from streptomycetes. Heterologous expression of this cluster
in Streptomyces coelicolor M512 resulted in the formation of
prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of
ppzP, only nonprenylated phenazine 1-carboxylic acid was formed.
Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble
protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate
dimethylallyltransferase, forming a C–C bond between C-1 of the
isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many
other prenyltransferases, the reaction of PpzP is independent of the presence
of magnesium or other divalent cations. The Km value for
dimethylallyl diphosphate was determined as 116 μm. For
dihydro-PCA, half-maximal velocity was observed at 35 μm.
Kcat was calculated as 0.435 s-1. PpzP shows
obvious sequence similarity to a recently discovered family of
prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The
present finding extends the substrate range of this family, previously limited
to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives
rise to an astounding diversity of secondary metabolites in bacteria, fungi,
and plants, including many compounds that are important in pharmacotherapy.
However, surprisingly little biochemical and genetic data are available on the
enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new
family of aromatic prenyltransferases was discovered in streptomycetes
(1), Gram-positive soil
bacteria that are prolific producers of antibiotics and other biologically
active compounds (2). The
members of this enzyme family show a new type of protein fold with a unique
α-β-β-α architecture
(3) and were therefore termed
ABBA prenyltransferases (1).
Only 13 members of this family can be identified by sequence similarity
searches in the data base at present, and only four of them have been
investigated biochemically
(3–6).
Up to now, only phenolic compounds have been identified as aromatic substrates
of ABBA prenyltransferases. We now report the discovery of a new member of the
ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl
moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate
(dihydro-PCA).2
Streptomyces strains produce many of prenylated phenazines as natural
products. For the first time, the present paper reports the identification of
a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of
different arthropods, produces several prenylated phenazines, among them
endophenazine A and B (Fig.
1A) (7).
We wanted to investigate which type of prenyltransferase might catalyze the
prenylation reaction in endophenazine biosynthesis. In streptomycetes and
other microorganisms, genes involved in the biosynthesis of a secondary
metabolite are nearly always clustered in a contiguous DNA region. Therefore,
the prenyltransferase of endophenazine biosynthesis was expected to be
localized in the vicinity of the genes for the biosynthesis of the phenazine
core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663.
B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG
is responsible for the biosynthesis of PCA
(8). The enzyme PhzC catalyzes
the condensation of phosphoenolpyruvate and erythrose-4-phosphate
(i.e. the first step of the shikimate pathway), and further enzymes
of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze
the conversion of chorismate to 2-amino-2-deoxyisochorismate and the
subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively.
These reactions are well established biochemically. Fewer data are available
about the following steps (i.e. dimerization of
2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a
decarboxylation, ultimately leading to PCA via several instable
intermediates). From Pseudomonas, experimental data on the role of
PhzF and PhzA/B have been published
(8,
9), whereas the role of PhzG is
yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis
described so far from streptomycetes
(10) was found not to contain
a phzF orthologue, raising the question of whether there may be
differences in the biosynthesis of phenazines between Pseudomonas and
Streptomyces.Screening of a genomic library of the endophenazine producer strain S.
anulatus now allowed the identification of the first complete gene
cluster of a prenylated phenazine, including the structural gene of
dihydro-PCA dimethylallyltransferase. 相似文献
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Haihong Zong Claire C. Bastie Jun Xu Reinhard Fassler Kevin P. Campbell Irwin J. Kurland Jeffrey E. Pessin 《The Journal of biological chemistry》2009,284(7):4679-4688
Integrin receptor plays key roles in mediating both inside-out and
outside-in signaling between cells and the extracellular matrix. We have
observed that the tissue-specific loss of the integrin β1 subunit in
striated muscle results in a near complete loss of integrin β1 subunit
protein expression concomitant with a loss of talin and to a lesser extent, a
reduction in F-actin content. Muscle-specific integrin β1-deficient mice
had no significant difference in food intake, weight gain, fasting glucose,
and insulin levels with their littermate controls. However, dynamic analysis
of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a
44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose
clearance, respectively. The whole body insulin resistance resulted from a
specific inhibition of skeletal muscle glucose uptake and glycogen synthesis
without any significant effect on the insulin suppression of hepatic glucose
output or insulin-stimulated glucose uptake in adipose tissue. The reduction
in skeletal muscle insulin responsiveness occurred without any change in GLUT4
protein expression levels but was associated with an impairment of the
insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not
threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation
occurred concomitantly with a decrease in integrin-linked kinase expression
but with no change in the mTOR·Rictor·LST8 complex (mTORC2).
These data demonstrate an in vivo crucial role of integrin β1
signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins
composed of a single α and β subunit assembled into a heterodimeric
complex. There are 19 α and 8 β mammalian subunit isoforms that
combine to form 25 distinct α,β heterodimeric receptors
(1-5).
These receptors play multiple critical roles in conveying extracellular
signals to intracellular responses (outside-in signaling) as well as altering
extracellular matrix interactions based upon intracellular changes (inside-out
signaling). Despite the large overall number of integrin receptor complexes,
skeletal muscle integrin receptors are limited to seven α subunit
subtypes (α1, α3, α4, α5, α6, α7, and
αν subunits), all associated with the β1 integrin subunit
(6,
7).Several studies have suggested an important cross-talk between
extracellular matrix and insulin signaling. For example, engagement of β1
subunit containing integrin receptors was observed to increase
insulin-stimulated insulin receptor substrate
(IRS)2
phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation
of protein kinase B/Akt
(8-11).
Integrin receptor regulation of focal adhesion kinase was reported to modulate
insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton
organization in cultured hepatocytes and myoblasts
(12,
13). Similarly, the
integrin-linked kinase (ILK) was suggested to function as one of several
potential upstream kinases that phosphorylate and activate Akt
(14-18).
In this regard small interfering RNA gene silencing of ILK in fibroblasts and
conditional ILK gene knockouts in macrophages resulted in a near complete
inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation
concomitant with an inhibition of Akt activity and phosphorylation of Akt
downstream targets (19).
However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2)
has been identified in several other studies as the Akt Ser-473 kinase
(20,
21). In addition to Ser-473,
Akt protein kinase activation also requires phosphorylation on threonine 308
Thr-30 by phosphoinositide-dependent protein kinase, PDK1
(22-24).In vivo, skeletal muscle is the primary tissue responsible for
postprandial (insulin-stimulated) glucose disposal that results from the
activation of signaling pathways leading to the translocation of the
insulin-responsive glucose transporter, GLUT4, from intracellular sites to the
cell surface membranes (25,
26). Dysregulation of any step
of this process in skeletal muscle results in a state of insulin resistance,
thereby predisposing an individual for the development of diabetes
(27-33).
Although studies described above have utilized a variety of tissue culture
cell systems to address the potential involvement of integrin receptor
signaling in insulin action, to date there has not been any investigation of
integrin function on insulin action or glucose homeostasis in vivo.
To address this issue, we have taken advantage of Cre-LoxP technology to
inactivate the β1 integrin receptor subunit gene in striated muscle. We
have observed that muscle creatine kinase-specific integrin β1 knock-out
(MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose
infusion rate and glucose clearance. The impairment of insulin-stimulated
skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease
in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK
expression. Together, these data demonstrate an important cross-talk between
integrin receptor function and insulin action and suggests that ILK may
function as an Akt Ser-473 kinase in skeletal muscle. 相似文献
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Roujian Lu Yong Li Youwen Zhang Yunjia Chen Angela D. Shields Danny G. Winder Timothy Angelotti Kai Jiao Lee E. Limbird Yi Zhou Qin Wang 《The Journal of biological chemistry》2009,284(19):13233-13243
Although ligand-selective regulation of G protein-coupled receptor-mediated
signaling and trafficking are well documented, little is known about whether
ligand-selective effects occur on endogenous receptors or whether such effects
modify the signaling response in physiologically relevant cells. Using a gene
targeting approach, we generated a knock-in mouse line, in which N-terminal
hemagglutinin epitope-tagged α2A-adrenergic receptor (AR)
expression was driven by the endogenous mouse α2AAR gene
locus. Exploiting this mouse line, we evaluated α2AAR
trafficking and α2AAR-mediated inhibition of Ca2+
currents in native sympathetic neurons in response to clonidine and
guanfacine, two drugs used for treatment of hypertension, attention deficit
and hyperactivity disorder, and enhancement of analgesia through actions on
the α2AAR subtype. We discovered a more rapid desensitization
of Ca2+ current suppression by clonidine than guanfacine, which
paralleled a more marked receptor phosphorylation and endocytosis of
α2AAR evoked by clonidine than by guanfacine.
Clonidine-induced α2AAR desensitization, but not receptor
phosphorylation, was attenuated by blockade of endocytosis with concanavalin
A, indicating a critical role for internalization of α2AAR in
desensitization to this ligand. Our data on endogenous receptor-mediated
signaling and trafficking in native cells reveal not only differential
regulation of G protein-coupled receptor endocytosis by different ligands, but
also a differential contribution of receptor endocytosis to signaling
desensitization. Taken together, our data suggest that these
HA-α2AAR knock-in mice will serve as an important model in
developing ligands to favor endocytosis or nonendocytosis of receptors,
depending on the target cell and pathophysiology being addressed.G protein-coupled receptors
(GPCRs)4 represent the
largest family of cell surface receptors mediating responses to hormones,
cytokines, neurotransmitters, and therapeutic agents
(1). In addition to regulating
downstream signaling, ligand binding to a receptor can initiate
phosphorylation of the active conformation of the receptor by G protein
receptor kinases (GRKs) and subsequent binding of arrestins, thus restricting
the magnitude and duration of the ligand-evoked signaling responses
(2,
3). Binding of arrestins to
GPCRs also leads to GPCR internalization
(4,
5), a process that has been
proposed as a means to desensitize receptor signaling at the cell surface,
resensitize receptors, and/or initiate intracellular signaling
(6,
7).Different ligands are able to induce distinct signaling and internalization
profiles of the same receptor
(8-14).
However, the lack of available tools to study trafficking of endogenous GPCRs
in native target cells has limited our understanding of ligand-selective
endocytosis profiles and the relative contribution of receptor endocytosis to
desensitization in native biological settings.To specifically test hypotheses regarding ligand-selective effects on GPCR
internalization, and functional consequences of this trafficking on signaling,
we utilized a homologous recombination gene targeting strategy to introduce a
hemagglutinin (HA) epitope-tagged wild type α2A-adrenergic
receptor (AR) into the mouse ADRA2A gene locus
(“knock-in”). The α2AAR is a prototypical GPCR
that couples to the Gi/o subfamily of G proteins
(15). Studies on genetically
engineered mice made null or mutant for the α2AAR have
revealed that this subtype mediates the therapeutic effects of
α2-adrenergic agents on blood pressure, pain perception,
volatile anesthetic sparing, analgesia, and working memory enhancement
(16-18).
Two classic α2-ligands, clonidine and guanfacine, have been
widely used to treat hypertension
(19), attention deficit and
hyperactivity disorder (20),
and to elicit analgesia (19,
21) mediated via the
α2AAR. Clinically guanfacine has a much longer duration of
action than clonidine
(22-24);
this longer duration of action cannot be accounted for by the pharmacokinetic
profile of these agents in human beings, as both drugs have a half-life of
12-14 h (25,
26). Because ligand-induced
desensitization and trafficking of GPCRs have been implicated as critical
mechanisms for modulating response duration in vivo
(3), one hypothesis underlying
the difference in duration between clonidine and guanfacine is that clonidine
provokes accelerated desensitization of the α2AAR via one or
several mechanisms, whereas guanfacine does not. Signaling desensitization in
response to these two agonists has not been compared under the same
experimental settings. To specifically test this hypothesis, we have exploited
our HA-α2AAR knock-in mice so that we could examine these
properties of guanfacine and clonidine in native target cells.We compared internalization of the α2AAR and inhibition of
Ca2+ currents induced by clonidine and guanfacine in primary
superior cervical ganglia (SCG) neurons, where the α2AAR is
the major adrenergic receptor subtype controlling norepinephrine release and
sympathetic tone (17,
27). Our data revealed a
differential regulation of α2AAR trafficking and signaling
duration by clonidine versus guanfacine, i.e. clonidine
induced a more dramatic desensitization of the α2AAR than
guanfacine, and this desensitization was largely because of
α2AAR internalization. These studies reveal the powerful tool
that the HA-α2AAR knock-in mice provide for identifying
endocytosis-dependent and -independent physiological phenomena for this
receptor subtype as a first step in defining novel loci for therapeutic
intervention in the α2AAR signaling/trafficking cascade. 相似文献
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Victoria L Alonso Carla Ritagliati Pamela Cribb Esteban C Serra 《Memórias do Instituto Oswaldo Cruz》2014,109(8):1081-1085
We present here three expression plasmids for Trypanosoma cruzi
adapted to the Gateway® recombination cloning system. Two of
these plasmids were designed to express trypanosomal proteins fused to a double tag
for tandem affinity purification (TAPtag). The TAPtag and Gateway®
cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid.
Both plasmids were assayed by introducing green fluorescent protein (GFP) by
recombination and the integrity of the double-tagged protein was determined by
western blotting and immunofluorescence microscopy. The third Gateway adapted vector
assayed was the inducible pTcINDEX. When tested with GFP,
pTcINDEX-GW showed a good response to tetracycline, being less
leaky than its precursor (pTcINDEX). 相似文献
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