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1.
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
2.
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
3.
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
4.
Yun Liu Yun-wu Zhang Xin Wang Han Zhang Xiaoqing You Francesca-Fang Liao Huaxi Xu 《The Journal of biological chemistry》2009,284(18):12145-12152
Excessive accumulation of β-amyloid peptides in the brain is a major
cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived
from β-amyloid precursor protein (APP) through sequential cleavages by
β- and γ-secretases, whose enzymatic activities are tightly
controlled by subcellular localization. Delineation of how intracellular
trafficking of these secretases and APP is regulated is important for
understanding Alzheimer disease pathogenesis. Although APP trafficking is
regulated by multiple factors including presenilin 1 (PS1), a major component
of the γ-secretase complex, and phospholipase D1 (PLD1), a
phospholipid-modifying enzyme, regulation of intracellular trafficking of
PS1/γ-secretase and β-secretase is less clear. Here we demonstrate
that APP can reciprocally regulate PS1 trafficking; APP deficiency results in
faster transport of PS1 from the trans-Golgi network to the cell
surface and increased steady state levels of PS1 at the cell surface, which
can be reversed by restoring APP levels. Restoration of APP in APP-deficient
cells also reduces steady state levels of other γ-secretase components
(nicastrin, APH-1, and PEN-2) and the cleavage of Notch by
PS1/γ-secretase that is more highly correlated with cell surface levels
of PS1 than with APP overexpression levels, supporting the notion that Notch
is mainly cleaved at the cell surface. In contrast, intracellular trafficking
of β-secretase (BACE1) is not regulated by APP. Moreover, we find that
PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell
surface accumulation of PS1 in an APP-independent manner. Our results clearly
elucidate a physiological function of APP in regulating protein trafficking
and suggest that intracellular trafficking of PS1/γ-secretase is
regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease
(AD)4 is the formation
of senile plaques in the brains of patients. The major components of those
plaques are β-amyloid peptides (Aβ), whose accumulation triggers a
cascade of neurodegenerative steps ending in formation of senile plaques and
intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible
brain regions (1,
2). Aβ is proteolytically
derived from the β-amyloid precursor protein (APP) through sequential
cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl
protease (3,
4), and by γ-secretase, a
high molecular weight complex consisting of at least four components:
presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and
presenilin enhancer-2 (PEN-2)
(5,
6). APP is a type I
transmembrane protein belonging to a protein family that includes APP-like
protein 1 (APLP1) and 2 (APLP2) in mammals
(7,
8). Full-length APP is
synthesized in the endoplasmic reticulum (ER) and transported through the
Golgi apparatus. Most secreted Aβ peptides are generated within the
trans-Golgi network (TGN), also the major site of steady state APP in
neurons
(9–11).
APP can be transported to the cell surface in TGN-derived secretory vesicles
if not proteolyzed to Aβ or an intermediate metabolite. At the cell
surface APP is either cleaved by α-secretase to produce soluble
sAPPα (12) or
reinternalized for endosomal/lysosomal degradation
(13,
14). Aβ may also be
generated in endosomal/lysosomal compartments
(15,
16). In contrast to neurotoxic
Aβ peptides, sAPPα possesses neuroprotective potential
(17,
18). Thus, the subcellular
distribution of APP and proteases that process it directly affect the ratio of
sAPPα to Aβ, making delineation of the mechanisms responsible for
regulating trafficking of all of these proteins relevant to AD
pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the
two mammalian PS gene homologues, PS1 and PS2, PS1
encodes the major form (PS1) in active γ-secretase
(19,
20). Nascent PSs undergo
endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a
carboxyl-terminal fragment (CTF) to form a functional PS heterodimer
(21). Based on observations
that PSs possess two highly conserved aspartate residues indispensable for
γ-secretase activity and that specific transition state analogue
γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers
(5,
22), PSs are believed to be
the catalytic component of the γ-secretase complex. PS assembles with
three other components, NCT, APH-1, and PEN-2, to form the functional
γ-secretase (5,
6). Strong evidence suggests
that PS1/γ-secretase resides principally in the ER, early Golgi, TGN,
endocytic and intermediate compartments, most of which (except the TGN) are
not major subcellular sites for APP
(23,
24). In addition to generating
Aβ and cleaving APP to release the APP intracellular domain,
PS1/γ-secretase cleaves other substrates such as Notch
(25), cadherin
(26), ErbB4
(27), and CD44
(28), releasing their
respective intracellular domains. Interestingly, PS1/γ-secretase
cleavage of different substrates seems to occur at different subcellular
compartments; APP is mainly cleaved at the TGN and early endosome domains,
whereas Notch is predominantly cleaved at the cell surface
(9,
11,
29). Thus, perturbing
intracellular trafficking of PS1/γ-secretase may alter interactions
between PS1/γ-secretase and APP, contributing to either abnormal Aβ
generation and AD pathogenesis or decreased access of PS1/γ-secretase to
APP such that Aβ production is reduced. However, mechanisms regulating
PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates
intracellular trafficking of several membrane proteins, including other
γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate
APP (reviewed in Ref. 30).
Intracellular APP trafficking is highly regulated and requires other factors
such as mint family members and SorLA
(2). Moreover, we recently
found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that
regulates membrane trafficking events, can interact with PS1, and can regulate
budding of APP-containing vesicles from the TGN and delivery of APP to the
cell surface (31,
32). Interestingly, Kamal
et al. (33)
identified an axonal membrane compartment that contains APP, BACE1, and PS1
and showed that fast anterograde axonal transport of this compartment is
mediated by APP and kinesin-I, implying a traffic-regulating role for APP.
Increased APP expression is also shown to decrease retrograde axonal transport
of nerve growth factor (34).
However, whether APP indeed regulates intracellular trafficking of proteins
including BACE1 and PS1/γ-secretase requires further validation. In the
present study we demonstrate that intracellular trafficking of PS1, as well as
that of other γ-secretase components, but not BACE1, is regulated by
APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase
complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In
addition, we find that PLD1 also regulates intracellular trafficking of PS1
through a different mechanism and more potently than APP. 相似文献
5.
6.
7.
8.
Kazuyuki Kitatani Kely Sheldon Vinodh Rajagopalan Viviana Anelli Russell W. Jenkins Ying Sun Gregory A. Grabowski Lina M. Obeid Yusuf A. Hannun 《The Journal of biological chemistry》2009,284(19):12972-12978
Activation of protein kinase C (PKC) promotes the salvage pathway of
ceramide formation, and acid sphingomyelinase has been implicated, in part, in
providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007)
J. Biol. Chem. 282, 11549–11561). In the present study, we
examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes
glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated
formation of ceramide from recycled sphingosine. Glucosylceramide levels
declined after treatment of MCF-7 cells with a potent PKC activator, phorbol
12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs
significantly attenuated acid glucocerebrosidase activity and decreased
PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced
degradation of glucosylceramide and generation of sphingosine, the source for
ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased
ceramide levels. These observations indicate that GBA1 activation can generate
the source (sphingosine) for PMA-induced formation of ceramide through the
salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in
the formation of ceramide was determined. By attenuating expression of
PKCδ, cells failed to trigger PMA-induced alterations in levels of
ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is
suggested to stimulate the degradation of both sphingomyelin and
glucosylceramide leading to the salvage pathway of ceramide formation.
Collectively, GBA1 is identified as a novel source of regulated formation of
ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which
are emerging as bioactive lipid mediators thought to play crucial roles in
cellular responses (1,
2). Ceramide, a central
sphingolipid, serves as the main precursor for various sphingolipids,
including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of
formation of ceramide has been demonstrated through the action of three major
pathways: the de novo pathway
(3,
4), the sphingomyelinase
pathway (5), and the salvage
pathway
(6–8).
The latter plays an important role in constitutive sphingolipid turnover by
salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that
serve as sphingolipid backbones for ceramide and dihydroceramide as well as
all complex sphingolipids (Fig.
1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and
inhibition of PMA induction of ceramide by fumonisin B1. A, the
scheme of the sphingosine salvage pathway of ceramide formation. B,
previously published data as to effects of fumonisin B1 on ceramide mass
profiles (23) are re-plotted
as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or
without 100 μm fumonisin B1 for 2 h followed by treatment with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Results are expressed as sum of
increased mass of ceramide species. Dotted or open columns
represents C16-ceramide or sum of other ceramide species
(C14-ceramide, C18-ceramide, C18:1-ceramide,
C20-ceramide, C24-ceramide, and
C24:1-ceramide), respectively. The data represent mean ±
S.E. of three to five values.Metabolically, ceramide is also formed from degradation of
glycosphingolipids (Fig.
1A) usually in acidic compartments, the lysosomes and/or
late endosomes (9). The
stepwise hydrolysis of complex glycosphingolipids eventually results in the
formation of glucosylceramide, which in turn is converted to ceramide by the
action of acid β-glucosidase 1
(GBA1)2
(9,
10). Severe defects in GBA1
activity cause Gaucher disease, which is associated with aberrant accumulation
of the lipid substrates
(10–14).
On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also
form ceramide (15,
16). Either process results in
the generation of lysosomal ceramide that can then be deacylated by acid
ceramidase (17), releasing
sphingosine that may escape the lysosome
(18). The released sphingosine
may become a substrate for either sphingosine kinases or ceramide synthases,
forming sphingosine 1-phosphate or ceramide, respectively
(3,
19–21).In a related line of investigation, our studies
(20,
22,
23) have begun to implicate
protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage
pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol
ester (PMA) was shown to stimulate the salvage pathway resulting in increases
in ceramide. All the induced ceramide was inhibited by pretreatment with a
ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating
acute activation of the de novo pathway and establishing a role for
ceramide synthesis (20,
23). Moreover, labeling
studies also implicated the salvage pathway because PMA induced turnover of
steady state-labeled sphingolipids but did not affect de novo labeled
ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream
molecule for the activation of acid sphingomyelinase in the salvage pathway
(22). Interestingly, the
PKCδ isoform induced the phosphorylation of acid sphingomyelinase at
serine 508, leading to its activation and consequent formation of ceramide.
The activation of acid sphingomyelinase appeared to contribute to ∼50% of
the salvage pathway-induced increase in ceramide
(28) (also, see
Fig. 4C). This raised
the possibility that distinct routes of ceramide metabolism may account for
the remainder of ceramide generation. In this study, we investigated
glucocerebrosidase GBA1 as a candidate for one of the other routes accounting
for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide
after PMA treatment. A, MCF-7 cells were transfected with 5
nm siRNAs of each of four individual sequences (SCR, GBA1-a,
GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA
for 1 h. Lipids were extracted, and then the levels of the
C16-ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to nine values. B, MCF-7 cells were transfected with 5
nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
individual ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to five values. C14-Cer,
C14-ceramide; C16-Cer,
C16-ceramide; C18-Cer;
C18-ceramide; C18:1-Cer,
C18:1-ceramide; C20-Cer,
C20-ceramide; C20-Cer,
C24-ceramide; C24:1-Cer,
C24:1-ceramide. C, MCF-7 cells were transfected with 5
nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a
(GBA1) for 48 h following stimulation with (PMA) or without
(Control) 100 nm PMA for 1 h. Lipids were extracted, and
then the levels of ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Levels of C16-ceramide are
shown. The data represent mean ± S.E. of four to five values.
Significant changes from SCR-transfected cells treated with PMA are shown in
A–C (*, p < 0.02; **,
p < 0.05; ***, p < 0.01). 相似文献
9.
Control of TANK-binding Kinase 1-mediated Signaling by the
��134.5 Protein of Herpes Simplex Virus
1
Dustin Verpooten Yijie Ma Songwang Hou Zhipeng Yan Bin He 《The Journal of biological chemistry》2009,284(2):1097-1105
TANK-binding kinase 1 (TBK1) is a key component of Toll-like
receptor-dependent and -independent signaling pathways. In response to
microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and
cytokine expression. Here we show that TBK1 is a novel target of the
γ134.5 protein, a virulence factor whose expression is
regulated in a temporal fashion. Remarkably, the γ134.5
protein is required to inhibit IRF3 phosphorylation, nuclear translocation,
and the induction of antiviral genes in infected cells. When expressed in
mammalian cells, the γ134.5 protein forms complexes with TBK1
and disrupts the interaction of TBK1 and IRF3, which prevents the induction of
interferon and interferon-stimulated gene promoters. Down-regulation of TBK1
requires the amino-terminal domain. In addition, unlike wild type virus, a
herpes simplex virus mutant lacking γ134.5 replicates
efficiently in TBK1-/- cells but not in TBK1+/+ cells.
Addition of exogenous interferon restores the antiviral activity in both
TBK1-/- and TBK+/+ cells. Hence, control of
TBK1-mediated cell signaling by the γ134.5 protein
contributes to herpes simplex virus infection. These results reveal that TBK1
plays a pivotal role in limiting replication of a DNA virus.Herpes simplex virus 1
(HSV-1)3 is a large
DNA virus that establishes latent or lytic infection, in which the virus
triggers innate immune responses. In HSV-infected cells, a number of antiviral
mechanisms operate in a cell type- and time-dependent manner
(1). In response to
double-stranded RNA (dsRNA), Toll-like receptor 3 (TLR3) recruits an adaptor
TIR domain-containing adaptor inducing IFN-β and stimulates cytokine
expression (2,
3). In the cytoplasm, RNA
helicases, RIG-I (retinoid acid-inducible gene-I), and MDA5 (melanoma
differentiation associated gene 5) recognize intracellular viral
5′-triphosphate RNA or dsRNA
(2,
4). Furthermore, a
DNA-dependent activator of IFN-regulatory factor (DAI) senses double-stranded
DNA in the cytoplasm and induces cytokine expression
(5). There is also evidence
that viral entry induces antiviral programs independent of TLR and RIG-I
pathways (6). While recognizing
distinct viral components, these innate immune pathways relay signals to the
two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB
kinase (IKKi) (2).The IKK-related kinases function as essential components that phosphorylate
IRF3 (interferon regulatory factor 3), as well as the closely related IRF7,
which translocates to the nucleus and induces antiviral genes, such as
interferon-α/β and ISG56 (interferon-stimulated gene 56)
(7,
8). TBK1 is constitutively
expressed, whereas IKKi is engaged as an inducible gene product of innate
immune signaling (9,
10). IRF3 activation is
attenuated in TBK1-deficient but not in IKKi-deficient cells
(11,
12). Its activation is
completely abolished in double-deficient cells
(12), suggesting a partially
redundant function of TBK1 and IKKi. Indeed, IKKi also negatively regulates
the STAT-signaling pathway
(13). TBK1/IKKi interacts with
several proteins, such as TRAF family member-associated NF-κB activator
(TANK), NAP1 (NAK-associated protein 1), similar to NAP1TBK1 adaptor
(SINTBAD), DNA-dependent activator of IFN-regulatory factors (DAI), and
secretory protein 5 (Sec5) in host cells
(5,
14–18).
These interactions are thought to regulate TBK1/IKKi, which delineates innate
as well as adaptive immune responses.Upon viral infection, expression of HSV proteins interferes with the
induction of antiviral immunity. When treated with UV or cycloheximide, HSV
induces an array of antiviral genes in human lung fibroblasts
(19,
20). Furthermore, an HSV
mutant, with deletion in immediate early protein ICP0, induces ISG56
expression (21). Accordingly,
expression of ICP0 inhibits the induction of antiviral programs mediated by
IRF3 or IRF7
(21–23).
However, although ICP0 negatively regulates IFN-β expression, it is not
essential for this effect
(24). In HSV-infected human
macrophages or dendritic cells, an immediate early protein ICP27 is required
to suppress cytokine induction involving IRF3
(25). In this context, it is
notable that an HSV mutant, lacking a leaky late gene γ134.5,
replicates efficiently in cells devoid of IFN-α/β genes
(26). Additionally, the
γ134.5 null mutant induces differential cytokine expression
as compared with wild type virus
(27). Thus, HSV modulation of
cytokine expression is a complex process that involves multiple viral
components. Currently, the molecular mechanism governing this event is
unclear. In this study, we show that HSV γ134.5 targets TBK1
and inhibits antiviral signaling. The data herein reveal a previously
unrecognized mechanism by which γ134.5 facilitates HSV
replication. 相似文献
10.
Johann Schredelseker Anamika Dayal Thorsten Schwerte Clara Franzini-Armstrong Manfred Grabner 《The Journal of biological chemistry》2009,284(2):1242-1251
The paralyzed zebrafish strain relaxed carries a null mutation for
the skeletal muscle dihydropyridine receptor (DHPR) β1a
subunit. Lack of β1a results in (i) reduced membrane
expression of the pore forming DHPR α1S subunit, (ii)
elimination of α1S charge movement, and (iii) impediment of
arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine
receptor (RyR1), a structural prerequisite for skeletal muscle-type
excitation-contraction (EC) coupling. In this study we used relaxed
larvae and isolated myotubes as expression systems to discriminate specific
functions of β1a from rather general functions of β
isoforms. Zebrafish and mammalian β1a subunits quantitatively
restored α1S triad targeting and charge movement as well as
intracellular Ca2+ release, allowed arrangement of DHPRs in
tetrads, and most strikingly recovered a fully motile phenotype in
relaxed larvae. Interestingly, the cardiac/neuronal
β2a as the phylogenetically closest, and the ancestral
housefly βM as the most distant isoform to β1a
also completely recovered α1S triad expression and charge
movement. However, both revealed drastically impaired intracellular
Ca2+ transients and very limited tetrad formation compared with
β1a. Consequently, larval motility was either only partially
restored (β2a-injected larvae) or not restored at all
(βM). Thus, our results indicate that triad expression and
facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common
features of all tested β subunits, whereas the efficient arrangement of
DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the
β1a isoform. Consequently, we postulate a model that presents
β1a as an allosteric modifier of α1S
conformation enabling skeletal muscle-type EC coupling.Excitation-contraction
(EC)3 coupling in
skeletal muscle is critically dependent on the close interaction of two
distinct Ca2+ channels. Membrane depolarizations of the myotube are
sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the
sarcolemma, leading to a rearrangement of charged amino acids (charge
movement) in the transmembrane segments S4 of the pore-forming DHPR
α1S subunit
(1,
2). This conformational change
induces via protein-protein interaction
(3,
4) the opening of the
sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca2+
influx through the DHPR (5).
The release of Ca2+ from the sarcoplasmic reticulum via RyR1
consequently induces muscle contraction. The protein-protein interaction
mechanism between DHPR and RyR1 requires correct ultrastructural targeting of
both channels. In Ca2+ release units (triads and peripheral
couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled
to every other RyR1 and hence are geometrically arranged following the
RyR-specific orthogonal arrays
(6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of
the voltage-sensing and pore-forming α1S subunit and
auxiliary subunits β1a, α2δ-1, and
γ1 (7). While
gene knock-out of the DHPR γ1 subunit
(8,
9) and small interfering RNA
knockdown of the DHPR α2δ-1 subunit
(10-12)
have indicated that neither subunit is essential for coupling of the DHPR with
RyR1, the lack of the α1S or of the intracellular
β1a subunit is incompatible with EC coupling and accordingly
null model mice die perinatally due to asphyxia
(13,
14). β subunits of
voltage-gated Ca2+ channels were repeatedly shown to be responsible
for the facilitation of α1 membrane insertion and to be
potent modulators of α1 current kinetics and voltage
dependence (15,
16). Whether the loss of EC
coupling in β1-null mice was caused by decreased DHPR membrane
expression or by the lack of a putative specific contribution of the β
subunit to the skeletal muscle EC coupling apparatus
(17,
18) was not clearly resolved.
Recently, other β-functions were identified in skeletal muscle using the
β1-null mutant zebrafish relaxed
(19,
20). Like the
β1-knock-out mouse
(14) zebrafish
relaxed is characterized by complete paralysis of skeletal muscle
(21,
22). While
β1-knock-out mouse pups die immediately after birth due to
respiratory paralysis (14),
larvae of relaxed are able to survive for several days because of
oxygen and metabolite diffusion via the skin
(23). Using highly
differentiated myotubes that are easy to isolate from these larvae, the lack
of EC coupling could be described by quantitative immunocytochemistry as a
moderate ∼50% reduction of α1S membrane expression
although α1S charge movement was nearly absent, and, most
strikingly, as the complete lack of the arrangement of DHPRs in tetrads
(19). Thus, in skeletal muscle
the β subunit enables EC coupling by (i) enhancing α1S
membrane targeting, (ii) facilitating α1S charge movement,
and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal
muscle β1a and which ones are rather general properties of
Ca2+ channel β subunits. Previous reconstitution studies made
in the β1-null mouse system
(24,
25) using different β
subunit constructs (26) did
not allow differentiation between β-induced enhancement of non-functional
α1S membrane expression and the facilitation of
α1S charge movement, due to the lack of information on
α1S triad expression levels. Furthermore, the β-induced
arrangement of DHPRs in tetrads was not detected as no ultrastructural
information was obtained.In the present study, we established zebrafish mutant relaxed as
an expression system to test different β subunits for their ability to
restore skeletal muscle EC coupling. Using isolated myotubes for in
vitro experiments (19,
27) and complete larvae for
in vivo expression studies
(28-31)
and freeze-fracture electron microscopy, a clear differentiation between the
major functional roles of β subunits was feasible in the zebrafish
system. The cloned zebrafish β1a and a mammalian (rabbit)
β1a were shown to completely restore all parameters of EC
coupling when expressed in relaxed myotubes and larvae. However, the
phylogenetically closest β subunit to β1a, the
cardiac/neuronal isoform β2a from rat, as well as the
ancestral βM isoform from the housefly (Musca
domestica), could recover functional α1S membrane
insertion, but led to very restricted tetrad formation when compared with
β1a, and thus to impaired DHPR-RyR1 coupling. This impairment
caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional
α1S membrane expression is a common function of all the
tested β subunits, from β1a to even the most distant
βM, whereas the effective formation of tetrads and thus proper
skeletal muscle EC coupling is an exclusive function of the skeletal muscle
β1a subunit. In context with previous studies, our results
suggest a model according to which β1a acts as an allosteric
modifier of α1S conformation. Only in the presence of
β1a, the α1S subunit is properly folded to
allow RyR1 anchoring and thus skeletal muscle-type EC coupling. 相似文献
11.
Gonzalo Izaguirre Alireza R. Rezaie Steven T. Olson 《The Journal of biological chemistry》2009,284(3):1550-1558
We have previously shown that residues Tyr-253 and Glu-255 in the serpin
antithrombin function as exosites to promote the inhibition of factor Xa and
factor IXa when the serpin is conformationally activated by heparin. Here we
show that functional exosites can be engineered at homologous positions in a
P1 Arg variant of the serpin α1-proteinase inhibitor
(α1PI) that does not require heparin for activation. The
combined effect of the two exosites increased the association rate constant
for the reactions of α1PI with factors Xa and IXa
11–14-fold, comparable with their rate-enhancing effects on the
reactions of heparin-activated antithrombin with these proteases. The effects
of the engineered exosites were specific, α1PI inhibitor
reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in
factor Xa, which interacts with the exosite residues in heparin-activated
antithrombin, abrogated the ability of the engineered exosites in
α1PI to promote factor Xa inhibition. Binding studies showed
that the exosites enhance the Michaelis complex interaction of
α1PI with S195A factor Xa as they do with the
heparin-activated antithrombin interaction. Replacement of the P4-P2 AIP
reactive loop residues in the α1PI exosite variant with a
preferred IEG substrate sequence for factor Xa modestly enhanced the
reactivity of the exosite mutant inhibitor with factor Xa by ∼2-fold but
greatly increased the selectivity of α1PI for inhibiting
factor Xa over thrombin by ∼1000-fold. Together, these results show that a
specific and selective inhibitor of factor Xa can be engineered by
incorporating factor Xa exosite and reactive site recognition determinants in
a serpin.The ubiquitous proteins of the serpin superfamily share a common structure
and mostly function as inhibitors of intracellular and extracellular serine
and cysteine-type proteases in a vast array of physiologic processes
(1,
2). Serpins inhibit their
target proteases by a suicide substrate inhibition mechanism in which an
exposed reactive loop of the serpin is initially recognized as a substrate by
the protease. Subsequent cleavage of the reactive loop by the protease up to
the acyl-intermediate stage of proteolysis triggers a massive conformational
change in the serpin that kinetically traps the acyl-intermediate
(3,
4). Although it is well
established that serpins recognize their cognate proteases through a specific
reactive loop “bait” sequence, it has more recently become clear
that serpin exosites outside the reactive loop provide crucial determinants of
protease specificity
(5–7).
In the case of the blood clotting regulator antithrombin and its target
proteases, physiological rates of protease inhibition are only possible with
the aid of exosites generated upon activation of the serpin by heparin binding
(5). Mutagenesis studies have
shown that the antithrombin exosites responsible for promoting the interaction
of heparin-activated antithrombin with factor Xa and factor IXa map to two key
residues, Tyr-253 and Glu-255, in strand 3 of β-sheet C
(8,
9). Parallel mutagenesis
studies of factor Xa and factor IXa have shown that the protease residues that
interact with the antithrombin exosites reside in the autolysis loop, arginine
150 in this loop being most important
(10,
11). The crystal structures of
the Michaelis complexes of heparin-activated antithrombin with catalytically
inactive S195A variants of thrombin and factor Xa have confirmed that these
complexes are stabilized by exosites in antithrombin and in heparin
(12–14).
In particular, the Michaelis complex with S195A factor Xa revealed that
Tyr-253 of antithrombin and Arg-150 of factor Xa comprise a critical
protein-protein interaction of the antithrombin exosite, in agreement with
mutagenesis studies. Binding studies of antithrombin interactions with S195A
proteases have shown that the exosites in heparin-activated antithrombin
increase the binding affinity for proteases minimally by ∼1000-fold in the
Michaelis complex (15,
16).In this study, we have grafted the two exosites in strand 3 of β-sheet
C of antithrombin onto their homologous positions in a P1 Arg variant of
α1-proteinase inhibitor
(α1PI)2
and shown that the exosites are functional in promoting α1PI
inhibition of factor Xa and factor IXa. The exosites specifically promote
factor Xa and factor IXa inhibition and do not affect the inhibition of
trypsin or thrombin. Moreover, mutation of the complementary exosite residue
in factor Xa, Arg-150, largely abrogates the rate-enhancing effect of the
engineered exosites in α1PI on factor Xa inhibition. Binding
studies show that the exosites function by promoting the binding of
α1PI and factor Xa in the Michaelis complex. Replacing the
P4-P2 residues of the P1 Arg α1PI with an IEG factor Xa
recognition sequence modestly enhances the reactivity of the exosite mutant of
α1PI with factor Xa and greatly increases the selectivity of
the mutant α1PI for inhibiting factor Xa over thrombin. These
findings demonstrate that a potent and selective inhibitor of factor Xa can be
engineered by grafting exosite and reactive site determinants for the protease
on a serpin scaffold. 相似文献
12.
Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase
: An Enzyme That Specifically Removes the Terminal Nonreducing
Galacturonosyl Residue in Rhamnogalacturonan Regions of
Pectin1
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Margien Mutter Gerrit Beldman Stuart M. Pitson Henk A. Schols Alphons G.J. Voragen 《Plant physiology》1998,117(1):153-163
A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg−1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h). 相似文献
13.
Lisa Placanica Leonid Tarassishin Guangli Yang Erica Peethumnongsin Seong-Hun Kim Hui Zheng Sangram S. Sisodia Yue-Ming Li 《The Journal of biological chemistry》2009,284(5):2967-2977
γ-Secretase is known to play a pivotal role in the pathogenesis of
Alzheimer disease through production of amyloidogenic Aβ42 peptides.
Early onset familial Alzheimer disease mutations in presenilin (PS), the
catalytic core of γ-secretase, invariably increase the
Aβ42:Aβ40 ratio. However, the mechanism by which these mutations
affect γ-secretase complex formation and cleavage specificity is poorly
understood. We show that our in vitro assay system recapitulates the
effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and
animal models. We have developed a series of small molecule affinity probes
that allow us to characterize active γ-secretase complexes. Furthermore
we reveal that the equilibrium of PS1- and PS2-containing active complexes is
dynamic and altered by overexpression of Pen2 or PS1 mutants and that
formation of PS2 complexes is positively correlated with increased
Aβ42:Aβ40 ratios. These data suggest that perturbations to
γ-secretase complex equilibrium can have a profound effect on enzyme
activity and that increased PS2 complexes along with mutated PS1 complexes
contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid
(Aβ)5 peptides
are believed to play a causative role in Alzheimer disease (AD). Aβ
peptides are generated from the processing of the amyloid precursor protein
(APP) by two proteases, β-secretase and γ-secretase. Although
γ-secretase generates heterogenous Aβ peptides ranging from 37 to
46 amino acids in length, significant work has focused mainly on the Aβ40
and Aβ42 peptides that are the major constituents of amyloid plaques.
γ-Secretase is a multisubunit membrane aspartyl protease comprised of at
least four known subunits: presenilin (PS), nicastrin (Nct), anterior
pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is
thought to contain the catalytic core of the complex
(1–4),
whereas Aph and Nct play critical roles in the assembly, trafficking, and
stability of γ-secretase as well as substrate recognition
(5,
6). Lastly Pen2 facilitates the
endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments
thereby yielding a catalytically competent enzyme
(5,
7–10).
All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for
γ-secretase activity in cell and animal models
(11,
12). There are two homologs of
PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At
least six active γ-secretase complexes have been reported (two
presenilins × three Aph1s)
(13,
14). The sum of apparent
molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120
kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active
γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa,
have been reported
(15–19).
Recently a study suggested that the γ-secretase complex contains only
one of each subunit (20).
Collectively these studies suggest that a four-protein complex around
200–250 kDa may be the minimal functional γ-secretase unit with
additional cofactors and/or varying stoichiometry of subunits existing in the
high molecular weight γ-secretase complexes. CD147 and TMP21 have been
found to be associated with the γ-secretase complex
(21,
22); however, their role in
the regulation of γ-secretase has been controversial
(23,
24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD),
although it is debatable whether these familial PS mutations act as
“gain or loss of function” alterations in regard to
γ-secretase activity
(25–27).
Regardless the overall outcome of these mutations is an increased ratio of
Aβ42:Aβ40. Clearly these mutations differentially affect
γ-secretase activity for the production of Aβ40 and Aβ42.
Despite intensive studies of Aβ peptides and γ-secretase, the
molecular mechanism controlling the specificity of γ-secretase activity
for Aβ40 and Aβ42 production has not been resolved. It has been
found that PS1 mutations affect the formation of γ-secretase complexes
(28). However, the precise
mechanism by which individual subunits alter the dynamics of γ-secretase
complex formation and activity is largely unresolved. A better mechanistic
understanding of γ-secretase activity associated with FAD mutations has
been hindered by the lack of suitable assays and probes that are necessary to
recapitulate the effect of these mutations seen in cell models and to
characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and
PS1 expression on the dynamics of PS1- and PS2-containing complexes and their
association with γ-secretase activity. Using newly developed
biotinylated small molecular probes and activity assays, we revealed that
expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of
PS1-containing active complexes to that of PS2-containing complexes and
results in an overall increase in the Aβ42:Aβ40 ratio in both stable
cell lines and animal models. Our studies indicate that perturbations to the
equilibrium of active γ-secretase complexes by an individual subunit can
greatly affect the activity of the enzyme. Moreover they serve as further
evidence that there are multiple and distinct γ-secretase complexes that
can exist within the same cells and that their equilibrium is dynamic.
Additionally the affinity probes developed here will facilitate further study
of the expression and composition of endogenous active γ-secretase from
a variety of model systems. 相似文献
14.
Ruqin Kou Juliano Sartoretto Thomas Michel 《The Journal of biological chemistry》2009,284(22):14734-14743
These studies explore the connections between simvastatin, Rac1, and
AMP-activated protein kinase (AMPK) pathways in cultured vascular endothelial
cells and in arterial preparations isolated from statin-treated mice. In
addition to their prominent effects on lipoprotein metabolism, statins can
regulate the small GTPase Rac1, and may also affect the phosphorylation of the
ubiquitous AMPK. We explored pathways of statin-modulated Rac1 and AMPK
activation both in arterial preparations from statin-treated mice as well as
in cultured endothelial cells. We treated adult mice with simvastatin daily
for 2 weeks and then harvested and analyzed arterial preparations. Simvastatin
treatment of mice led to a significant increase in AMPK and LKB1
phosphorylation and to a decrease in protein kinase A activity relative to
control animals, associated with a marked increase in Rac1 activation.
Exposure of bovine aortic endothelial cells to simvastatin for 24 h strikingly
increased GTP-bound Rac1 and led to increased phosphorylation of AMPK as well
as the AMPK kinase LKB1. These responses to simvastatin were blocked by
mevalonate or geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate.
Small interfering RNA (siRNA)-mediated knockdown of AMPK abrogated
simvastatin-induced Rac1 activation and LKB1 phosphorylation. Importantly,
siRNA-mediated knockdown of the key AMPK kinase, calcium/calmodulin-dependent
protein kinase kinase β, completely blocked simvastatin-induced
endothelial cell migration and also abrogated statin-promoted phosphorylation
of AMPK and LKB1, as did pharmacological inhibition with the specific
calcium/calmodulin-dependent protein kinase β inhibitor STO-609.
Moreover, siRNA-mediated knockdown of Rac1 completely blocked
simvastatin-induced LKB1 phosphorylation, but without affecting
simvastatin-induced AMPK phosphorylation. These findings establish a key role
for simvastatin in activation of a novel Rac1-dependent signaling pathway in
the vascular wall.HMG-CoA2 reductase
inhibitors, commonly known as statins, are widely prescribed for the
prevention and treatment of hypercholesterolemia and cardiovascular diseases
(1,
2). The salutary clinical
effects of these drugs derive in part from their effects on the levels of
serum lipoproteins, yet other statin responses appear to be mediated by
alterations in vascular function involving the endothelial isoform of
nitric-oxide synthase (3) and
related signaling pathways. Inhibition of HMG-CoA reductase suppresses the
cellular levels of its enzymatic product mevalonate, thereby attenuating
formation both of cholesterol as well as the synthesis of distinct isoprenoid
compounds such as farnesyl pyrophosphate (Fpp) and geranylgeranyl
pyrophosphate (GGpp). Many key signaling proteins are covalently modified by
these isoprenoids, which are the products of a metabolic pathway that diverges
from the pathway that leads to cholesterol synthesis downstream of HMG-CoA
reductase. These isoprenoid compounds can provide lipophilic anchors that
facilitate membrane targeting and modulate protein-protein interactions of
many key signaling proteins. One such iso-prenylated signaling protein is the
GTP-binding cytoskeletonassociated protein Rac1, a member of the Rho GTPase
small G protein family that undergoes geranylgeranylation at its C terminus.
Statins also affect post-translational modification of another small GTPase,
RhoA, that, like Rac1, is a geranylgeranylated protein that is an important
determinant of vascular signaling
(4–8).
Rac1 has particularly important roles in vascular endothelial cells, where
this cytoskeleton regulatory protein modulates activity of the endothelial
isoform of nitric-oxide synthase (eNOS), a key determinant of vascular
homeostasis (9). Rac1
activation in endothelial cells is influenced by the AMP-activated protein
kinase (AMPK) (6), which itself
is phosphorylated by the protein kinase LKB1 and by the
calcium-calmodulin-dependent protein kinase β (CaMKKβ) (see review
(10)). In recent years,
numerous reports have described effects of statins on variety of these
signaling proteins in different experimental systems
(11–14).Statins have been shown to promote the phosphorylation of AMPK
(13), a heterotrimeric enzyme
involved in the modulation of cellular energy pathways that has also been
implicated in eNOS regulation
(3,
15–17).
AMPK was originally discovered and characterized as a cellular “energy
sensor” that can be activated by increases in the intracellular AMP:ATP
ratio (18). However, in recent
years, it has become clear that AMPK is also regulated through AMP-independent
pathways involving enzyme phosphorylation on threonine 172 of the enzyme''s
α subunit, leading to marked enzyme activation
(19). Protein kinases that
phosphorylate AMPK include the tumor suppressor LKB1 and the
calcium/calmodulin-dependent kinase CaMKKβ. LKB1 itself is a
phosphoprotein. The pathways that regulate LKB1 are incompletely understood,
and a variety of upstream protein kinases have been implicated in LKB1
regulation (see review (20)).
CaMKKβ is principally regulated by calcium binding, but this kinase may
also be phosphorylated by the cAMP-dependent protein kinase PKA
(21,
22). Another substrate for PKA
in vascular cells is the actin-binding phosphoprotein VASP
(23,
24); the phosphorylation state
of VASP at its PKA site can serve as a surrogate marker for the activity of
cAMP-dependent signaling pathways in the vascular wall
(25). CaMKKβ has been
shown to be involved in AMPK regulation in endothelial cells in response to
receptor tyrosine kinase activation and via G protein-coupled receptor
pathways (6). Activated AMPK
directly phosphorylates eNOS, and this kinase thereby appears be an important
determinant of NO-dependent signaling in endothelial cells. However, much
remains to be learned about the molecular mechanisms whereby statins enhance
AMPK activation.In cultured cells, statins have been shown to inhibit the
geranylgeranylation of Rac1, associated with an increase in Rac1 GTP binding
and activation (26). The
activation of Rac1 is a key step in eNOS activation: siRNA-mediated Rac1
“knockdown” in endothelial cells markedly suppresses receptor
signaling to eNOS (5,
7). siRNA-mediated AMPK
knockdown suppresses Rac1 activation, again leading to the attenuation of
receptor-dependent activation of eNOS
(6). The relationships among
these various statin-modulated signaling pathways are incompletely
characterized. The present studies identify CaMKKβ and LKB1 as critical
determinants of simvastatin-dependent activation of AMPK- and Rac1-modulated
signaling and reveal that Rac1 in turn regulates LKB1 phosphorylation. 相似文献
15.
Yong Chen Chongguang Chen Evangelia Kotsikorou Diane L. Lynch Patricia H. Reggio Lee-Yuan Liu-Chen 《The Journal of biological chemistry》2009,284(3):1673-1685
We demonstrated previously that the protein GEC1 (glandular epithelial cell
1) bound to the human κ opioid receptor (hKOPR) and promoted cell
surface expression of the receptor by facilitating its trafficking along the
secretory pathway. Here we showed that three hKOPR residues
(Phe345, Pro346, and Met350) and seven GEC1
residues (Tyr49, Val51, Leu55,
Thr56, Val57, Phe60, and Ile64)
are indispensable for the interaction. Modeling studies revealed that the
interaction was mediated via direct contacts between the kinked hydrophobic
fragment in hKOPR C-tail and the curved hydrophobic surface in GEC1 around the
S2 β-strand. Intramolecular Leu44-Tyr109
interaction in GEC1 was important, likely by maintaining its structural
integrity. Microtubule binding mediated by the GEC1 N-terminal domain was
essential for the GEC1 effect. Expression of GEC1 also increased cell surface
levels of the GluR1 subunit and the prostaglandin EP3.f receptor, which have
FPXXM and FPXM sequences, respectively. With its widespread
distribution in the nervous system and its predominantly hydrophobic
interactions, GEC1 may have chaperone-like effects for many cell surface
proteins along the biosynthesis pathway.κ opioid receptor
(KOPR)2 is one of the
three major types of opioid receptors mediating effects of opioid drugs and
endogenous opioid peptides. Stimulation of KOPR generates many effects in
vivo, for example antinociception (especially for visceral chemical pain,
antipruritis, and water diuresis
(1). The KOPR agonist
nalfurafine (TRK-820) is used clinically in Sweden for the treatment of uremic
pruritus in kidney dialysis patients
(2). Because KOPR agonists
produce profound sedative effects, it has been proposed that KOPR agonists may
be useful in treating mania, antagonists as anti-depressants, and partial
agonists for the management of mania depression
(3). KOPR antagonists may also
be useful for curbing cocaine craving and as anti-anxiety drugs
(4,
5).KOPR, a member of the rhodopsin subfamily of the seven-transmembrane
receptor superfamily, is coupled preferentially to pertussis toxin-sensitive G
proteins, namely Gi/o proteins
(6). KOPR has been found to
interact with several non-G protein-binding partners, such as
Na+,H+-exchanger regulatory
factor-1/ezrin-radixin-moesin-binding phosphoprotein-50 and the δ opioid
receptor. These interactions have influence on signal transduction and
trafficking of the receptor
(7–9).
By yeast two-hybrid (Y2H) assay using the hKOPR C-tail to screen a human brain
cDNA library, we identified GEC1, also named GABAA
receptor-associated protein like 1 (GABARAPL1), to be a binding partner of
hKOPR (10).GEC1 cDNA was first cloned as an early estrogen-regulated mRNA from guinea
pig endometrial glandular epithelial cells by Pellerin et al.
(11). Subsequently, it was
cloned from other species, including human and house mouse
(12). Interestingly, the amino
acid sequences of GEC1 are completely conserved among all these species except
orangutan, in which Arg99 substitutes for His99.
Northern blot and immunoblotting analyses revealed that it has widespread
tissue distribution
(12–14).
In particular, GEC1 was found to be abundant in the central nervous system and
expressed throughout the rat brain
(14,
15). This wide tissue
distribution and the high sequence identity across species strongly suggest
that GEC1 has important biological functions in mammalian cells.Based on sequence similarity, GEC1 is classified as a member of
microtubule-associated proteins (MAPs), which also include GABAA
receptor-associated protein (GABARAP), Golgi-associated ATPase enhancer of 16
kDa (GATE16), GABARAP-like 3 (GABARAPL3), light chain 3 (LC3) of MAP 1A/1B,
and the yeast autophagy protein 8 (Atg8)
(12,
13). Among these homologues,
GEC1 share the highest identity with GABARAPL3 (93%), followed by GABARAP
(86%), GATE16 (61%), Atg8 (55%), and LC3 (∼30%).A growing body of evidence shows that this protein family is closely
related to two distinct biological functions. Studies mainly on GABARAP,
GATE16, and GEC1 indicate that they promote intracellular protein trafficking
by enhancing vesicle fusion
(10,
16–21).
In addition, they facilitate degradation of proteins and intracellular
organelles via autophagy-related pathways, which is bolstered largely by
research on Atg8 and LC3 (22,
23).We previously reported that GEC1 interacted with the hKOPR C-tail and
enhanced cell surface levels of hKOPR stably expressed in CHO cells. GEC1
expression enhances hKOPR expression through facilitating its anterograde
trafficking along the protein biosynthesis pathway without affecting
degradation of the receptor
(10). This represented the
first biological function reported for GEC1. Mansuy et al.
(24) demonstrated that GEC1
interacted with tubulin and promoted microtubule bundling in vitro,
and that green fluorescence protein-tagged GEC1 was localized in the
perinuclear vesicles with a scattered pattern. Our electron microscopic
studies in the rat brain showed that GEC1 was associated with ER, Golgi
apparatus, endosome-like vesicles, and plasma membranes and scattered in
cytoplasm in neurons (14). In
addition, N-ethylmaleimide-sensitive factor, a protein critical for
intracellular membrane-trafficking events, binds directly to GEC1
(10).In this study, we employed Y2H techniques to determine the amino acid
residues in both GEC1 and hKOPR C-tail involved in the interaction. Further
studies were then carried out in mammalian cells to examine if elimination of
the interaction affected the effect of GEC1 on hKOPR expression. In addition,
we generated a molecular model of GEC1 based on the x-ray crystal structure of
GABARAP and found that the residues involved in hKOPR binding formed
hydrophobic patches on the exterior surface of GEC1. Moreover, we found that
the cytosolic tail of AMPA receptor subunit GluR1 has the same FPXXM
motif as that found in the hKOPR C-tail to be involved in GEC1 binding and
that GEC1 expression up-regulated GluR1. 相似文献
16.
17.
Annamari Paino Tuuli Ahlstrand Jari Nuutila Indre Navickaite Maria Lahti Heidi Tuominen Hannamari V?limaa Urpo Lamminm?ki Marja T. P?ll?nen Riikka Ihalin 《PloS one》2013,8(7)
Aggregatibacter
actinomycetemcomitans
is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1β also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1β-binding surface-exposed lipoprotein that may be part of the bacterial IL-1β-sensing system. 相似文献
18.
19.
20.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献