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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.
5.
6.
7.
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
8.
Carl van Walraven 《CMAJ》2013,185(16):E755-E762
Background:
Changes in the long-term survival of people admitted to hospital is unknown. This study examined trends in 1-year survival of patients admitted to hospital adjusted for improved survival in the general population.Methods:
One-year survival after admission to hospital was determined for all adults admitted to hospital in Ontario in 1994, 1999, 2004, or 2009 by linking to vital statistics datasets. Annual survival in the general population was determined from life tables for Ontario.Results:
Between 1994 and 2009, hospital use decreased (from 8.8% to 6.3% of the general adult population per year), whereas crude 1-year mortality among people with hospital admissions increased (from 9.2% to 11.6%). During this time, patients in hospital became significantly older (median age increased from 51 to 58 yr) and sicker (the proportion with a Charlson comorbidity index score of 0 decreased from 68.2% to 60.0%), and were more acutely ill on admission (elective admissions decreased from 47.4% to 42.0%; proportion brought to hospital by ambulance increased from 16.1% to 24.8%). Compared with 1994, the adjusted odds ratio (OR) for death at 1 year in 2009 was 0.78 (95% confidence interval [CI] 0.77–0.79). However, 1-year risk of death in the general population decreased by 24% during the same time. After adjusting for improved survival in the general population, risk of death at 1 year for people admitted to hospital remained significantly lower in 2009 than in 1994 (adjusted relative excess risk 0.81, 95% CI 0.80–0.82).Interpretation:
After accounting for both the increased burden of patient sickness and improved survival in the general population, 1-year survival for people admitted to hospital increased significantly from 1994 to 2009. The reasons for this improvement cannot be determined from these data. Hospitals have a special place in most health care systems. Hospital staff care for the people with the most serious illnesses and the most vulnerable. They are frequently the location of many life-defining moments — including birth, surgery, acute medical illness and death — of many people and their families. Hospitals serve as a focus in the training of most physicians. In addition, they consume a considerable proportion of health care expenditures worldwide. 1 Given the prominence of hospitals in health care systems, measuring outcomes related to hospital care is important. In particular, the measurement of trends for outcomes of hospital care can help us to infer whether the care provided to hospital patients is improving. Previous such studies have focused on survival trends for specific diseases or patients who received treatment in specific departments. 2 – 12 None of these studies have adjusted for survival trends in the general population, the adjustment for which is important to determine whether changes in survival of patients in hospital merely reflect changes in the overall population. In this study, whether or not patient outcomes have changed over time was determined by examining trends in 1-year survival in all patients admitted to hospital, adjusting for improved survival in the general population. 相似文献9.
10.
Christian Rosker Gargi Meur Emily J. A. Taylor Colin W. Taylor 《The Journal of biological chemistry》2009,284(8):5186-5194
Ryanodine receptors (RyR) are Ca2+ channels that mediate
Ca2+ release from intracellular stores in response to diverse
intracellular signals. In RINm5F insulinoma cells, caffeine, and
4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca2+
entry that was independent of store-operated Ca2+ entry, and
blocked by prior incubation with a concentration of ryanodine that inactivates
RyR. Patch-clamp recording identified small numbers of large-conductance
(γK = 169 pS) cation channels that were activated by
caffeine, 4CmC or low concentrations of ryanodine. Similar channels were
detected in rat pancreatic β-cells. In RINm5F cells, the channels were
blocked by cytosolic, but not extracellular, ruthenium red. Subcellular
fractionation showed that type 3 IP3 receptors (IP3R3)
were expressed predominantly in endoplasmic reticulum, whereas RyR2 were
present also in plasma membrane fractions. Using RNAi selectively to reduce
expression of RyR1, RyR2, or IP3R3, we showed that RyR2 mediates
both the Ca2+ entry and the plasma membrane currents evoked by
agonists of RyR. We conclude that small numbers of RyR2 are selectively
expressed in the plasma membrane of RINm5F pancreatic β-cells, where they
mediate Ca2+ entry.Ryanodine receptors
(RyR)3 and inositol
1,4,5-trisphosphate receptors (IP3R)
(1,
2) are the archetypal
intracellular Ca2+ channels. Both are widely expressed, although
RyR are more restricted in their expression than IP3R
(3,
4). In common with many cells,
pancreatic β-cells and insulin-secreting cell lines express both
IP3R (predominantly IP3R3)
(5,
6) and RyR (predominantly RyR2)
(7). Both RyR and
IP3R are expressed mostly within membranes of the endoplasmic (ER),
where they mediate release of Ca2+. Functional RyR are also
expressed in the secretory vesicles
(8,
9) or, and perhaps more likely,
in the endosomes of β-cells
(10). Despite earlier
suggestions (11),
IP3R are probably not present in the secretory vesicles of
β-cells (8,
12,
13).All three subtypes of IP3R are stimulated by IP3 with
Ca2+ (1), and the
three subtypes of RyR are each directly regulated by Ca2+. However,
RyR differ in whether their most important physiological stimulus is
depolarization of the plasma membrane (RyR1), Ca2+ (RyR2) or
additional intracellular messengers like cyclic ADP-ribose. The latter
stimulates both Ca2+ release and insulin secretion in β-cells
(8,
14). The activities of both
families of intracellular Ca2+ channels are also modulated by many
additional signals that act directly or via phosphorylation
(15,
16). Although they commonly
mediate release of Ca2+ from the ER, both IP3R and RyR
select rather poorly between Ca2+ and other cations (permeability
ratio, PCa/PK ∼7)
(1,
17). This may allow
electrogenic Ca2+ release from the ER to be rapidly compensated by
uptake of K+ (18),
and where RyR or IP3R are expressed in other membranes it may allow
them to affect membrane potential.Both Ca2+ entry and release of Ca2+ from
intracellular stores contribute to the oscillatory increases in cytosolic
Ca2+ concentration ([Ca2+]i) that
stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells
(7). Glucose rapidly
equilibrates across the plasma membrane (PM) of β-cells and its oxidative
metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing
KATP channels to close
(19). This allows an
unidentified leak current to depolarize the PM
(20) and activate
voltage-gated Ca2+ channels, predominantly L-type Ca2+
channels (21). The resulting
Ca2+ entry is amplified by Ca2+-induced Ca2+
release from intracellular stores
(7), triggering exocytotic
release of insulin-containing dense-core vesicles
(22). The importance of this
sequence is clear from the widespread use of sulfonylurea drugs, which close
KATP channels, in the treatment of type 2 diabetes. Ca2+
uptake by mitochondria beneath the PM further stimulates ATP production,
amplifying the initial response to glucose and perhaps thereby contributing to
the sustained phase of insulin release
(23). However, neither the
increase in [Ca2+]i nor the insulin release
evoked by glucose or other nutrients is entirely dependent on Ca2+
entry (7,
24) or closure of
KATP channels (25).
This suggests that glucose metabolism may also more directly activate RyR
(7,
26) and/or IP3R
(27) to cause release of
Ca2+ from intracellular stores. A change in the ATP/ADP ratio is
one means whereby nutrient metabolism may be linked to opening of
intracellular Ca2+ channels because both RyR
(28) and IP3R
(1) are stimulated by ATP.The other major physiological regulators of insulin release are the
incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic
hormone (29). These hormones,
released by cells in the small intestine, stimulate synthesis of cAMP in
β-cells and thereby potentiate glucose-evoked insulin release
(30). These pathways are also
targets of drugs used successfully to treat type 2 diabetes
(29). The responses of
β-cells to cAMP involve both cAMP-dependent protein kinase and epacs
(exchange factors activated by cAMP)
(31,
32). The effects of the latter
are, at least partly, due to release of Ca2+ from intracellular
stores via RyR
(33–35)
and perhaps also via IP3R
(36). The interplays between
Ca2+ and cAMP signaling generate oscillatory changes in the
concentrations of both messengers
(37). RyR and IP3R
are thus implicated in mediating responses to each of the major physiological
regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores,
which probably include both the ER and secretory vesicles and/or endosomes,
functional RyR2 are also expressed in small numbers in the PM of RINm5F
insulinoma cells and rat pancreatic β-cells. 相似文献
11.
12.
Omar A. Ramírez René L. Vidal Judith A. Tello Karina J. Vargas Stefan Kindler Steffen H?rtel Andrés Couve 《The Journal of biological chemistry》2009,284(19):13077-13085
Understanding the mechanisms that control synaptic efficacy through the
availability of neurotransmitter receptors depends on uncovering their
specific intracellular trafficking routes. γ-Aminobutyric acid type B
(GABAB) receptors (GABABRs) are obligatory heteromers
present at dendritic excitatory and inhibitory postsynaptic sites. It is
unknown whether synthesis and assembly of GABABRs occur in the
somatic endoplasmic reticulum (ER) followed by vesicular transport to
dendrites or whether somatic synthesis is followed by independent transport of
the subunits for assembly and ER export throughout the somatodendritic
compartment. To discriminate between these possibilities we studied the
association of GABABR subunits in dendrites of hippocampal neurons
combining live fluorescence microscopy, biochemistry, quantitative
colocalization, and bimolecular fluorescent complementation. We demonstrate
that GABABR subunits are segregated and differentially mobile in
dendritic intracellular compartments and that a high proportion of
non-associated intracellular subunits exist in the brain. Assembled heteromers
are preferentially located at the plasma membrane, but blockade of ER exit
results in their intracellular accumulation in the cell body and dendrites. We
propose that GABABR subunits assemble in the ER and are exported
from the ER throughout the neuron prior to insertion at the plasma membrane.
Our results are consistent with a bulk flow of segregated subunits through the
ER and rule out a post-Golgi vesicular transport of preassembled
GABABRs.The efficacy of synaptic transmission depends on the intracellular
trafficking of neurotransmitter receptors
(1,
2). The trafficking of
glutamatergic and
GABAA6
receptors has been extensively studied, and their implications for synaptic
plasticity have been well documented
(3,
4). For example, differential
trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptors modifies synaptic strength and influences
experience-dependent plasticity in vivo
(5). The molecular mechanisms
that govern the trafficking of metabotropic GABABRs and their
consequences for synaptic inhibition remain less clear. In particular, limited
information is available regarding the relationship between the trafficking of
GABABRs and the topological complexity of the secretory pathway in
neurons.GABABRs mediate the slow component of synaptic inhibition by
acting on pre- and postsynaptic targets
(6–8).
They are implicated in epilepsy, anxiety, stress, sleep disorders,
nociception, depression, and cognition
(9). They also represent
attractive targets for the treatment of withdrawal symptoms from drugs of
addiction such as cocaine
(10). They are obligatory
heteromers composed of GABABR1 and GABABR2 subunits.
GABABR1 contains an RXR-type sequence in the intracellular
C-terminal domain that functions as an ER retention motif
(11,
12). The ER retention sequence
is masked upon assembly with GABABR2 resulting in the appearance of
functional receptors at the plasma membrane. Only GABABR1 binds
GABA with high affinity, whereas G protein signaling is exclusively mediated
by the second and third intracellular loops of GABABR2
(13–15).
GABABRs are located in dendrites and axons, but their distribution
does not coincide with the active zone or the postsynaptic density. Rather,
they are adjacent to both compartments constituting perisynaptic receptors
(16,
17).If GABABR subunits are synthesized in the soma, at least two
possibilities exist for their anterograde transport, assembly, and insertion
in dendrites. First, the subunits may be synthesized in the cell body,
assembled in the somatic ER, and targeted preassembled in post-Golgi vesicles
to their site of insertion in dendrites. Alternatively, they may be
synthesized in the soma and transported through the ER membrane as
non-heteromeric subunits. In the latter scenario, newly assembled receptors
may exit the ER throughout the somatodendritic compartment prior to insertion
at the plasma membrane and diffuse laterally for retention at functional
sites. No evidence exists to discriminate between these possibilities. We
reasoned that a prevalence of associated subunits in post-Golgi vesicles in
dendrites would favor the first alternative, whereas the existence of
non-associated subunits in intracellular compartments would support a
somatodendritic assembly mechanism. Here we explore the presence of associated
GABABR subunits using fluorescence recovery after photobleaching
(FRAP), biochemistry, and quantitative colocalization. In addition, we report
for the first time the use of BiFC
(18) to study
GABABR assembly in neurons. Our results demonstrate that
GABABR subunits are differentially mobile in dendrites and that a
high proportion of non-associated subunits prevail in an intracellular
fraction of the adult brain. They also show that GABABR subunits
are heteromeric at the plasma membrane but segregated in intracellular
compartments of dendrites of hippocampal neurons. Importantly, treatment with
brefeldin A (BFA) or interference of the coatomer protein complex II impair ER
export and result in the accumulation of assembled subunits in intracellular
compartments throughout the somatodendritic arbor. We conclude that
GABABR subunits are synthesized in the soma and remain segregated
in intracellular compartments prior to somatodendritic assembly. Our
observations rule out a post-Golgi vesicular transport of preassembled
GABABRs and suggest an alternative mechanism of receptor
targeting. 相似文献
13.
14.
15.
Rosanna Pescini Gobert Monique van den Eijnden Cedric Szyndralewiez Catherine Jorand-Lebrun Dominique Swinnen Linfeng Chen Corine Gillieron Fiona Pixley Pierre Juillard Patrick Gerber Caroline Johnson-L��ger Serge Halazy Montserrat Camps Agnes Bombrun Margaret Shipp Pierre-Alain Vitte Vittoria Ardissone Chiara Ferrandi Dominique Perrin Christian Rommel Rob Hooft van Huijsduijnen 《The Journal of biological chemistry》2009,284(17):11385-11395
16.
Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase
: An Enzyme That Specifically Removes the Terminal Nonreducing
Galacturonosyl Residue in Rhamnogalacturonan Regions of
Pectin1 下载免费PDF全文
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
17.
Ana PM Oliveira Rosana Gentile Arnaldo Maldonado Júnior Eduardo J Lopes Torres Silvana C Thiengo 《Memórias do Instituto Oswaldo Cruz》2015,110(6):739-744
The aim of this study was to analyse the infection dynamics ofAngiostrongylus
cantonensis in its possible intermediate hosts over two years in an urban
area in the state of Rio de Janeiro where the presence ofA.
cantonensis had been previously recorded in molluscs. Four of the seven
mollusc species found in the study were exotic.Bradybaena similaris
was the most abundant, followed byAchatina fulica,
Streptaxis sp., Subulina octona,
Bulimulus tenuissimus, Sarasinula linguaeformis
and Leptinaria unilamellata. Only A. fulica
and B. similaris were parasitised by A.
cantonensis and both presented co-infection with other helminths. The
prevalence of A. cantonensisin A. fulica was more
than 50% throughout the study. There was an inverse correlation between the
population size ofA. fulica and the prevalence of A.
cantonensis and abundance of the latter was negatively related to
rainfall. The overall prevalence of A. cantonensis in B.
similariswas 24.6%. A. fulica was the most important
intermediary host of A. cantonensis in the studied area
andB. similaris was secondary in importance for A.
cantonensis transmission dynamics. 相似文献
18.
Daniel Beltr��n-Valero de Bernab�� Kei-ichiro Inamori Takako Yoshida-Moriguchi Christine J. Weydert Hollie A. Harper Tobias Willer Michael D. Henry Kevin P. Campbell 《The Journal of biological chemistry》2009,284(17):11279-11284
The interaction between epithelial cells and the extracellular matrix is
crucial for tissue architecture and function and is compromised during cancer
progression. Dystroglycan is a membrane receptor that mediates interactions
between cells and basement membranes in various epithelia. In many
epithelium-derived cancers, β-dystroglycan is expressed, but
α-dystroglycan is not detected. Here we report that α-dystroglycan
is correctly expressed and trafficked to the cell membrane but lacks laminin
binding as a result of the silencing of the like-acetylglucosaminyltransferase
(LARGE) gene in a cohort of highly metastatic epithelial cell lines
derived from breast, cervical, and lung cancers. Exogenous expression of LARGE
in these cancer cells restores the normal glycosylation and laminin binding of
α-dystroglycan, leading to enhanced cell adhesion and reduced cell
migration in vitro. Our findings demonstrate that LARGE repression is
responsible for the defects in dystroglycan-mediated cell adhesion that are
observed in epithelium-derived cancer cells and point to a defect of
dystroglycan glycosylation as a factor in cancer progression.Normal epithelial cells are tightly associated with one another and with
the underlying basement membrane to maintain tissue architecture and function.
During cancer progression, primitive cancer cells escape from this control by
modifying the binding affinities of their cell membrane receptors. Several
receptors have been described as important for this process. Of these, the
integrins are the best studied
(1). The receptor dystroglycan
has been reported to be required for the development and maintenance of
epithelial tissues (2,
3). A direct requirement for
dystroglycan in epithelia is further demonstrated by the profound effect that
loss of dystroglycan expression has on cell polarity and laminin binding in
cultured mammary epithelial cells
(4,
5). However, dystroglycan is
not only important in the establishment and maintenance of epithelial
structure. Associations have also been made between the loss of
α-dystroglycan immunoreactivity and cancer progression in tumors of
epithelial origin, including breast, colon, cervix, and prostate cancers
(4,
6–9).
The dystroglycan loss of function could thus serve as an effective means by
which cancerous cells modify their adhesion to the extracellular matrix
(ECM).2Dystroglycan is a ubiquitously expressed cell membrane protein that plays a
key function in cellular integrity, linking the intracellular cytoskeleton to
the extracellular matrix. The dystroglycan gene encodes a preprotein that is
cleaved into two peptides
(10). The C-terminal
component, known as β-dystroglycan, is embedded within the cell membrane,
whereas the N-terminal component, α-dystroglycan, is present within the
extracellular periphery but remains associated with β-dystroglycan
through non-covalent bonds. β-Dystroglycan binds to actin
(11), dystrophin
(11), utrophin
(11), and Grb2
(12) through its C-terminal
intracellular domain. α-Dystroglycan, on the other hand, binds to ECM
proteins that contain laminin globular domains including laminins
(13,
14), agrin
(15), and perlecan
(16), as well as to the
transmembrane protein neurexin
(17). α-Dystroglycan is
extensively decorated by three different types of glycan modifications: mucin
type O-glycosylation, O-mannosylation, and
N-glycosylation. The state of α-dystroglycan glycosylation has
been shown to be critical for the ability of the protein to bind to laminin
globular domain-containing proteins of the ECM
(18).Previous studies of epithelium-derived cancers
(4,
9) demonstrated that the loss
of immunoreactivity of α-dystroglycan antibodies correlates with tumor
grade and poor prognosis. This reduced detection of α-dystroglycan,
however, is based on a loss of α-dystroglycan reactivity to antibodies
(known as IIH6 and VIA4-1) that recognize the laminin-binding glyco-epitope of
α-dystroglycan, i.e. the protein is only functional when it is
glycosylated in such a way (henceforth, referred to as functional
glycosylation). However, in most of the cancer samples that have been studied
to date, β-dystroglycan is expressed at normal levels at the cell
membrane. Thus, the aforementioned cancer-associated loss of
α-dystroglycan expression may reflect a failure in the
post-translational processing of dystroglycan rather than in the synthesis of
α-dystroglycan itself.A similar defect in dystroglycan has been reported in a group of congenital
muscular dystrophies (19).
This spectrum of human developmental syndromes involves the brain, eye, and
skeletal muscle and shows a dramatic gradient of phenotypic severity that
ranges from the most devastating in Walker-Warburg syndrome to the least
severe in limb-girdle muscular dystrophy. Six distinct known and putative
glycosyltransferases have been shown to underlie these syndromes: protein
O-mannosyltransferase 1 (POMT1), protein
O-mannosyltransferase 2 (POMT2), protein O-mannose
β-1,2-acetylglucosaminyltransferase 1 (POMGnT1), like
acetylglucosaminyltransferase (LARGE), Fukutin, and Fukutin-related protein
(FKRP)
(20–25).
Indeed, all muscular dystrophy patients with mutations in any of these genes
fail to express the functionally glycosylated α-dystroglycan epitope
that is recognized by the IIH6 and VIA4-1 antibodies.To investigate the molecular mechanism responsible for the loss of
α-dystroglycan in epithelium-derived cancers and its role in metastatic
progression, we examined the expression and glycosylation status of
α-dystroglycan in a group of breast, cervical, and lung cancer cell
lines. Here we report that although α-dystroglycan is expressed in the
metastatic cell lines MDA-MB-231, HeLa, H1299, and H2030, it is not
functionally glycosylated. In screening these cell lines for expression of the
six known α-dystroglycan-modifying proteins, we observed that only one,
LARGE, was extensively down-regulated. We also report that the ectopic
restoration of LARGE expression in these cell lines led not only to the
production of a functional dystroglycan but also to the reversion of certain
characteristics associated with invasiveness, namely cell attachment to ECM
proteins and cell migration. 相似文献
19.
20.
Nobuchika Suzuki Kouhei Tsumoto Nicole Hajicek Kenji Daigo Reiko Tokita Shiro Minami Tatsuhiko Kodama Takao Hamakubo Tohru Kozasa 《The Journal of biological chemistry》2009,284(8):5000-5009
The transient protein-protein interactions induced by guanine
nucleotide-dependent conformational changes of G proteins play central roles
in G protein-coupled receptor-mediated signaling systems. Leukemia-associated
RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS
homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and
acts both as a GTPase-activating protein (GAP) and an effector for
Gα13. However, the molecular mechanism of LARG activation
upon Gα13 binding is not yet well understood. In this study,
we analyzed the Gα13-LARG interaction using cellular and
biochemical methods, including a surface plasmon resonance (SPR) analysis. The
results obtained using various LARG fragments demonstrated that active
Gα13 interacts with LARG through the RH domain, DH/PH
domains, and C-terminal region. However, an alanine substitution at the RH
domain contact position in Gα13 resulted in a large decrease
in affinity. Thermodynamic analysis revealed that binding of
Gα13 proceeds with a large negative heat capacity change
(ΔCp°), accompanied by a positive entropy change
(ΔS°). These results likely indicate that the binding of
Gα13 with the RH domain triggers conformational
rearrangements between Gα13 and LARG burying an exposed
hydrophobic surface to create a large complementary interface, which
facilitates complex formation through both GAP and effector interfaces, and
activates the RhoGEF. We propose that LARG activation is regulated by an
induced-fit mechanism through the GAP interface of Gα13.Heterotrimeric G
proteins3 serve as key
molecular switches to transduce a large array of extracellular signals into
cells by actively alternating their conformations between GDP-bound inactive
and GTP-bound active forms. In the current model, the ligand-activated G
protein-coupled receptors (GPCRs) catalyze the exchange of GDP for GTP on
Gα subunits (1). Upon
activation, three switch regions in the Gα subunit undergo significant
conformational changes, followed by dissociation of the GTP-bound Gα
subunit from the Gβγ subunits. Both Gα-GTP and free
Gβγ interact with diverse downstream effectors to transmit
intracellular signals. The Gα subunit hydrolyzes bound GTP to GDP by its
intrinsic GTPase activity. This deactivation process is further accelerated by
GTPase-activating proteins (GAPs) such as regulator of G protein signaling
(RGS) proteins (2,
3). Gα-GDP dissociates
from effectors and re-associates with Gβγ to terminate the
signal.Although this model explains the basic concept of G protein signaling, the
molecular dynamics of interactions among GPCR, G protein, RGS protein, and
effector during the signaling process is not well understood. It has been
suggested that the GPCR signals are integrated into the intracellular
signaling network at the level of G proteins
(4). Accumulating evidence
suggests that the Gα subunit acts as the core of the signaling complex
at the membrane, which is formed through the transient protein-protein
interactions of multiple signaling components
(5,
6). Thus, the quantitative
analysis of the dynamic molecular interactions in the GPCR signaling complex
will be crucial to understanding various cellular processes.Gα12 and Gα13 subunits have been
demonstrated to regulate the activity of Rho GTPase through RhoGEFs, which
contain an N-terminal RGS homology domain (RH-RhoGEFs)
(7–10).
RH-RhoGEFs, which consist of p115RhoGEF/Lsc, PDZ-Rho-GEF/GTRAP48, and LARG in
mammalian species, directly link the activation of GPCRs by extracellular
ligands to the regulation of Rho activity in cells
(10–14).
All three RH-RhoGEFs contain an N-terminal RH domain, which specifically
recognizes the active form of Gα12 or Gα13
and central DH/PH domains characteristic of GEFs for Rho GTPases. It has been
demonstrated in vitro that LARG and p115RhoGEF serve as specific GAPs
for Gα12/13 through their RH domains and also as their
effectors to regulate Rho GTPase activation
(11–13).
A structural study has demonstrated that the interface of the RH domain of
p115RhoGEFs and a Gα13/i1 chimera is different from that of
the RGS domain of RGS4 and Gαi1
(7). The N-terminal small
element in the RH domain, which is required for GAP activity toward
Gα13, contacts the switch regions and the helical domain of
the Gα13/i1 chimera. The core module of the p115RhoGEF RH
domain binds to the region of Gα13/i1, which is
conventionally used for effector binding. These results suggest roles for the
RH domain in the stimulation of GEF activity by Gα13 in
addition to GAP activity. On the other hand, several studies have also
indicated that regions outside of RH domain of RH-RhoGEFs, particularly the
DH/PH domains, interact directly with activated Gα13
(11,
14,
15). In addition, we have
demonstrated recently that p115RhoGEF interacts with distinct surfaces of
Gα13 for the GAP reaction or GEF activity regulation
(16). However, the molecular
mechanism of LARG activation upon Gα13 binding is not clearly
understood.In this study, we have developed a quantitative method for the kinetic and
thermodynamic analysis of Gα13-effector interaction using
surface plasmon resonance (SPR) with sensor chips on which
Gα13 was immobilized. We examined the kinetics and
thermodynamics of the Gα13-LARG interaction and assessed LARG
activation using both in vitro and cell-based approaches. We present
evidence that, in addition to the interaction with the RH domain, the DH/PH
domains and C-terminal region of LARG also interact with Gα13
to form the high affinity Gα13-LARG complex and activate
RhoGEF activity. We further propose that LARG adopts the active conformation
using an induced-fit mechanism through association with the GAP interface of
Gα13. A similar mechanism may also be used with other
Gα-effector interactions. 相似文献