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1.
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
2.
3.
4.
Tamer M. A. Mohamed Delvac Oceandy Sukhpal Prehar Nasser Alatwi Zeinab Hegab Florence M. Baudoin Adam Pickard Aly O. Zaki Raja Nadif Elizabeth J. Cartwright Ludwig Neyses 《The Journal of biological chemistry》2009,284(18):12091-12098
The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a
modulator of cardiac contractility. We have demonstrated previously that
isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart
and that this complex regulates β-adrenergic signal transmission in
vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a
specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG)
showed a significant reduction in nNOS and total NOS activities as well as in
cGMP levels in the heart compared with their wild type (WT) littermates. In
contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with
the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a
3-fold increase in Ser16 phospholamban (PLB) phosphorylation as
well as Ser22 and Ser23 cardiac troponin I (cTnI)
phosphorylation at base line compared with the WT. In addition, the relative
induction of PLB phosphorylation and cTnI phosphorylation following
isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining
the blunted physiological response to the β-adrenergic stimulation. In
keeping with the data from the transgenic animals, neonatal rat cardiomyocytes
overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP
levels. This was accompanied by an increase in cAMP levels, which led to an
increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP
levels were likely due to the modulation of cardiac phosphodiesterase, which
determined the balance between cGMP and cAMP following PMCA4b overexpression.
In conclusion, these results showed that the nNOS-PMCA4b complex regulates
contractility via cAMP and phosphorylation of both PLB and cTnI.Neuronal nitric-oxide synthase
(nNOS)5 is involved in
a number of key processes in cardiomyocytes including calcium cycling
(1), the β-adrenergic
contractile response (2,
3), post-infarct left
ventricular remodeling (4), and
the regulation of redox equilibrium
(5). Moreover, a polymorphism
in an nNOS-interacting protein, CAPON, has been found to form a quantitative
trait for the determination of the QT interval in humans
(6), whereas a mutation in
α1-syntrophin (SNTA1), another interacting partner of nNOS, has been
associated with long QT syndrome
(7). The signaling events
downstream of the nNOS-CAPON
(8) and nNOS-SNTA1
(7) complexes, which are
responsible for mediating cardiac repolarization and sodium current
respectively, have been elucidated. The nNOS-containing protein complex is
therefore of immediate relevance to human pathology.In recent years, we have shown that the sarcolemmal calcium pump, which
ejects calcium to the extracellular compartment (reviewed in Refs.
9 and
10), is an important molecule
involved in signal regulation and transmission in the heart
(11). We have demonstrated
that isoform 4b of the sarcolemmal calcium pump (also known as PMCA4b for
plasma membrane calcium/calmodulin-dependent
ATPase 4b) modulates signaling through a tight molecular
interaction with nNOS, leading to the modulation of β-adrenergic
responsiveness in the heart
(12). However, the events
following signaling through the PMCA4b-nNOS complex remain unknown.In myocardial cells, nNOS has been localized to the sarcolemma
(13), sarcoplasmic reticulum
(2), and mitochondria
(14), and translocation
between compartments has been demonstrated
(15). It has been speculated
that these various localizations provide specificity to NO signaling, but the
exact mechanisms have yet to be elucidated. In this study, we show a mechanism
by which one fraction of nNOS serves highly specific functions through binding
to PMCA4b. As PMCA4b is confined to the sarcolemma and is a calcium pump, it
is the first identified protein to fulfill these aggregate functions. 1) It
acts as an anchoring protein; 2) it regulates nNOS activity; and 3) it
modulates a process at the plasma membrane, i.e. β-adrenergic
signaling. 相似文献
5.
Kulandaivelu S. Vetrivel Xavier Meckler Ying Chen Phuong D. Nguyen Nabil G. Seidah Robert Vassar Philip C. Wong Masaki Fukata Maria Z. Kounnas Gopal Thinakaran 《The Journal of biological chemistry》2009,284(6):3793-3803
Alzheimer disease β-amyloid (Aβ) peptides are generated via
sequential proteolysis of amyloid precursor protein (APP) by BACE1 and
γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane
microdomains, termed lipid rafts. BACE1 processing in raft microdomains of
cultured cells and neurons was characterized in previous studies by disrupting
the integrity of lipid rafts by cholesterol depletion. These studies found
either inhibition or elevation of Aβ production depending on the extent
of cholesterol depletion, generating controversy. The intricate interplay
between cholesterol levels, APP trafficking, and BACE1 processing is not
clearly understood because cholesterol depletion has pleiotropic effects on
Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this
study, we used an alternate strategy to explore the function of BACE1 in
membrane microdomains without altering the cellular cholesterol level. We
demonstrate that BACE1 undergoes S-palmitoylation at four Cys
residues at the junction of transmembrane and cytosolic domains, and Ala
substitution at these four residues is sufficient to displace BACE1 from lipid
rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null
fibroblasts and neuroblastoma cells revealed that S-palmitoylation
neither contributes to protein stability nor subcellular localization of
BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1
did not have discernible influence on BACE1 processing of APP or secretion of
Aβ. These results indicate that post-translational
S-palmitoylation of BACE1 is not required for APP processing, and
that BACE1 can efficiently cleave APP in both raft and non-raft
microdomains.Alzheimer disease-associated β-amyloid
(Aβ)3 peptides
are derived from the sequential proteolysis of β-amyloid precursor
protein (APP) by β- and γ-secretases. The major β-secretase is
an aspartyl protease, termed BACE1 (β-site
APP-cleaving enzyme 1)
(1–4).
BACE1 cleaves APP within the extracellular domain of APP, generating the N
terminus of Aβ. In addition, BACE1 also cleaves to a lesser extent within
the Aβ domain between Tyr10 and Glu11
(β′-cleavage site). Processing of APP at these sites results in the
shedding/secretion of the large ectodomain (sAPPβ) and generating
membrane-tethered C-terminal fragments +1 and +11 (β-CTF)
(5). The multimeric
γ-secretase cleaves at multiple sites within the transmembrane domain of
β-CTF, generating C-terminal heterogeneous Aβ peptides (ranging in
length between 38 and 43 residues) that are secreted, as well as cytosolic APP
intracellular domains (6). In
addition to BACE1, APP can be cleaved by α-secretase within the Aβ
domain between Lys16 and Leu17, releasing sAPPα
and generating α-CTF. γ-Secretase cleavage of α-CTF
generates N-terminal truncated Aβ, termed p3.Genetic ablation of BACE1 completely abolishes Aβ production,
establishing BACE1 as the major neuronal enzyme responsible for initiating
amyloidogenic processing of APP
(4,
7). Interestingly, both the
expression and activity of BACE1 is specifically elevated in neurons adjacent
to senile plaques in brains of individuals with Alzheimer disease
(8). In the past few years
additional substrates of BACE1 have been identified that include APP
homologues APLP1 and APLP2 (9),
P-selectin glycoprotein ligand-1
(10), β-galactoside
α2,6-sialyltransferase
(11), low-density lipoprotein
receptor-related protein (12),
β-subunits of voltage-gated sodium channels
(13), and neuregulin-1
(14,
15), thus extending the
physiological function of BACE1 beyond Alzheimer disease pathogenesis.BACE1 is a type I transmembrane protein with a long extracellular domain
harboring a catalytic domain and a short cytoplasmic tail. BACE1 is
synthesized as a proenzyme, which undergoes post-translational modifications
that include removal of a pro-domain by a furin-like protease,
N-glycosylation, phosphorylation, S-palmitoylation, and
acetylation, during the transit in the secretory pathway
(16–20).
In non-neuronal cells the majority of BACE1 localizes to late Golgi/TGN and
endosomes at steady-state and a fraction of BACE1 also cycles between the cell
surface and endosomes (21).
The steady-state localization of BACE1 is consistent with the acidic pH
optimum of BACE1 in vitro, and BACE1 cleavage of APP is observed in
the Golgi apparatus, TGN, and endosomes
(22–25).
BACE1 endocytosis and recycling are mediated by the GGA family of adaptors
binding to a dileucine motif (496DISLL) in its cytoplasmic tail
(21,
26–31).
Phosphorylation at Ser498 within this motif modulates GGA-dependent
retrograde transport of BACE1 from endosomes to TGN
(21,
26–31).Over the years, a functional relationship between cellular cholesterol
level and Aβ production has been uncovered, raising the intriguing
possibility that cholesterol levels may determine the balance between
amyloidogenic and non-amyloidogenic processing of APP
(32–34).
Furthermore, several lines of evidence from in vitro and in
vivo studies indicate that cholesterol- and sphingolipid-rich membrane
microdomains, termed lipid rafts, might be the critical link between
cholesterol levels and amyloidogenic processing of APP. Lipid rafts function
in the trafficking of proteins in the secretory and endocytic pathways in
epithelial cells and neurons, and participate in a number of important
biological functions (35).
BACE1 undergoes S-palmitoylation
(19), a reversible
post-translational modification responsible for targeting a variety of
peripheral and integral membrane proteins to lipid rafts
(36). Indeed, a significant
fraction of BACE1 is localized in lipid raft microdomains in a
cholesterol-dependent manner, and addition of glycosylphosphatidylinositol
(GPI) anchor to target BACE1 exclusively to lipid rafts increases APP
processing at the β-cleavage site
(37,
38). Antibody-mediated
co-patching of cell surface APP and BACE1 has provided further evidence for
BACE1 processing of APP in raft microdomains
(33,
39). Components of the
γ-secretase complex also associate with detergent-resistant membrane
(DRM) fractions enriched in raft markers such as caveolin, flotillin, PrP, and
ganglioside GM1 (40). The
above findings suggest a model whereby APP is sequentially processed by BACE1
and γ-secretase in lipid rafts.Despite the accumulating evidence, cleavage of APP by BACE1 in non-raft
membrane regions cannot be unambiguously ruled out because of the paucity of
full-length APP (APP FL) and BACE1 in DRM isolated from adult brain and
cultured cells (41). Moreover,
it was recently reported that moderate reduction of cholesterol (<25%)
displaces BACE1 from raft domains, and increases BACE1 processing by promoting
the membrane proximity of BACE1 and APP in non-raft domains
(34). Nevertheless, this study
also found that BACE1 processing of APP is inhibited with further loss of
cholesterol (>35%), consistent with earlier studies
(32,
33). Nevertheless, given the
pleiotropic effects of cholesterol depletion on membrane properties and
vesicular trafficking of secretory and endocytic proteins
(42–47),
unequivocal conclusions regarding BACE1 processing of APP in lipid rafts
cannot be reached based on cholesterol depletion studies.In this study, we explored the function of BACE1 in lipid raft microdomains
without manipulating cellular cholesterol levels. In addition to the
previously reported S-palmitoylation sites
(Cys478/Cys482/Cys485) within the cytosolic
tail of BACE1 (19), we have
identified a fourth site (Cys474) within the transmembrane domain
of BACE1 that undergoes S-palmitoylation. A BACE1 mutant with Ala
substitution of all four Cys residues (BACE1-4C/A) fails to associate with DRM
in cultured cells, but is not otherwise different from wtBACE1 in terms of
protein stability, maturation, or subcellular localization. Surprisingly, APP
processing and Aβ generation were unaffected in cells stably expressing
the BACE1-4C/A mutant. Finally, we observed an increase in the levels of APP
CTFs in detergent-soluble fractions of BACE1-4C/A as compared with wtBACE1
cells. Thus, our data collectively indicate a non-obligatory role of
S-palmitoylation and lipid raft localization of BACE1 in
amyloidogenic processing of APP. 相似文献
6.
Rachel M. Smith Alistair J. Jacklin Jacqueline J. T. Marshall Frank Sobott Stephen E. Halford 《Nucleic acids research》2013,41(1):405-417
The Type IIB restriction–modification protein BcgI contains A and B subunits in a
2:1 ratio: A has the active sites for both endonuclease and methyltransferase functions
while B recognizes the DNA. Like almost all Type IIB systems, BcgI needs two unmethylated
sites for nuclease activity; it cuts both sites upstream and downstream of the recognition
sequence, hydrolyzing eight phosphodiester bonds in a single synaptic complex. This
complex may incorporate four A2B protomers to give the eight catalytic centres
(one per A subunit) needed to cut all eight bonds. The BcgI recognition sequence contains
one adenine in each strand that can be N6-methylated. Although most DNA
methyltransferases operate at both unmethylated and hemi-methylated sites, BcgI
methyltransferase is only effective at hemi-methylated sites, where the nuclease component
is inactive. Unlike the nuclease, the methyltransferase acts at solitary sites,
functioning catalytically rather than stoichiometrically. Though it transfers one methyl
group at a time, presumably through a single A subunit, BcgI methyltransferase can be
activated by adding extra A subunits, either individually or as part of A2B
protomers, which indicates that it requires an assembly containing at least two
A2B units. 相似文献
7.
Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
8.
Lei Zhang Hui Zhao Yu Qiu Horace H. Loh Ping-Yee Law 《The Journal of biological chemistry》2009,284(4):1990-2000
Recent studies have revealed that in G protein-coupled receptor signalings
switching between G protein- and β-arrestin (βArr)-dependent
pathways occurs. In the case of opioid receptors, the signal is switched from
the initial inhibition of adenylyl cyclase (AC) to an increase in AC activity
(AC activation) during prolonged agonist treatment. The mechanism of such AC
activation has been suggested to involve the switching of G proteins activated
by the receptor, phosphorylation of signaling molecules, or receptor-dependent
recruitment of cellular proteins. Using protein kinase inhibitors, dominant
negative mutant studies and mouse embryonic fibroblast cells isolated from Src
kinase knock-out mice, we demonstrated that μ-opioid receptor
(OPRM1)-mediated AC activation requires direct association and activation of
Src kinase by lipid raft-located OPRM1. Such Src activation was independent of
βArr as indicated by the ability of OPRM1 to activate Src and AC after
prolonged agonist treatment in mouse embryonic fibroblast cells lacking both
βArr-1 and -2. Instead the switching of OPRM1 signals was dependent on
the heterotrimeric G protein, specifically Gi2 α-subunit.
Among the Src kinase substrates, OPRM1 was phosphorylated at Tyr336
within NPXXY motif by Src during AC activation. Mutation of this Tyr
residue, together with mutation of Tyr166 within the DRY motif to
Phe, resulted in the complete blunting of AC activation. Thus, the recruitment
and activation of Src kinase by OPRM1 during chronic agonist treatment, which
eventually results in the receptor tyrosine phosphorylation, is the key for
switching the opioid receptor signals from its initial AC inhibition to
subsequent AC activation.Classical G protein-coupled receptor
(GPCR)2 signaling
involves the activation of specific heterotrimeric G proteins and the
subsequent dissociation of α- and βγ-subunits. These G
protein subunits serve as the activators and/or inhibitors of several effector
systems, including adenylyl cyclases, phospholipases, and ion channels
(1). However, recent studies
have shown that GPCR signaling deviates from such a classical linear model.
For example, in kidney and colonic epithelial cells, protease-activated
receptor 1 can transduce its signals through either Gαi/o or
Gαq subunits via inhibition of small GTPase RhoA or
activation of RhoD. Thus, RhoA and RhoD act as molecular switches between the
negative and positive signaling activity of protease-activated receptor 1
(2). Another example is the
ability of β2-adrenergic receptor to switch from
Gs-dependent pathways to non-classical signaling pathways by
coupling to pertussis toxin-sensitive Gi proteins in a
cAMP-dependent protein kinase/protein kinase C phosphorylation-dependent
manner. In this case, the phosphorylation-induced switch in G protein coupling
provides the receptor access to alternative signaling pathways. For
β2-adrenergic receptors, this leads to a
Gi-dependent activation of MAP kinase
(3,
4). Furthermore the involvement
of protein scaffolds, such as β-arrestins in the MAP kinase cascade,
could also alter the GPCR signaling
(5–8).
Hence the formation of “signaling units” or
“receptosomes” would influence the GPCR signaling process and
destination.For opioid receptors, which are members of the rhodopsin GPCR subfamily
receptors, signal switching is also observed. Normally opioid receptors
inhibit AC activity, activate the MAP kinases and Kir3 K+ channels,
inhibit the voltage-dependent Ca2+ channels, and regulate other
effectors such as phospholipase C
(9). However, during prolonged
agonist treatment, not only is there a blunting of these cellular responses
but also a compensatory increase in intracellular cAMP level, which is
particularly significant upon the removal of the agonist or the addition of an
antagonist such as naloxone
(10–12).
This compensatory adenylyl cyclase activation phenomenon has been postulated
to be responsible for the development of drug tolerance and dependence
(13). The observed change from
receptor-mediated AC inhibition to receptor-mediated AC activation reflects
possible receptor signal switching. Although the exact mechanism for such
signal changes has yet to be elucidated, activation of specific protein
kinases and subsequent phosphorylation of AC isoforms
(14,
15) and other signaling
molecules (16) have been
suggested to be the key for observed AC activation. Among all the protein
kinases studied, involvement of protein kinase C, MAP kinase, and Raf-1 has
been implicated in the activation of AC
(17–19).
Alternative mechanisms, such as agonist-induced receptor internalization and
the increase in the constitutive activities of the receptor, also have been
suggested to play a role in increased AC activity after prolonged opioid
agonist treatment (20).
Earlier studies also implicated the switching of the opioid receptor from
Gi/Go to Gs coupling during chronic agonist
treatment (21). Regardless of
the mechanism, the exact molecular events that lead to the switching of opioid
receptor from an inhibitory response to a stimulatory response remain
elusive.Src kinases, which are members of the nonreceptor tyrosine kinase family,
have been implicated in GPCR function because several Src family members such
as cSrc, Fyn, and Yes have been reported to be activated by several GPCRs,
including β2-
(22) and β3
(23)-adrenergic,
M2- (24) and
M3 (25)-muscarinic,
and bradykinin receptors (26).
The GPCRs that are capable of activating Src predominantly couple to
Gi/o family G proteins
(27). Src kinases appear to
associate with, and be activated by, GPCRs themselves either through direct
interaction with intracellular receptor domains or by binding to
GPCR-associated proteins, such as G protein subunits or β-arrestins
(27). Src kinase has been
reported to be activated by κ-
(28) and δ
(29)-opioid receptors and
regulate the c-Jun kinase and MAP kinase activities. Src kinase within the
nucleus accumbens has been implicated in the rewarding effect and
hyperlocomotion induced by morphine in mice
(30). However, it is not clear
whether the Src kinase is activated and involved in the signal transduction in
AC activation after chronic opioid agonist administration.Previously we reported that the lipid raft location of the receptor and the
Gαi2 proteins are two prerequisites for the observed increase
in AC activity during prolonged agonist treatment
(31,
32). Because various protein
kinases including Src kinases and G proteins have been shown to be enriched in
lipid rafts (33), the roles of
these cellular proteins in the eventual switching of opioid receptor signals
from inhibition to stimulation of AC activity were examined in the current
studies. We were able to demonstrate that the association with and subsequent
activation of Src kinase by the μ-opioid receptor (OPRM1), which leads to
eventual tyrosine phosphorylation of OPRM1, are the cellular events required
for the switching of opioid receptor signaling upon chronic agonist
treatment. 相似文献
9.
Zbynek Heger Petr Michalek Roman Guran Barbora Havelkova Marketa Kominkova Natalia Cernei Lukas Richtera Miroslava Beklova Vojtech Adam Rene Kizek 《PloS one》2015,10(12)
Background
The environmental impacts of various substances on all levels of organisms are under investigation. Among these substances, endocrine-disrupting compounds (EDCs) present a threat, although the environmental significance of these compounds remains largely unknown. To shed some light on this field, we assessed the effects of 17β-oestradiol on the growth, reproduction and formation of free radicals in Eisenia fetida.Methodology/Principal Findings
Although the observed effects on growth and survival were relatively weak, a strong impact on reproduction was observed (50.70% inhibition in 100 μg/kg of E2). We further demonstrated that the exposure of the earthworm Eisenia fetida to a contaminant of emerging concern, 17β-oestradiol (E2), significantly affected the molecules involved in antioxidant defence. Exposure to E2 results in the production of reactive oxygen species (ROS) and the stimulation of antioxidant systems (metallothionein and reduced oxidized glutathione ratio) but not phytochelatins at both the mRNA and translated protein levels. Matrix-assisted laser desorption/ionization (MALDI)-imaging revealed the subcuticular bioaccumulation of oestradiol-3,4-quinone, altering the levels of local antioxidants in a time-dependent manner.Conclusions/Significance
The present study illustrates that although most invertebrates do not possess oestrogen receptors, these organisms can be affected by oestrogen hormones, likely reflecting free diffusion into the cellular microenvironment with subsequent degradation to molecules that undergo redox cycling, producing ROS, thereby increasing environmental contamination that also perilously affects keystone animals, forming lower trophic levels. 相似文献10.
11.
Saija Kiljunen Neeta Datta Svetlana V. Dentovskaya Andrey P. Anisimov Yuriy A. Knirel Jos�� A. Bengoechea Otto Holst Mikael Skurnik 《Journal of bacteriology》2011,193(18):4963-4972
φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis. 相似文献
12.
13.
Karen Vanhoorelbeke Simon F. De Meyer Inge Pareyn Chantal Melchior Sebastien Plan?on Christiane Margue Olivier Pradier Pierre Fondu Nelly Kieffer Timothy A. Springer Hans Deckmyn 《The Journal of biological chemistry》2009,284(22):14914-14920
Three heterozygous mutations were identified in the genes encoding platelet
integrin receptor αIIbβ3 in a patient with an ill defined platelet
disorder: one in the β3 gene (S527F) and two in the αIIb gene
(R512W and L841M). Five stable Chinese hamster ovary cell lines were
constructed expressing recombinant αIIbβ3 receptors bearing the
individual R512W, L841M, or S527F mutation; both the R512W and L841M
mutations; or all three mutations. All receptors were expressed on the cell
surface, and mutations R512W and L841M had no effect on integrin function.
Interestingly, the β3 S527F mutation produced a constitutively active
receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound
to non-activated αIIbβ3 receptors carrying the S527F mutation,
indicating that the conformation of this receptor was altered and corresponded
to the high affinity ligand binding state. In addition, the conformational
change induced by S527F was evident from basal anti-ligand-induced binding
site antibody binding to the receptor. A molecular model bearing this mutation
was constructed based on the crystal structure of αIIbβ3 and
revealed that the S527F mutation, situated in the third integrin epidermal
growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor
from adopting a wild type-like bent conformation. Movement of I-EGF3 into a
cleft in the bent conformation may be hampered both by steric hindrance
between Phe527 in β3 and the calf-1 domain in αIIb and
by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin
receptors that consist of noncovalently linked α/β-heterodimers.
They are cell-surface receptors that play a role in cell-cell and cell-matrix
interactions. Under resting conditions, integrin receptors adopt the low
affinity conformation and do not interact with their ligands. Inside-out
signaling turns the receptor into a high affinity conformation capable of
ligand binding. Ligand binding itself induces additional conformational
changes resulting in exposure of neoantigenic sites called ligand-induced
binding sites (LIBS)3
and generates in turn outside-in signaling, which triggers a range of
downstream signals (1,
2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In
flowing blood under resting conditions, αIIbβ3 does not interact
with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere
at sites of vascular injury and become activated. As a consequence,
αIIbβ3 adopts the high affinity conformation and binds fibrinogen.
This results in platelet aggregation and thrombus formation, which eventually
will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal
ligand-binding head domain (the β-propeller domain in the αIIb
chain and the βI domain in the β3 chain) standing on two long legs
or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb
chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial
growth factor-like (I-EGF), and β-tail domains in the β3 chain),
followed by transmembrane and cytoplasmic domains
(1,
2). X-ray crystal structures of
the extracellular domain of non-activated αVβ3 revealed that the
legs are severely bent, putting the head domain next to the membrane-proximal
portions of the legs (4,
5). The bending occurs between
I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1
domains in the α-subunit. This bent conformation represents the low
affinity state of the receptor. The high affinity state of the receptor is
induced by activation and is associated with a large-scale conformational
rearrangement in which the integrin extends with a switchblade-like motion
(2). Recently, the crystal
structure of the entire extracellular domain of αIIbβ3 in its low
affinity conformation was resolved and revealed that this integrin also adopts
the bent conformation under resting conditions
(6). Structural rearrangements
in αIIbβ3 between the bent and extended conformations are similar
to what has been reported for other integrins
(7).We report here that the S527F mutation in the I-EGF3 region of the β3
polypeptide chain of the αIIbβ3 receptor induces a constitutively
active receptor adopting an extended high affinity conformation. This was
evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding.
These data were further corroborated by modeling the replacement of
Ser527 with Phe in the crystal structure of the extracellular
domain of αIIbβ3. In this model, the S527F mutation decreases the
flexibility of I-EGF3 and appears to prevent movement of the lower β-leg
into the cleft between the upper β-leg and the lower α-leg. As a
consequence, formation of the bent conformation of the non-activated receptor
is hampered. 相似文献
14.
Sophie Jamet Jaclyn Bubnell Patrick Pfister Delia Tomoiaga Matthew E. Rogers Paul Feinstein 《PloS one》2015,10(10)
Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs. 相似文献
15.
Guo-Dong Li David C. Chiara Jonathan B. Cohen Richard W. Olsen 《The Journal of biological chemistry》2009,284(18):11771-11775
Photoaffinity labeling of γ-aminobutyric acid type A
(GABAA)-receptors (GABAAR) with an etomidate analog and
mutational analyses of direct activation of GABAAR by neurosteroids
have each led to the proposal that these structurally distinct general
anesthetics bind to sites in GABAARs in the transmembrane domain at
the interface between the β and α subunits. We tested whether the
two ligand binding sites might overlap by examining whether neuroactive
steroids inhibited etomidate analog photolabeling. We previously identified
(Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and
Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate
photolabeling of GABAAR α1Met-236 and βMet-286 (in
αM1 and βM3). Positioning these two photolabeled amino acids in a
single type of binding site at the interface of β and α subunits
(two copies per pentamer) is consistent with a GABAAR homology
model based upon the structure of the nicotinic acetylcholine receptor and
with recent αM1 to βM3 cross-linking data. Biologically active
neurosteroids enhance rather than inhibit azietomidate photolabeling, as
assayed at the level of GABAAR subunits on analytical SDS-PAGE, and
protein microsequencing establishes that the GABAAR-modulating
neurosteroids do not inhibit photolabeling of GABAAR
α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus
modulatory steroids do not bind at the same site as etomidate, and neither of
the amino acids identified as neurosteroid activation determinants (Hosie, A.
M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature
444, 486–489) are located at the subunit interface defined by our
etomidate site model.GABAA3
receptors (GABAAR) are major mediators of brain inhibitory
neurotransmission and participate in most circuits and behavioral pathways
relevant to normal and pathological function
(1). GABAAR are
subject to modulation by endogenous neurosteroids, as well as myriad
clinically important central nervous system drugs including general
anesthetics, benzodiazepines, and possibly ethanol
(1,
2). The mechanism of
GABAAR modulation by these different classes of drugs is of major
interest, including identification of the receptor amino acid residues
involved in binding and action of the drugs.In the absence of high resolution crystal structures of drug-receptor
complexes, the locations of anesthetic binding sites in GABAARs
have been predicted based upon analyses of functional properties of point
mutant receptors, which identified residues in the α and β subunit
M1–M4 transmembrane helices important for modulation by volatile
anesthetics (primarily α subunit) and by intravenous agents, including
etomidate and propofol (β subunit)
(3–5).
Position βM2–15, numbered relative to the N terminus of the helix,
functions as a major determinant of etomidate and propofol potency as GABA
modulators in vitro and in vivo
(6–8).
By contrast, this residue is not implicated for modulation by the
neurosteroids, potent endogenous modulators of GABAAR
(9).Photoaffinity labeling, which allows the identification of residues in
proximity to drug binding sites
(10,
11), has been used to identify
two GABAAR amino acids covalently modified by the etomidate analog
[3H]azietomidate
(12): α1Met-236 within
αM1 and βMet-286 within βM3. Photolabeling of these residues
was inhibited equally by nonradioactive etomidate and enhanced proportionately
by GABA present in the assay, consistent with the presence of these two
residues in a common drug binding pocket that would be located at the
interface between the β and α subunits in the transmembrane domain
(12). Mutational analyses
identify these positions as etomidate and propofol sensitivity determinants
(13–15).A recent mutagenesis study
(16) identified two other
residues in GABAAR αM1 and βM3 as critical for direct
activation by neurosteroids, αThr-236 (rat numbering, corresponding to
α1Thr-237, bovine numbering used here and by Li et al.
(12))4
and βTyr-284. These residues were also proposed to contribute to a
neurosteroid binding pocket in the transmembrane domain at the interface
between β and α subunits, based upon their location in an
alternative GABAAR structural model that positioned those amino
acids, and not α1Met-236 or βMet-286, at the subunit interface. For
GABAARs and other members of the Cys-loop superfamily of
neurotransmitter-gated ion channels, the transmembrane domain of each subunit
is made up of a loose bundle of four α helices (M1–M4), with M2
from each subunit contributing to the lumen of the ion channel and M4
positioned peripherally in greatest contact with lipid, as seen in the
structures of the Torpedo nicotinic acetylcholine receptor (nAChR)
(17) and in distantly related
prokaryote homologs (18).
However, uncertainties in the alignment of GABAAR subunit sequences
relative to those of the nAChR result in alternative GABAAR
homology models (12,
19,
20) that differ in the
location of amino acids in the M3 and M4 membrane-spanning helices and in the
M1 helix in some models (16,
21).If etomidate and neurosteroids both bind at the same β/α
interface in the GABAAR transmembrane domain, the limited space
available for ligand binding suggests that their binding pockets might overlap
and that ligand binding would be mutually exclusive. To address this question,
we photolabeled purified bovine brain GABAAR with
[3H]azietomidate in the presence of different neuroactive steroids
and determined by protein microsequencing whether active neurosteroids
inhibited labeling of α1Met-236 and βMet-286, as expected for
mutually exclusive binding, or resulted in [3H]azietomidate
photolabeling of other amino acids, a possible consequence of allosteric
interactions. Active steroids failed to inhibit labeling and enhanced labeling
of α1Met-236, clearly indicating that the neurosteroid and the etomidate
sites are distinct. Our GABAAR homology model that positions
α1Met-236 and βMet-286 at the β/α interface, but not
that of Hosie et al.
(16), is also consistent with
cysteine substitution cross-linking studies
(20,
22), which define the
proximity relations between amino acids in the αM1, αM2,
αM3, and βM3 helices, and these results support the interpretation
that the two residues photolabeled by [3H]azietomidate are part of
a single subunit interface binding pocket, whereas the steroid sensitivity
determinants identified by mutagenesis neither are at the β/α
subunit interface nor are contributors to a common binding pocket. 相似文献
16.
Giovanni Maga Barbara van Loon Emmanuele Crespan Giuseppe Villani Ulrich H��bscher 《The Journal of biological chemistry》2009,284(21):14267-14275
Abasic (AP) sites are very frequent and dangerous DNA lesions. Their
ability to block the advancement of a replication fork has been always viewed
as a consequence of their inhibitory effect on the DNA synthetic activity of
replicative DNA polymerases (DNA pols). Here we show that AP sites can also
affect the strand displacement activity of the lagging strand DNA pol δ,
thus preventing proper Okazaki fragment maturation. This block can be overcome
through a polymerase switch, involving the combined physical and functional
interaction of DNA pol β and Flap endonuclease 1. Our data identify a
previously unnoticed deleterious effect of the AP site lesion on normal cell
metabolism and suggest the existence of a novel repair pathway that might be
important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage
in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic)
lesions occur spontaneously in DNA; in eukaryotes it has been estimated that
about 104 depurination and 102 depyrimidation events
occur per genome per day. An equally important source of abasic DNA lesions
results from the action of DNA glycosylases, such as uracil glycosylase, which
excises uracil arising primarily from spontaneous deamination of cytosines
(1). Although most AP sites are
removed by the base excision repair
(BER)5 pathway, a
small fraction of lesions persists, and DNA with AP lesions presents a strong
block to DNA synthesis by replicative DNA polymerases (DNA pols)
(2,
3). Several studies have been
performed to address the effects of AP sites on the template DNA strand on the
synthetic activity of a variety of DNA pols. The major replicative enzyme of
eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP
lesion, but only in the presence of the auxiliary factor proliferating cell
nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared
with an undamaged DNA template
(4). On the other hand, the
family X DNA pols β and λ were shown to bypass an AP site but in a
very mutagenic way (5). Recent
genetic evidence in Saccharomyces cerevisiae cells showed that DNA
pol δ is the enzyme replicating the lagging strand
(6). According to the current
model for Okazaki fragment synthesis
(7–9),
the action of DNA pol δ is not only critical for the extension of the
newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA
segment of about 30 nucleotides on the pre-existing downstream Okazaki
fragment to create an intermediate Flap structure that is the target for the
subsequent action of the Dna2 endonuclease and the Flap endonuclease 1
(Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid
fragment synthesized by the DNA pol α/primase, potentially containing
nucleotide misincorporations caused by the lack of a proofreading exonuclease
activity of DNA pol α/primase. This results in a more accurate copy
synthesized by DNA pol δ. The intrinsic strand displacement activity of
DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A
(RP-A), has been also proposed to be essential for the S phase-specific long
patch BER pathway (10,
11). Although it is clear that
an AP site on the template strand is a strong block for DNA pol
δ-dependent synthesis on single-stranded DNA, the functional
consequences of such a lesion on the ability of DNA pol δ to carry on
strand displacement synthesis have never been investigated so far. Given the
high frequency of spontaneous hydrolysis and/or cytidine deamination events,
any detrimental effect of an AP site on the strand displacement activity of
DNA pol δ might have important consequences both for lagging strand DNA
synthesis and for long patch BER. In this work, we addressed this issue by
constructing a series of synthetic gapped DNA templates with a single AP site
at different positions with respect to the downstream primer to be displaced
by DNA pol δ (see Fig.
1A). We show that an AP site immediately upstream of a
single- to double-strand DNA junction constitutes a strong block to the strand
displacement activity of DNA pol δ, even in the presence of RP-A and
PCNA. Such a block could be resolved only through a “polymerase
switch” involving the concerted physical and functional interaction of
DNA pol β and Fen-1. The closely related DNA pol λ could only
partially substitute for DNA pol β. Based on our data, we propose that
stalling of a replication fork by an AP site not only is a consequence of its
ability to inhibit nucleotide incorporation by the replicative DNA pols but
can also stem from its effects on strand displacement during Okazaki fragment
maturation. In summary, our data suggest the existence of a novel repair
pathway that might be important in preventing replication fork stalling and
identify a previously unnoticed deleterious effect of the AP site lesion on
normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region
inhibits the strand displacement activity of DNA polymerase δ. The
reactions were performed as described under “Experimental
Procedures.” A, schematic representation of the various DNA
templates used. The size of the resulting gaps is indicated in nt. The
position of the AP site on the 100-mer template strand is indicated relative
to the 3′ end. Base pairs in the vicinity of the lesion are indicated by
dashes. The size of the gaps (35–38 nt) is consistent with the
size of ssDNA covered by a single RP-A molecule, which has to be released
during Okazaki fragment synthesis when the DNA pol is approaching the
5′-end of the downstream fragment. When the AP site is covered by the
downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the
nucleotide placed on the opposite strand is C to mimic the situation generated
by spontaneous loss of a guanine or excision of an oxidized guanine, whereas
when the AP site is covered by the primer (nicked AP template), the nucleotide
placed on the opposite strand is A to mimic the most frequent incorporation
event occurring opposite an AP site. B, human PCNA was titrated in
the presence of 15 nm (lanes 2–4 and
10–12) or 30 nm (lanes 6–8 and
14–16) recombinant human four subunit DNA pol δ, on a
linear control (lanes 1–8) or a 38-nt gap control (lanes
9–16) template. Lanes 1, 5, 9, and 13, control
reactions in the absence of PCNA. C, human PCNA was titrated in the
presence of 60 nm DNA pol δ, on a linear AP (lanes
2–4) or 38-nt gap AP (lanes 6–9) template. Lanes
1 and 5, control reactions in the absence of PCNA. 相似文献
17.
Regulated nuclear entry of clock proteins is a conserved feature of eukaryotic circadian clocks and serves to separate the phase of mRNA activation from mRNA repression in the molecular feedback loop. In Drosophila, nuclear entry of the clock proteins, PERIOD (PER) and TIMELESS (TIM), is tightly controlled, and impairments of this process produce profound behavioral phenotypes. We report here that nuclear entry of PER-TIM in clock cells, and consequently behavioral rhythms, require a specific member of a classic nuclear import pathway, Importin α1 (IMPα1). In addition to IMPα1, rhythmic behavior and nuclear expression of PER-TIM require a specific nuclear pore protein, Nup153, and Ran-GTPase. IMPα1 can also drive rapid and efficient nuclear expression of TIM and PER in cultured cells, although the effect on PER is mediated by TIM. Mapping of interaction domains between IMPα1 and TIM/PER suggests that TIM is the primary cargo for the importin machinery. This is supported by attenuated interaction of IMPα1 with TIM carrying a mutation previously shown to prevent nuclear entry of TIM and PER. TIM is detected at the nuclear envelope, and computational modeling suggests that it contains HEAT-ARM repeats typically found in karyopherins, consistent with its role as a co-transporter for PER. These findings suggest that although PER is the major timekeeper of the clock, TIM is the primary target of nuclear import mechanisms. Thus, the circadian clock uses specific components of the importin pathway with a novel twist in that TIM serves a karyopherin-like role for PER. 相似文献
18.
Sufang Zhang Yajing Zhou Ali Sarkeshik John R. Yates III Timothy M. Thomson Zhongtao Zhang Ernest Y. C. Lee Marietta Y. W. T. Lee 《The Journal of biological chemistry》2013,288(5):2941-2950
DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin-conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA damage response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage-induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8−/− mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage-induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways. 相似文献
19.
Plants are not passive victims of the myriad attackers that rely on them for nutrition. They have a suite of physical and chemical defences, and are even able to take advantage of the enemies of their enemies. These strategies are often only deployed upon attack, so may lead to indirect interactions between herbivores and phytopathogens. In this study we test for induced responses in wild populations of an alpine plant (Adenostyles alliariae) that possesses constitutive chemical defence (pyrrolizidine alkaloids) and specialist natural enemies (two species of leaf beetle, Oreina elongata and Oreina cacaliae, and the phytopathogenic rust Uromyces cacaliae). Plants were induced in the field using chemical elicitors of the jasmonic acid (JA) and salicylic acid (SA) pathways and monitored for one month under natural conditions. There was evidence for induced resistance, with lower probability and later incidence of attack by beetles in JA-induced plants and of rust infection in SA-induced plants. We also demonstrate ecological cross-effects, with reduced fungal attack following JA-induction, and a cost of SA-induction arising from increased beetle attack. As a result, there is the potential for negative indirect effects of the beetles on the rust, while in the field the positive indirect effect of the rust on the beetles appears to be over-ridden by direct effects on plant nutritional quality. Such interactions resulting from induced susceptibility and resistance must be considered if we are to exploit plant defences for crop protection using hormone elicitors or constitutive expression. More generally, the fact that induced defences are even found in species that possess constitutively-expressed chemical defence suggests that they may be ubiquitous in higher plants. 相似文献