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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
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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
6.
Transit Peptide Mutations That Impair in Vitro and in Vivo
Chloroplast Protein Import Do Not Affect Accumulation of the
γ-Subunit of Chloroplast ATPase
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We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ. 相似文献
7.
Background
Recent analyses have suggested an accelerated decline in child mortality in Ghana since 2000. This study examines the long-term child mortality trends in the country, relates them to changes in the key drivers of mortality decline, and assesses the feasibility of the country''s MDG 4 attainment.Methodology
Data from five Demographic and Health Surveys (DHS) between 1988 and 2008 and the Maternal Health Survey 2007 were used to generate two-year estimates of under-five mortality rates back to 1967. Lowess regression fitted past and future trends towards 2015. A modified Poisson approach was applied on the person-period data created from the DHS 2003 and 2008 to examine determinants of under-five mortality and their contributions to the change in mortality. A policy-modelling system assessed the feasibility of the country''s MDG 4 attainment.Findings
The under-five mortality rate has steadily declined over the past 40 years with acceleration since 2000, and is projected to reach between 45 and 69 per 1000 live births in 2015. Preceding birth interval (reference: 36+ months, relative risk [RR] increased as the interval shortened), bed net use (RR 0.71, 95% confidence interval [CI]: 0.52–0.95), maternal education (reference: secondary/higher, RR 1.71, 95% CI: 1.18–2.47 for primary), and maternal age at birth (reference: 17+ years, RR 2.13, 95% CI: 1.12–4.05) were primarily associated with under-five mortality. Increased bed-net use made a substantial contribution to the mortality decline. The scale-up of key interventions will allow the possibility of Ghana''s MDG 4 attainment.Conclusions
National and global efforts for scaling up key child survival interventions in Ghana are paying off ― these concerted efforts need to be sustained in order to achieve MDG 4. 相似文献8.
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
9.
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
10.
Gonzalo Izaguirre Alireza R. Rezaie Steven T. Olson 《The Journal of biological chemistry》2009,284(3):1550-1558
We have previously shown that residues Tyr-253 and Glu-255 in the serpin
antithrombin function as exosites to promote the inhibition of factor Xa and
factor IXa when the serpin is conformationally activated by heparin. Here we
show that functional exosites can be engineered at homologous positions in a
P1 Arg variant of the serpin α1-proteinase inhibitor
(α1PI) that does not require heparin for activation. The
combined effect of the two exosites increased the association rate constant
for the reactions of α1PI with factors Xa and IXa
11–14-fold, comparable with their rate-enhancing effects on the
reactions of heparin-activated antithrombin with these proteases. The effects
of the engineered exosites were specific, α1PI inhibitor
reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in
factor Xa, which interacts with the exosite residues in heparin-activated
antithrombin, abrogated the ability of the engineered exosites in
α1PI to promote factor Xa inhibition. Binding studies showed
that the exosites enhance the Michaelis complex interaction of
α1PI with S195A factor Xa as they do with the
heparin-activated antithrombin interaction. Replacement of the P4-P2 AIP
reactive loop residues in the α1PI exosite variant with a
preferred IEG substrate sequence for factor Xa modestly enhanced the
reactivity of the exosite mutant inhibitor with factor Xa by ∼2-fold but
greatly increased the selectivity of α1PI for inhibiting
factor Xa over thrombin by ∼1000-fold. Together, these results show that a
specific and selective inhibitor of factor Xa can be engineered by
incorporating factor Xa exosite and reactive site recognition determinants in
a serpin.The ubiquitous proteins of the serpin superfamily share a common structure
and mostly function as inhibitors of intracellular and extracellular serine
and cysteine-type proteases in a vast array of physiologic processes
(1,
2). Serpins inhibit their
target proteases by a suicide substrate inhibition mechanism in which an
exposed reactive loop of the serpin is initially recognized as a substrate by
the protease. Subsequent cleavage of the reactive loop by the protease up to
the acyl-intermediate stage of proteolysis triggers a massive conformational
change in the serpin that kinetically traps the acyl-intermediate
(3,
4). Although it is well
established that serpins recognize their cognate proteases through a specific
reactive loop “bait” sequence, it has more recently become clear
that serpin exosites outside the reactive loop provide crucial determinants of
protease specificity
(5–7).
In the case of the blood clotting regulator antithrombin and its target
proteases, physiological rates of protease inhibition are only possible with
the aid of exosites generated upon activation of the serpin by heparin binding
(5). Mutagenesis studies have
shown that the antithrombin exosites responsible for promoting the interaction
of heparin-activated antithrombin with factor Xa and factor IXa map to two key
residues, Tyr-253 and Glu-255, in strand 3 of β-sheet C
(8,
9). Parallel mutagenesis
studies of factor Xa and factor IXa have shown that the protease residues that
interact with the antithrombin exosites reside in the autolysis loop, arginine
150 in this loop being most important
(10,
11). The crystal structures of
the Michaelis complexes of heparin-activated antithrombin with catalytically
inactive S195A variants of thrombin and factor Xa have confirmed that these
complexes are stabilized by exosites in antithrombin and in heparin
(12–14).
In particular, the Michaelis complex with S195A factor Xa revealed that
Tyr-253 of antithrombin and Arg-150 of factor Xa comprise a critical
protein-protein interaction of the antithrombin exosite, in agreement with
mutagenesis studies. Binding studies of antithrombin interactions with S195A
proteases have shown that the exosites in heparin-activated antithrombin
increase the binding affinity for proteases minimally by ∼1000-fold in the
Michaelis complex (15,
16).In this study, we have grafted the two exosites in strand 3 of β-sheet
C of antithrombin onto their homologous positions in a P1 Arg variant of
α1-proteinase inhibitor
(α1PI)2
and shown that the exosites are functional in promoting α1PI
inhibition of factor Xa and factor IXa. The exosites specifically promote
factor Xa and factor IXa inhibition and do not affect the inhibition of
trypsin or thrombin. Moreover, mutation of the complementary exosite residue
in factor Xa, Arg-150, largely abrogates the rate-enhancing effect of the
engineered exosites in α1PI on factor Xa inhibition. Binding
studies show that the exosites function by promoting the binding of
α1PI and factor Xa in the Michaelis complex. Replacing the
P4-P2 residues of the P1 Arg α1PI with an IEG factor Xa
recognition sequence modestly enhances the reactivity of the exosite mutant of
α1PI with factor Xa and greatly increases the selectivity of
the mutant α1PI for inhibiting factor Xa over thrombin. These
findings demonstrate that a potent and selective inhibitor of factor Xa can be
engineered by grafting exosite and reactive site determinants for the protease
on a serpin scaffold. 相似文献
11.
Control of TANK-binding Kinase 1-mediated Signaling by the
��134.5 Protein of Herpes Simplex Virus
1
Dustin Verpooten Yijie Ma Songwang Hou Zhipeng Yan Bin He 《The Journal of biological chemistry》2009,284(2):1097-1105
TANK-binding kinase 1 (TBK1) is a key component of Toll-like
receptor-dependent and -independent signaling pathways. In response to
microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and
cytokine expression. Here we show that TBK1 is a novel target of the
γ134.5 protein, a virulence factor whose expression is
regulated in a temporal fashion. Remarkably, the γ134.5
protein is required to inhibit IRF3 phosphorylation, nuclear translocation,
and the induction of antiviral genes in infected cells. When expressed in
mammalian cells, the γ134.5 protein forms complexes with TBK1
and disrupts the interaction of TBK1 and IRF3, which prevents the induction of
interferon and interferon-stimulated gene promoters. Down-regulation of TBK1
requires the amino-terminal domain. In addition, unlike wild type virus, a
herpes simplex virus mutant lacking γ134.5 replicates
efficiently in TBK1-/- cells but not in TBK1+/+ cells.
Addition of exogenous interferon restores the antiviral activity in both
TBK1-/- and TBK+/+ cells. Hence, control of
TBK1-mediated cell signaling by the γ134.5 protein
contributes to herpes simplex virus infection. These results reveal that TBK1
plays a pivotal role in limiting replication of a DNA virus.Herpes simplex virus 1
(HSV-1)3 is a large
DNA virus that establishes latent or lytic infection, in which the virus
triggers innate immune responses. In HSV-infected cells, a number of antiviral
mechanisms operate in a cell type- and time-dependent manner
(1). In response to
double-stranded RNA (dsRNA), Toll-like receptor 3 (TLR3) recruits an adaptor
TIR domain-containing adaptor inducing IFN-β and stimulates cytokine
expression (2,
3). In the cytoplasm, RNA
helicases, RIG-I (retinoid acid-inducible gene-I), and MDA5 (melanoma
differentiation associated gene 5) recognize intracellular viral
5′-triphosphate RNA or dsRNA
(2,
4). Furthermore, a
DNA-dependent activator of IFN-regulatory factor (DAI) senses double-stranded
DNA in the cytoplasm and induces cytokine expression
(5). There is also evidence
that viral entry induces antiviral programs independent of TLR and RIG-I
pathways (6). While recognizing
distinct viral components, these innate immune pathways relay signals to the
two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB
kinase (IKKi) (2).The IKK-related kinases function as essential components that phosphorylate
IRF3 (interferon regulatory factor 3), as well as the closely related IRF7,
which translocates to the nucleus and induces antiviral genes, such as
interferon-α/β and ISG56 (interferon-stimulated gene 56)
(7,
8). TBK1 is constitutively
expressed, whereas IKKi is engaged as an inducible gene product of innate
immune signaling (9,
10). IRF3 activation is
attenuated in TBK1-deficient but not in IKKi-deficient cells
(11,
12). Its activation is
completely abolished in double-deficient cells
(12), suggesting a partially
redundant function of TBK1 and IKKi. Indeed, IKKi also negatively regulates
the STAT-signaling pathway
(13). TBK1/IKKi interacts with
several proteins, such as TRAF family member-associated NF-κB activator
(TANK), NAP1 (NAK-associated protein 1), similar to NAP1TBK1 adaptor
(SINTBAD), DNA-dependent activator of IFN-regulatory factors (DAI), and
secretory protein 5 (Sec5) in host cells
(5,
14–18).
These interactions are thought to regulate TBK1/IKKi, which delineates innate
as well as adaptive immune responses.Upon viral infection, expression of HSV proteins interferes with the
induction of antiviral immunity. When treated with UV or cycloheximide, HSV
induces an array of antiviral genes in human lung fibroblasts
(19,
20). Furthermore, an HSV
mutant, with deletion in immediate early protein ICP0, induces ISG56
expression (21). Accordingly,
expression of ICP0 inhibits the induction of antiviral programs mediated by
IRF3 or IRF7
(21–23).
However, although ICP0 negatively regulates IFN-β expression, it is not
essential for this effect
(24). In HSV-infected human
macrophages or dendritic cells, an immediate early protein ICP27 is required
to suppress cytokine induction involving IRF3
(25). In this context, it is
notable that an HSV mutant, lacking a leaky late gene γ134.5,
replicates efficiently in cells devoid of IFN-α/β genes
(26). Additionally, the
γ134.5 null mutant induces differential cytokine expression
as compared with wild type virus
(27). Thus, HSV modulation of
cytokine expression is a complex process that involves multiple viral
components. Currently, the molecular mechanism governing this event is
unclear. In this study, we show that HSV γ134.5 targets TBK1
and inhibits antiviral signaling. The data herein reveal a previously
unrecognized mechanism by which γ134.5 facilitates HSV
replication. 相似文献
12.
13.
Chloroplast to chromoplast
development involves new synthesis and plastid localization of
nuclear-encoded proteins, as well as changes in the organization of
internal plastid membrane compartments. We have demonstrated that
isolated red bell pepper (Capsicum annuum) chromoplasts
contain the 75-kD component of the chloroplast outer envelope
translocon (Toc75) and are capable of importing chloroplast precursors
in an ATP-dependent fashion, indicating a functional general import
apparatus. The isolated chromoplasts were able to further localize the
33- and 17-kD subunits of the photosystem II O2-evolution
complex (OE33 and OE17, respectively), lumen-targeted precursors that
utilize the thylakoidal Sec and ΔpH pathways, respectively, to the
lumen of an internal membrane compartment. Chromoplasts contained the
thylakoid Sec component protein, cpSecA, at levels comparable to
chloroplasts. Routing of OE17 to the lumen was abolished by ionophores,
suggesting that routing is dependent on a transmembrane ΔpH. The
chloroplast signal recognition particle pathway precursor major
photosystem II light-harvesting chlorophyll a/b protein
failed to associate with chromoplast membranes and instead accumulated
in the stroma following import. The Pftf (plastid
fusion/translocation factor), a
chromoplast protein, integrated into the internal membranes of
chromoplasts during in vitro assays, and immunoblot analysis indicated
that endogenous plastid fusion/translocation factor was also an
integral membrane protein of chromoplasts. These data demonstrate that
the internal membranes of chromoplasts are functional with respect to
protein translocation on the thylakoid Sec and ΔpH
pathways.Plastids are developmentally related organelles capable of
interconversion among a variety of structurally and biochemically
distinct forms in response to both environmental and tissue-specific
cues (Whatley, 1978; Thomson and Whatley, 1980). Formation of
chromoplasts in many fruits is one striking example of this plasticity.
Heavily pigmented, photosynthetically inactive chromoplasts frequently
develop from chloroplasts during ripening. This conversion involves
dramatic changes in the organization and composition of the internal
plastid compartment, which include the loss of proteins involved in
carbon fixation in the stroma and replacement with
chromoplast-specific proteins, the breakdown of the photosynthetic
thylakoid membranes and loss of proteins involved in light capture and
electron transfer, and, in some cases, the formation of new membranes
(Spurr and Harris, 1968; Camara and Brangeon, 1981; Piechulla et al.,
1987; Kuntz et al., 1989).Chromoplast formation is an active rather than simply a degradative
process. New proteins, specific to or enhanced in chromoplasts, are
synthesized and compartmentalized in the plastid (Camara et al., 1995;
Price et al., 1995). Most chromoplast proteins are predicted to be
nuclear encoded, translated on cytoplasmic ribosomes, and
posttranslationally imported into the plastid, as are nuclear-encoded
chloroplast proteins. Import of chloroplast proteins occurs via a
general import machinery that appears to mediate translocation of most
or all proteins that are delivered to the chloroplast stroma, either as
a final destination or as an intermediate location (Cline and Henry,
1996; Robinson and Mant, 1997; Schnell, 1998). Proteins are targeted to
the general import pathway by an N-terminal extension that is
cleaved upon import, resulting in the appearance of a processed
protein of reduced Mr. Presumably, the
import of proteins into chromoplasts is accomplished by the same
machinery that is responsible for import of proteins into chloroplasts,
although this has never been directly examined.In some chromoplasts an extensive set of internal membranes
accumulates, replacing the thylakoids. For example, in the
fibrillar-type chromoplast of bell pepper (Capsicum annuum),
the photosynthetic membranes are replaced by membranous sheets and
vesicles in addition to the carotenoid-rich plastoglobules and fibrils
(Spurr and Harris, 1968; Laborde and Spurr, 1973; Camara and Brangeon,
1981; Deruere et al., 1994). The often extensive internal membranes are
the site of synthesis of keto-xanthophylls, which constitute the major
carotenoids of red fruit (Bouvier et al., 1994).Our interests are in the biogenesis of the internal membranes of
plastids, in particular the proteins that are integral to the bilayer,
as well as those located in the luminal compartment formed by the
bilayer. In chloroplasts, proteins destined for the thylakoid membrane
or lumen are routed from the stroma into the thylakoid membrane and
lumen by one of at least four distinct mechanisms: the ΔpH,
chloroplast SRP, thylakoid Sec pathways, and an apparently spontaneous
insertion mechanism (for review, see Cline and Henry, 1996; Robinson
and Mant, 1997; Schnell, 1998). In view of the extensive internal
membrane system of bell pepper chromoplasts, one would expect the
presence of proteins and accompanying translocation machinery in these
membranes. However, no chromoplast-specific proteins have been
conclusively demonstrated to be either integral or luminal to these
membranes.One protein, Pftf (plastid
fusion/translocation factor),
predicted to be membrane anchored by sequence analysis, has been
purified from the stromal compartment of pepper chromoplasts (Hugueney
et al., 1995). This raised the possibility that mature chromoplasts
lack the ability to localize proteins into/across internal membranes.
To address this question we developed a method for isolating protein
import-competent chromoplasts from bell peppers. Immunoblotting
confirmed that these chromoplasts contain known translocation machinery
components. Chromoplasts were assayed in vitro for their ability to
import and localize passenger proteins from the three known
protein-machinery-dependent thylakoid-targeting pathways. We found
mature chromoplasts to be capable of membrane targeting of proteins
that utilize the thylakoidal Sec and ΔpH pathways but not capable of
inserting a membrane protein, LHCP, which utilizes the chloroplast SRP
pathway. Pftf was inserted into the membranes of these chromoplasts in
a manner similar to that observed in chloroplasts, and resident Pftf
was also found to be integrally associated with chromoplast membranes.
The precise role of these pathways in the formation of bell pepper
chromoplasts remains to be fully elucidated. 相似文献
14.
15.
Black, Latinx, and Indigenous people have contracted the SARS-CoV-2 virus and died of COVID-19 at higher rates than White people. Individuals rated public transit, taxis, and ride-hailing as the modes of transportation putting them at greatest risk of COVID-19 infection. Cycling may thus be an attractive alternative for commuting. Amid the increase in bikeshare usage during the early months of the pandemic, bikeshare companies made changes to membership requirements to increase accessibility, targeting especially essential workers. Essential workers in the United States are disproportionately Black and Latinx, underpaid, and reliant on public transit to commute to work. We document changes made by bikeshare companies, including benefits to various groups of essential workers, and we discuss such changes in the context of longstanding racial disparities in bikeshare access. While well intended, the arbitrary delineation in eligibility for such benefits by class of essential workers unwittingly curtailed access for many who may have benefited most. Given that equity in bikeshare is an important tool to improve access to safe transportation, critical changes in the distribution, accessibility, and usability of bikeshare networks is essential. Bikeshare companies, city planners, and policy makers should collaborate with community-based bike advocates to implement changes, as vocalized by those most in need of alternative forms of transportation. 相似文献
16.
17.
Specimens were studied of 65 samples of the genus Aphidura (Aphididae, Aphidinae, Macrosiphini) from the collection of the Muséum national d’Histoire naturelle (Paris). The possible synonymies of three pairs of species are discussed. New aphid host plant relationships are reported for Aphidura bozhkoae, Aphidura delmasi, Aphidura ornata, Aphidura pannonica and Aphidura picta; this last species is recorded for first time from Afghanistan. The record of Aphidura pujoli from Pakistan is refuted. The fundatrices, oviparous females and males of Aphidura delmasi are described. Six new species are established: Aphidura gallica
sp. n. and Aphidura amphorosiphon
sp. n. from specimens caught on species of Silene (Caryophyllaceae) from France and Iran, respectively, Aphidura pakistanensis
sp. n., Aphidura graeca
sp. n. and Aphidura urmiensis
sp. n. from specimens caught on species of Dianthus, Gypsophila and Spergula (Caryophyllaceae) from Pakistan, Greece and Iran, respectively, and Aphidura iranensis
sp. n. from specimens caught on Prunus sp. from Iran. Modifications are made to the keys by Blackman and Eastop to aphids living on Dianthus, Gypsophyla, Silene, Spergula and Prinsepia and Prunus (Rosaceae). An identification key to apterous viviparous females of species of Aphidura is also provided. 相似文献
18.
In this paper, we describe the new genus Pauropygus
gen. n. which includes three minute species, blind and unpigmented, living in interstitial littoral habitats in tropical or subtropical countries. Two of these species are new to science (type species Pauropygus projectus
sp. n. from New Caledonia and Pauropygus pacificus
sp. n. from China); the third one, originally described in the genus Cryptopygus (Cryptopygus caussaneli Thibaud, 1996), has a larger pantropical distribution. We synonymize here Cryptopygus riebi Barra, 1997 from South Africa with Pauropygus caussaneli. Two paratypes of the Mexican species Cryptopygus axayacatl Palacios & Thibaud, 2001 turned also to be Pauropygus caussaneli, while the holotype and remaining paratypes of this species support its placement in Proisotomodes. Among the Cryptopygus complex, Pauropygus gen. n. is easily recognized by characters of mouthparts (presence of two large projections on pleural fold, basolateral field with 6 chaetae, modified mouthparts) and reduced sensillar chaetotaxy (tergal sensilla 2-3,0-1/0-1,0-1,1-2,1-2,1-3, microsensilla reduced in number: 00/0-100, with sensilla situated in p-row on the abdomen). Small size, absence of eyes and pigment are also shared by all its species. The three species belonging to the genus differ by sensillar chaetotaxy. 相似文献
19.
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
20.
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