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1.
Yamini S. Bynagari Bela Nagy Jr. Florin Tuluc Kamala Bhavaraju Soochong Kim K. Vinod Vijayan Satya P. Kunapuli 《The Journal of biological chemistry》2009,284(20):13413-13421
The novel class of protein kinase C (nPKC) isoform η is expressed in
platelets, but not much is known about its activation and function. In this
study, we investigated the mechanism of activation and functional implications
of nPKCη using pharmacological and gene knock-out approaches. nPKCη
was phosphorylated (at Thr-512) in a time- and concentration-dependent manner
by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1
receptor antagonist, or YM-254890, a Gq blocker, abolished
2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to
activate nPKCη in platelets isolated from P2Y1 and
Gq knock-out mice. However, pretreatment of platelets with
P2Y12 receptor antagonist, AR-C69331MX did not interfere with
ADP-induced nPKCη phosphorylation. In addition, when platelets were
activated with 2MeSADP under stirring conditions, although nPKCη was
phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by
activated integrin αIIbβ3 mediated outside-in
signaling. Moreover, in the presence of SC-57101, a
αIIbβ3 receptor antagonist, nPKCη
dephosphorylation was inhibited. Furthermore, in murine platelets lacking
PP1cγ, a catalytic subunit of serine/threonine phosphatase,
αIIbβ3 failed to dephosphorylate nPKCη.
Thus, we conclude that ADP activates nPKCη via P2Y1 receptor
and is subsequently dephosphorylated by PP1γ phosphatase activated by
αIIbβ3 integrin. In addition, pretreatment of
platelets with η-RACK antagonistic peptides, a specific inhibitor of
nPKCη, inhibited ADP-induced thromboxane generation. However, these
peptides had no affect on ADP-induced aggregation when thromboxane generation
was blocked. In summary, nPKCη positively regulates agonist-induced
thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis
(1). Vascular injury exposes
subendothelial collagen that activates platelets to change shape, secrete
contents of granules, generate thromboxane, and finally aggregate via
activated αIIbβ3 integrin, to prevent further
bleeding (2,
3). ADP is a physiological
agonist of platelets secreted from dense granules and is involved in feedback
activation of platelets and hemostatic plug stabilization
(4). It activates two distinct
G-protein-coupled receptors (GPCRs) on platelets, P2Y1 and
P2Y12, which couple to Gq and Gi,
respectively
(5–8).
Gq activates phospholipase Cβ (PLCβ), which leads to
diacyl glycerol (DAG)2
generation and calcium mobilization
(9,
10). On the other hand,
Gi is involved in inhibition of cAMP levels and PI 3-kinase
activation (4,
6). Synergistic activation of
Gq and Gi proteins leads to the activation of the
fibrinogen receptor integrin αIIbβ3.
Fibrinogen bound to activated integrin αIIbβ3
further initiates feed back signaling (outside-in signaling) in platelets that
contributes to the formation of a stable platelet plug
(11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate
various platelet functional responses such as dense granule secretion and
integrin αIIbβ3 activation
(12,
13). Based on their structure
and cofactor requirements, PKCs are divided in to three classes: classical
(cofactors: DAG, Ca2+), novel (cofactors: DAG) and atypical
(cofactors: PIP3) PKC isoforms
(14). All the members of the
novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ,
η, and ε, are expressed in platelets
(15), and they require DAG for
activation. Among all the nPKCs, PKCδ
(15,
16) and PKCθ
(17–19)
are fairly studied in platelets. Whereas nPKCδ is reported to regulate
protease-activated receptor (PAR)-mediated dense granule secretion
(15,
20), nPKCθ is activated
by outside-in signaling and contributes to platelet spreading on fibrinogen
(18). On the other hand, the
mechanism of activation and functional role of nPKCη is not addressed as
yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via
three mechanisms (14,
21): 1) cofactor binding: upon
cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as
DAG, Ca2+, or PIP3, release autoinhibition, and attain an active
conformation exposing catalytic domain of the enzyme. 2) phosphorylations:
3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates
conserved threonine residues on activation loop of catalytic domain; this is
followed by autophosphorylations of serine/threonine residues on turn motif
and hydrophobic region. These series of phosphorylations maintain an active
conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind
receptors for activated C kinases (RACKs) and are lead to various subcellular
locations to access the substrates
(22,
23). Although various leading
laboratories have elucidated the activation of PKCs, the mechanism of
down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein
phosphorylations by kinases and dephosphorylations by phosphatases has gained
immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the
serine/threonine phosphatases reported to date. Among them PP1 and PP2
phosphatases are known to regulate various platelet functional responses
(24,
25). Furthermore, PP1c, is the
catalytic unit of PP1 known to constitutively associate with
αIIb and is activated upon integrin engagement with
fibrinogen and subsequent outside-in signaling
(26). Among various PP1
isoforms, recently PP1γ is shown to positively regulate platelet
functional responses (27).
Thus, in this study we investigated if the above-mentioned phosphatases are
involved in down-regulation of nPKCη. Furthermore, reports from other cell
systems suggest that nPKCη regulates ERK/JNK pathways
(28). In platelets ERK is
known to regulate agonist induced thromboxane generation
(29,
30). Thus, we also
investigated if nPKCη regulates ERK phosphorylation and thereby
agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP
receptors and its inactivation by an integrin-associated phosphatase
PP1γ. We also studied if nPKCη regulates functional responses in
platelets and found that this isoform regulates ADP-induced thromboxane
generation, but not fibrinogen receptor activation in platelets. 相似文献
2.
3.
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
4.
Victoria L Alonso Carla Ritagliati Pamela Cribb Esteban C Serra 《Memórias do Instituto Oswaldo Cruz》2014,109(8):1081-1085
We present here three expression plasmids for Trypanosoma cruzi
adapted to the Gateway® recombination cloning system. Two of
these plasmids were designed to express trypanosomal proteins fused to a double tag
for tandem affinity purification (TAPtag). The TAPtag and Gateway®
cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid.
Both plasmids were assayed by introducing green fluorescent protein (GFP) by
recombination and the integrity of the double-tagged protein was determined by
western blotting and immunofluorescence microscopy. The third Gateway adapted vector
assayed was the inducible pTcINDEX. When tested with GFP,
pTcINDEX-GW showed a good response to tetracycline, being less
leaky than its precursor (pTcINDEX). 相似文献
5.
Hong Cao Jing Chen Eugene W. Krueger Mark A. McNiven 《Molecular and cellular biology》2010,30(3):781-792
The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.Iron is an essential element for all mammalian organisms that plays essential roles in hemoglobin and myoglobin production (23). Altered iron transport can lead to disease states such as hemochromatosis (23), anemia (5, 23), and neuronal disorders (23). The transferrin receptor (TfR) is an important component of iron regulation in cells. There are two distinct TfRs in humans sharing 45% identity that are homodimeric and bind iron-associated transferrin (Tf) at markedly different affinities (26). While significant attention has been paid toward understanding the basic endocytic machinery that supports the efficient internalization and recycling of the TfR1 and its associated iron-bound ligand, it has been assumed that this transport process is constitutive in nature. This is in direct contrast to the highly regulated internalization pathway used by members of the receptor tyrosine kinase family (RTKs) and the family of G-coupled protein receptors (GPCRs) that utilize phosphorylation and/or ubiquination as signaling modules to regulate internalization.To test if TfR1 internalization might be regulated in a similar fashion, we focused on two essential components of the endocytic machinery: the large GTPase Dyn2 that mediates endocytic vesicle scission (35) and Cort that binds to Dyn2 via an SH3-PRD interaction and has been postulated to regulate actin dynamics to facilitate vesicle invagination and release (36, 40). Both Dyn2 and Cort have shown to be phosphorylated in vivo and in vitro by a variety of kinases (51, 58). Dyn1 interacts with (17) and is phosphorylated by Src in neuronal cells and in other excitable cells in response to activation of GPCRs and epidermal growth factor (EGF) (1, 2). While the Src phosphorylation motifs of dynamin are conserved in the epithelial expressed form of Dyn2, it is unclear if Dyn2 is phosphorylated in response to ligands that induce clathrin-based endocytosis.Cort possesses a series of C-terminal tyrosines that are heavily Src-phosphorylated and implicated in regulating actin remodeling during cell motility (20). In this study, we demonstrate that addition of Tf to cultured epithelial cells results in an internalization of the TfR1 mediated by a Src kinase-dependent phosphoactivation of the Dyn2-Cort-based endocytic machinery. In support of these findings, dominant negative forms of c-Src kinase, when expressed in a hepatocyte-derived cell line (Clone 9), attenuate Tf internalization. Remarkably, cells exposed to Tf showed a 3- to 4-fold increase in Dyn2 and Cort phosphorylation compared to that shown by untreated cells, an increase exceeding that observed in cells treated with EGF. These findings provide new insights into the regulation of what was thought to be a constitutive endocytic process. 相似文献
6.
Adrien W. Schmid Diego Chiappe V��r��ne Pignat Valerie Grimminger Ivan Hang Marc Moniatte Hilal A. Lashuel 《The Journal of biological chemistry》2009,284(19):13128-13142
Tissue transglutaminase (tTG) has been implicated in the pathogenesis of
Parkinson disease (PD). However, exactly how tTG modulates the structural and
functional properties of α-synuclein (α-syn) and contributes to
the pathogenesis of PD remains unknown. Using site-directed mutagenesis
combined with detailed biophysical and mass spectrometry analyses, we sought
to identify the exact residues involved in tTG-catalyzed cross-linking of
wild-type α-syn and α-syn mutants associated with PD. To better
understand the structural consequences of each cross-linking reaction, we
determined the effect of tTG-catalyzed cross-linking on the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Our
findings show that tTG-catalyzed cross-linking of monomeric α-syn
involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed
cross-linked monomeric α-syn composed of either wild-type or Gln →
Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping
by mass spectrometry. Using this approach, we identified the glutamine and
lysine residues involved in tTG-catalyzed intramolecular cross-linking of
α-syn. These studies demonstrate for the first time that
Gln79 and Gln109 serve as the primary tTG reactive
sites. Mutating both residues to asparagine abolishes tTG-catalyzed
cross-linking of α-syn and tTG-induced inhibition of α-syn
fibrillization in vitro. To further elucidate the sequence and
structural basis underlying these effects, we identified the lysine residues
that form isopeptide bonds with Gln79 and Gln109. This
study provides mechanistic insight into the sequence and structural basis of
the inhibitory effects of tTG on α-syn fibrillogenesis in vivo,
and it sheds light on the potential role of tTG cross-linking on modulating
the physiological and pathogenic properties of α-syn.Parkinson disease
(PD)2 is a progressive
movement disorder that is caused by the loss of dopaminergic neurons in the
substantia nigra, the part of the brain responsible for controlling movement.
Clinically, PD is manifested in symptoms that include tremors, rigidity, and
difficulty in initiating movement (bradykinesia). Pathologically, PD is
characterized by the presence of intraneuronal, cytoplasmic inclusions known
as Lewy bodies (LB), which are composed primarily of the protein
“α-synuclein” (α-syn)
(1) and are seen in the
post-mortem brains of PD patients with the sporadic or familial forms of the
disease (2). α-Syn is a
presynaptic protein of 140 residues with a “natively” unfolded
structure (3). Three missense
point mutations in α-syn (A30P, E46K, and A53T) are associated with the
early-onset, dominant, inherited form of PD
(4,
5). Moreover, duplication or
triplication of the α-syn gene has been linked to the familial
form of PD, suggesting that an increase in α-syn expression is
sufficient to cause PD. Together, these findings suggest that α-syn
plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn
oligomerization and fibrillogenesis in vivo remain poorly understood.
In vitro aggregation studies have shown that the mutations associated
with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but
only E46K and A53T α-syn show higher propensity to fibrillize than
wild-type (WT) α-syn
(6-8).
This suggests that oligomerization, rather than fibrillization, is linked to
early-onset familial PD (9).
Our understanding of the molecular composition and biochemical state of
α-syn in LBs has provided important clues about protein-protein
interactions and post-translational modifications that may play a role in
modulating oligomerization, fibrillogenesis, and LB formation of the protein.
In addition to ubiquitination
(10), phosphorylation
(11,
12), nitration
(13,
14), and C-terminal truncation
(15,
16), analysis of post-mortem
brain tissues from PD and Lewy bodies in dementia patients has confirmed the
colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked
α-syn monomers and higher molecular aggregates in LBs within
dopaminergic neurons (17,
18). Tissue transglutaminase
catalyzes a calcium-dependent transamidating reaction involving glutamine and
lysine residues, which results in the formation of a covalent cross-link via
ε-(γ-glutamyl) lysine bonds
(Fig. 2F). To date,
seven different isoforms of tTGs have been reported, of which only tTG2 seems
to be expressed in the human brain
(19), whereas tTG1 and tTG3
are more abundantly found in stratified squamous epithelia
(20). Subsequent
immuno-histochemical, colocalization, and immunoprecipitation studies have
shown that the levels of tTG and cross-linked α-syn species are
increased in the substantia nigra of PD brains
(17). These findings, combined
with the known role of tTG in cross-linking and stabilizing bimolecular
assemblies, led to the hypothesis that tTG plays an important role in the
initiation and propagation of α-syn fibril formation and that it
contributes to fibril stability in LBs. This hypothesis was initially
supported by in vitro studies demonstrating that tTG catalyzes the
polymerization of the α-syn-derived non-amyloid component (NAC) peptide
via intermolecular covalent cross-linking of residues Gln79 and
Lys80 (21) and by
other studies suggesting that tTG promotes the fibrillization of amyloidogenic
proteins implicated in the pathogenesis of other neurodegenerative diseases
such as Alzheimer disease, supranuclear palsy, Huntington disease, and other
polyglutamine diseases
(22-24).
However, recent in vitro studies with full-length α-syn have
shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn
and inhibits, rather than promotes, its fibrillization in vitro
(25,
26). The structural basis of
this inhibitory effect and the exact residues involved in tTG-mediated
cross-linking of α-syn, as well as structural and functional
consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three
intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native
(—) and cross-linked (- - -) α-syn, showing that most
tTG-catalyzed cross-linking products of WT or disease-associated mutant forms
of α-syn are intramolecularly linked (predominant peak with two
cross-links), and up to three intramolecular cross-links can occur (left
shoulder). The abbreviations M and m/cl are
used to designate native and cross-linked α-synuclein, respectively.
D and E, kinetic analysis of α-syn (A30P)
cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic
depiction of the tTG-catalyzed chemical reaction (isodipeptide formation)
between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues
involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn
and investigated how cross-linking these residues affects the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Using
single-site mutagenesis and mass spectrometry applied to exhaustive
proteolytic digests of native and cross-linked monomeric α-syn, we
identified Gln109 and Gln79 as the major tTG substrates.
We demonstrate that the altered electrophoretic mobility of the
intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of
tTG-catalyzed cross-linking of Gln109 to lysine residues in the N
terminus of α-syn, which leads to the formation of more compact
monomers. Consistent with previous studies, we show that intramolecularly
cross-linked α-syn forms off-pathway oligomers that are distinct from
those formed by the wild-type protein and that do not convert to fibrils
within the time scale of our experiments (3-5 days). We also show that
membrane-bound α-syn is a substrate of tTG and that intramolecular
cross-linking does not interfere with the ability of monomeric α-syn to
adopt an α-helical conformation upon binding to synthetic membranes.
These studies provide novel mechanistic insight into the sequence and
structural basis of events that allow tTG to inhibit α-syn
fibrillogenesis, and they shed light on the potential role of tTG-catalyzed
cross-linking in modulating the physiological and pathogenic properties of
α-syn. 相似文献
7.
Intracellular β-Carbonic Anhydrase of the Unicellular Green
Alga Coccomyxa
: Cloning of the cDNA and Characterization of the Functional
Enzyme Overexpressed in Escherichia coli 下载免费PDF全文
Thomas Hiltonen Harry Bj?rkbacka Cecilia Forsman Adrian K. Clarke G?ran Samuelsson 《Plant physiology》1998,117(4):1341-1349
Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic. 相似文献
8.
Nelli Mnatsakanyan Arathianand M. Krishnakumar Toshiharu Suzuki Joachim Weber 《The Journal of biological chemistry》2009,284(17):11336-11345
ATP synthase uses a unique rotational mechanism to convert chemical energy
into mechanical energy and back into chemical energy. The helix-turn-helix
motif, termed “DELSEED-loop,” in the C-terminal domain of the
β subunit was suggested to be involved in coupling between catalysis and
rotation. Here, the role of the DELSEED-loop was investigated by functional
analysis of mutants of Bacillus PS3 ATP synthase that had 3–7
amino acids within the loop deleted. All mutants were able to catalyze ATP
hydrolysis, some at rates several times higher than the wild-type enzyme. In
most cases ATP hydrolysis in membrane vesicles generated a transmembrane
proton gradient, indicating that hydrolysis occurred via the normal rotational
mechanism. Except for two mutants that showed low activity and low abundance
in the membrane preparations, the deletion mutants were able to catalyze ATP
synthesis. In general, the mutants seemed less well coupled than the wild-type
enzyme, to a varying degree. Arrhenius analysis demonstrated that in the
mutants fewer bonds had to be rearranged during the rate-limiting catalytic
step; the extent of this effect was dependent on the size of the deletion. The
results support the idea of a significant involvement of the DELSEED-loop in
mechanochemical coupling in ATP synthase. In addition, for two deletion
mutants it was possible to prepare an
α3β3γ subcomplex and measure nucleotide
binding to the catalytic sites. Interestingly, both mutants showed a severely
reduced affinity for MgATP at the high affinity site.F1F0-ATP synthase catalyzes the final step of
oxidative phosphorylation and photophosphorylation, the synthesis of ATP from
ADP and inorganic phosphate. F1F0-ATP synthase consists
of the membrane-embedded F0 subcomplex, with, in most bacteria, a
subunit composition of ab2c10, and the peripheral
F1 subcomplex, with a subunit composition of
α3β3γδε. The energy
necessary for ATP synthesis is derived from an electrochemical transmembrane
proton (or, in some organisms, a sodium ion) gradient. Proton flow down the
gradient through F0 is coupled to ATP synthesis on F1 by
a unique rotary mechanism. The protons flow through (half) channels at the
interface of the a and c subunits, which drives rotation of the ring of c
subunits. The c10 ring, together with F1 subunits
γ and ε, forms the rotor. Rotation of γ leads to
conformational changes in the catalytic nucleotide binding sites on the β
subunits, where ADP and Pi are bound. The conformational changes
result in the formation and release of ATP. Thus, ATP synthase converts
electrochemical energy, the proton gradient, into mechanical energy in the
form of subunit rotation and back into chemical energy as ATP. In bacteria,
under certain physiological conditions, the process runs in reverse. ATP is
hydrolyzed to generate a transmembrane proton gradient, which the bacterium
requires for such functions as nutrient import and locomotion (for reviews,
see Refs.
1–6).F1 (or F1-ATPase) has three catalytic nucleotide
binding sites located on the β subunits at the interface to the adjacent
α subunit. The catalytic sites have pronounced differences in their
nucleotide binding affinity. During rotational catalysis, the sites switch
their affinities in a synchronized manner; the position of γ determines
which catalytic site is the high affinity site
(Kd1 in the nanomolar range), which site is the
medium affinity site (Kd2 ≈ 1
μm), and which site is the low affinity site
(Kd3 ≈ 30–100 μm; see
Refs. 7 and
8). In the original crystal
structure of bovine mitochondrial F1
(9), one of the three catalytic
sites, was filled with the ATP analog
AMP-PNP,2 a second was
filled with ADP (plus azide) (see Ref.
10), and the third site was
empty. Hence, the β subunits are referred to as βTP,
βDP, and βE. The occupied β subunits,
βTP and βDP, were in a closed conformation,
and the empty βE subunit was in an open conformation. The main
difference between these two conformations is found in the C-terminal domain.
Here, the “DELSEED-loop,” a helix-turn-helix structure containing
the conserved DELSEED motif, is in an “up” position when the
catalytic site on the respective β subunit is filled with nucleotide and
in a “down” position when the site is empty
(Fig. 1A). When all
three catalytic sites are occupied by nucleotide, the previously open
βE subunit assumes an intermediate, half-closed
(βHC) conformation. It cannot close completely because of
steric clashes with γ
(11).Open in a separate windowFIGURE 1.The βDELSEED-loop. A, interaction of the
βTP and βE subunits with theγ
subunit.β subunits are shown in yellow andγ in
blue. The DELSEED-loop (shown in orange, with the DELSEED
motif itself in green)of βTP interacts with the
C-terminal helixγ and the short helix that runs nearly perpendicular to
the rotation axis. The DELSEED-loop of βE makes contact with
the convex portion of γ, formed mainly by the N-terminal helix. A
nucleotide molecule (shown in stick representation) occupies the catalytic
site of βTP, and the subunit is in the closed conformation.
The catalytic site on βE is empty, and the subunit is in the
open conformation. This figure is based on Protein Data Bank file 1e79
(32). B, deletions in
the βDELSEED-loop. The loop was “mutated” in silico
to represent the PS3 ATP synthase. The 3–4-residue segments that are
removed in the deletion mutants are color-coded as follows:
380LQDI383, pink;
384IAIL387, green;
388GMDE391, yellow;
392LSD394, cyan;
395EDKL398, orange;
399VVHR402, blue. Residues that are the most
involved in contacts with γ are labeled. All figures were generated
using the program PyMOL (DeLano Scientific, San Carlos, CA).The DELSEED-loop of each of the three β subunits makes contact with
the γ subunit. In some cases, these contacts consist of hydrogen bonds
or salt bridges between the negatively charged residues of the DELSEED motif
and positively charged residues on γ. The interactions of the
DELSEED-loop with γ, its movement during catalysis, the conservation of
the DELSEED motif (see 12–14).
Thus, the finding that an AALSAAA mutant in the
α3β3γ complex of ATP synthase from the
thermophilic Bacillus PS3, where several hydrogen bonds/salt bridges
to γ are removed simultaneously, could drive rotation of γ with
the same torque as the wild-type enzyme
(14) came as a surprise. On
the other hand, it seems possible that it is the bulk of the DELSEED-loop,
more so than individual interactions, that drives rotation of γ.
According to a model favored by several authors
(6,
15,
16) (see also Refs.
17–19),
binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site
on βE and the subsequent closure of this site, accompanied by
its conversion into the high affinity site, are responsible for driving the
large (80–90°) rotation substep during ATP hydrolysis, with the
DELSEED-loop acting as a “pushrod.” A recent molecular dynamics
(20) study supports this model
and implicates mainly the region around several hydrophobic residues upstream
of the DELSEED motif (specifically βI386 and
βL387)3 as being
responsible for making contact with γ during the large rotation
substep.
TABLE 1
Conservation of residues in the DELSEED-loop Amino acids found in selected species in the turn region of the DELSEED-loop. Listed are all positions subjected to deletions in the present study. Residue numbers refer to the PS3 enzyme. Consensus annotation: p, polar residue; s, small residue; h, hydrophobic residue; –, negatively charged residue; +, positively charged residue.Open in a separate windowIn the present study, we investigated the function of the DELSEED-loop using an approach less focused on individual residues, by deleting stretches of 3–7 amino acids between positions β380 and β402 of ATP synthase from the thermophilic Bacillus PS3. We analyzed the functional properties of the deletion mutants after expression in Escherichia coli. The mutants showed ATPase activities, which were in some cases surprisingly high, severalfold higher than the activity of the wild-type control. On the other hand, in all cases where ATP synthesis could be measured, the rates where below or equal to those of the wild-type enzyme. In Arrhenius plots, the hydrolysis rates of the mutants were less temperature-dependent than those of wild-type ATP synthase. In those cases where nucleotide binding to the catalytic sites could be tested, the deletion mutants had a much reduced affinity for MgATP at high affinity site 1. The functional role of the DELSEED-loop will be discussed in light of the new information. 相似文献9.
10.
Omar Ramadan Yongxia Qu Raj Wadgaonkar Ghayath Baroudi Eddy Karnabi Mohamed Chahine Mohamed Boutjdir 《The Journal of biological chemistry》2009,284(8):5042-5049
The novel α1D L-type Ca2+ channel is expressed
in supraventricular tissue and has been implicated in the pacemaker activity
of the heart and in atrial fibrillation. We recently demonstrated that PKA
activation led to increased α1D Ca2+ channel
activity in tsA201 cells by phosphorylation of the channel protein. Here we
sought to identify the phosphorylated PKA consensus sites on the
α1 subunit of the α1D Ca2+
channel by generating GST fusion proteins of the intracellular loops, N
terminus, proximal and distal C termini of the α1 subunit of
α1D Ca2+ channel. An in vitro PKA kinase
assay was performed for the GST fusion proteins, and their phosphorylation was
assessed by Western blotting using either anti-PKA substrate or
anti-phosphoserine antibodies. Western blotting showed that the N terminus and
C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus
sites, were phosphorylated by PKA and identified by mass spectrometry. Site
directed mutagenesis and patch clamp studies revealed that serines 1743 and
1816 were major functional PKA consensus sites. Altogether, biochemical and
functional data revealed that serines 1743 and 1816 are major functional PKA
consensus sites on the α1 subunit of α1D
Ca2+ channel. These novel findings provide new insights into the
autonomic regulation of the α1D Ca2+ channel in
the heart.L-type Ca2+ channels are essential for the generation of normal
cardiac rhythm, for induction of rhythm propagation through the
atrioventricular node and for the contraction of the atrial and ventricular
muscles
(1–5).
L-type Ca2+ channel is a multisubunit complex including
α1, β and α2/δ subunits
(5–7).
The α1 subunit contains the voltage sensor, the selectivity
filter, the ion conduction pore, and the binding sites for all known
Ca2+ channel blockers
(6–9).
While α1C Ca2+ channel is expressed in the atria
and ventricles of the heart
(10–13),
expression of α1D Ca2+ channel is restricted to
the sinoatrial (SA)2
and atrioventricular (AV) nodes, as well as in the atria, but not in the adult
ventricles (2,
3,
10).Only recently it has been realized that α1D along with
α1C Ca2+ channels contribute to L-type
Ca2+ current (ICa-L) and they both play important but
unique roles in the physiology/pathophysiology of the heart
(6–9).
Compared with α1C, α1D L-type
Ca2+ channel activates at a more negative voltage range and shows
slower current inactivation during depolarization
(14,
15). These properties may
allow α1D Ca2+ channel to play critical roles in
SA and AV nodes function. Indeed, α1D Ca2+ channel
knock-out mice exhibit significant SA dysfunction and various degrees of AV
block (12,
16–19).The modulation of α1C Ca2+ channel by
cAMP-dependent PKA phosphorylation has been extensively studied, and the C
terminus of α1 was identified as the site of the modulation
(20–22).
Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a
membrane-permeable cAMP analog, increased α1D Ca2+
channel activity using patch clamp studies
(2). However, very little is
known about potential PKA phosphorylation consensus motifs on the
α1D Ca2+ channel. We therefore hypothesized that
the C terminus of the α1 subunit of the α1D
Ca2+ channel mediates its modulation by cAMP-dependent PKA
pathway. 相似文献
11.
12.
13.
Vladimir I. Razinkov Grigory B. Melikyan Richard M. Epand Raquel F. Epand Fredric S. Cohen 《The Journal of general physiology》1998,112(4):409-422
Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores.
R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that
these probes facilitate the formation of negative curvature structures. The probes are known to translocate from
one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of
the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the
open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis
monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe
symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of
the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer
promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate.
When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare.
The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass
shape from which the pore flickers shut. 相似文献
14.
Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[3H]Glc, [3H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-32P]UDP-Glc, [β-32P]UDP-Glc, and [3H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter. 相似文献
15.
Identification and Partial Characterization of the Pectin
Methyltransferase “Homogalacturonan-Methyltransferase” from
Membranes of Tobacco Cell Suspensions 下载免费PDF全文
A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA. 相似文献
16.
17.
18.
19.
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