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1.
Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
Calcium-binding protein 1 (CaBP1), a neuron-specific member of the
calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of
inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present
NMR structures of CaBP1 in both Mg2+-bound and
Ca2+-bound states and their structural interaction with
InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The
N-domain consists of EF1 and EF2 in a closed conformation with Mg2+
bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits
a Ca2+-induced closed to open transition like that of CaM. The
Ca2+-bound C-domain contains exposed hydrophobic residues
(Leu132, His134, Ile141, Ile144,
and Val148) that may account for selective binding to
InsP3Rs. Isothermal titration calorimetry analysis reveals a
Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region
of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did
not show appreciable binding. CaBP1 binding to InsP3Rs requires
both the suppressor and ligand-binding core domains, but has no effect on
InsP3 binding to the receptor. We propose that CaBP1 may regulate
Ca2+-dependent activity of InsP3Rs by promoting
structural contacts between the suppressor and core domains.Calcium ion (Ca2+) in the cell functions as an important
messenger that controls neurotransmitter release, gene expression, muscle
contraction, apoptosis, and disease processes
(1). Receptor stimulation in
neurons promotes large increases in intracellular Ca2+ levels
controlled by Ca2+ release from intracellular stores through
InsP3Rs (2). The
neuronal type-1 receptor
(InsP3R1)2
is positively and negatively regulated by cytosolic Ca2+
(3-6),
important for the generation of repetitive Ca2+ transients known as
Ca2+ spikes and waves
(1). Ca2+-dependent
activation of InsP3R1 contributes to the fast rising phase of
Ca2+ signaling known as Ca2+-induced Ca2+
release (7).
Ca2+-induced inhibition of InsP3R1, triggered at higher
cytosolic Ca2+ levels, coordinates the temporal decay of
Ca2+ transients (6).
The mechanism of Ca2+-dependent regulation of InsP3Rs is
complex (8,
9), and involves direct
Ca2+ binding sites
(5,
10) as well as remote sensing
by extrinsic Ca2+-binding proteins such as CaM
(11,
12), CaBP1
(13,
14), CIB1
(15), and NCS-1
(16).Neuronal Ca2+-binding proteins (CaBP1-5
(17)) represent a new
sub-branch of the CaM superfamily
(18) that regulate various
Ca2+ channel targets. Multiple splice variants and isoforms of
CaBPs are localized in different neuronal cell types
(19-21)
and perform specialized roles in signal transduction. CaBP1, also termed
caldendrin (22), has been
shown to modulate the Ca2+-sensitive activity of InsP3Rs
(13,
14). CaBP1 also regulates
P/Q-type voltage-gated Ca2+ channels
(23), L-type channels
(24), and the transient
receptor potential channel, TRPC5
(25). CaBP4 regulates
Ca2+-dependent inhibition of L-type channels in the retina and may
be genetically linked to retinal degeneration
(26). Thus, the CaBP proteins
are receiving increased attention as a family of Ca2+ sensors that
control a variety of Ca2+ channel targets implicated in neuronal
degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in
CaM and troponin C (18)
(Fig. 1). By analogy to CaM
(27), the four EF-hands are
grouped into two domains connected by a central linker that is four residues
longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain
non-conserved amino acids within the N-terminal region that may confer target
specificity. Another distinguishing property of CaBPs is that the second
EF-hand lacks critical residues required for high affinity Ca2+
binding (17). CaBP1 binds
Ca2+ only at EF3 and EF4, whereas it binds Mg2+ at EF1
that may serve a functional role
(28). Indeed, changes in
cytosolic Mg2+ levels have been detected in cortical neurons after
treatment with neurotransmitter
(29). Other neuronal
Ca2+-binding proteins such as DREAM
(30), CIB1
(31), and NCS-1
(32) also bind Mg2+
and exhibit Mg2+-induced physiological effects. Mg2+
binding in each of these proteins helps stabilize their Ca2+-free
state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary
structural elements (α-helices and β-strands) were derived from NMR
analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted
green, red, cyan, and yellow. Residues in the 12-residue
Ca2+-binding loops are underlined and chelating residues
are highlighted bold. Non-conserved residues in the hydrophobic patch
are colored red.Despite extensive studies on CaBP1, little is known about its structure and
target binding properties, and regulation of InsP3Rs by CaBP1 is
somewhat controversial and not well understood. Here, we present the NMR
solution structures of both Mg2+-bound and Ca2+-bound
conformational states of CaBP1 and their structural interactions with
InsP3R1. These CaBP1 structures reveal important
Ca2+-induced structural changes that control its binding to
InsP3R1. Our target binding analysis demonstrates that the C-domain
of CaBP1 exhibits Ca2+-induced binding to the N-terminal cytosolic
region of InsP3R1. We propose that CaBP1 may regulate
Ca2+-dependent channel activity in InsP3Rs by promoting
a structural interaction between the N-terminal suppressor and ligand-binding
core domains that modulates Ca2+-dependent channel gating
(8,
33,
34). 相似文献
2.
Qinli Wang Bo Chen Peng Liu Maozhong Zheng Yuqing Wang Sujuan Cui Daye Sun Xiaohong Fang Chun-Ming Liu William J. Lucas Jinxing Lin 《The Journal of biological chemistry》2009,284(18):12000-12007
Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In
plants, CaM also appears to be present in the apoplasm, and application of
exogenous CaM has been shown to influence a number of physiological functions
as a polypeptide signal; however, the existence and localization of its
corresponding apoplasmic binding sites remain controversial. To identify the
site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for
single molecule level detection at the surface of plant cells. Using this
approach, we show that QD-CaM binds selectively to sites on the outer surface
of the plasma membrane, which was further confirmed by high resolution
transmission electron microscopy. Measurements of Ca2+ fluxes
across the plasma membrane, using ion-selective microelectrodes, demonstrated
that exogenous CaM induces a net influx into protoplasts. Consistent with
these flux studies, calcium-green-dextran and FRET experiments confirmed that
applied CaM/QD-CaM elicited an increase in cytoplasmic Ca2+ levels.
These results support the hypothesis that apoplasmic CaM can act as a
signaling agent. These findings are discussed in terms of CaM acting as an
apoplasmic peptide ligand to mediate transmembrane signaling in the plant
kingdom.Calmodulin (CaM)2
is a conserved multifunctional calcium sensor that mediates intracellular
Ca2+ signaling and regulates diverse cellular processes by
interacting with calmodulin-binding proteins
(1–3).
Interestingly, in both animals and plants, CaM may also act as an
extracellular agent to regulate physiological events
(4). Consistent with this
notion, extracellular CaM has been detected within the cell walls of a broad
range of plant species (4,
5).Functional studies have established that exogenously applied CaM can
stimulate the proliferation of suspension-cultured plant cells
(6) as well as affect
intracellular activities of heterotrimeric G proteins and phospholipases in
protoplasts (7,
8). Based on these findings, it
has been proposed that, in plants, extracellular CaM may function as a
signaling agent involved in the regulation of cell growth and development
(4). However, as a 17-kDa
hydrophilic protein, exogenously applied CaM could well be retrieved from the
apoplasmic space and then exert its effects on components within the
cytoplasm. Evidence against this hypothesis was provided by studies with
Arabidopsis thaliana suspension-cultured cells in which it was shown
that 24 h of incubation in exogenous CaM did not result in protein uptake or
degradation (4).To exert an effect from the apoplasm, it would seem logical to assume that
a protein(s) within the plant plasma membrane would have a CaM-binding site
exposed to the apoplasm. Although a number of studies have addressed the
molecular mechanism(s) by which extracellular CaM might act as a signal
(6,
9) and attempts have been made
to identify extracellular CaM-binding proteins
(4,
6), currently there is no
direct evidence in support of the hypothesis that specific CaM-binding sites
exist at the surface of plant cells.To address this question, a CaM-conjugated quantum dot (QD) system was
employed for single molecule level detection
(10–13)
at the surface of plant cells. These nanoparticles have several advantages
over conventional fluorophores for light microscopic imaging, including their
higher brightness and photostability
(14,
15). In addition, because of
their electron dense nature, QDs can be used for single labeling studies at
the transmission electron microscope level
(16,
17). Using this QD-CaM system,
we demonstrate that QD-CaM binds selectively to sites on the outer surface of
the plant plasma membrane. We also show by three independent methods that
applied CaM can modulate Ca2+ fluxes across the plasma membrane,
leading to alterations in cytoplasmic Ca2+ status. These findings
support the hypothesis that, in plants, apoplasmic CaM can act as a signaling
agent. 相似文献
3.
Quang-Kim Tran Jared Leonard D. J. Black Owen W. Nadeau Igor G. Boulatnikov Anthony Persechini 《The Journal of biological chemistry》2009,284(18):11892-11899
We have investigated the possible biochemical basis for enhancements in NO
production in endothelial cells that have been correlated with agonist- or
shear stress-evoked phosphorylation at Ser-1179. We have found that a
phosphomimetic substitution at Ser-1179 doubles maximal synthase activity,
partially disinhibits cytochrome c reductase activity, and lowers the
EC50(Ca2+) values for calmodulin binding and enzyme
activation from the control values of 182 ± 2 and 422 ± 22
nm to 116 ± 2 and 300 ± 10 nm. These are
similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q.
K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry
47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179
has no additional effect on maximal synthase activity, cooperativity between
the two substitutions completely disinhibits reductase activity and further
reduces the EC50(Ca2+) values for calmodulin binding and
enzyme activation to 77 ± 2 and 130 ± 5 nm. We have
confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179
and phosphomimetic substitutions at these positions have similar functional
effects. Changes in the biochemical properties of eNOS produced by combined
phosphorylation at Ser-617 and Ser-1179 are predicted to substantially
increase synthase activity in cells at a typical basal free Ca2+
concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and
l-citrulline from l-arginine and O2, with
NADPH as the electron donor
(1). The role of NO generated
by endothelial nitricoxide synthase
(eNOS)2 in the
regulation of smooth muscle tone is well established and was the first of
several physiological roles for this small molecule that have so far been
identified (2). The
nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each
subunit contains a reductase and oxygenase domain
(1). A significant difference
between the reductase domains in eNOS and nNOS and the homologous P450
reductases is the presence of inserts in these synthase isoforms that appear
to maintain them in their inactive states
(3,
4). A calmodulin (CaM)-binding
domain is located in the linker that connects the reductase and oxygenase
domains, and the endothelial and neuronal synthases both require
Ca2+ and exogenous CaM for activity
(5,
6). When CaM is bound, it
somehow counteracts the effects of the autoinhibitory insert(s) in the
reductase. The high resolution structure for the complex between
(Ca2+)4-CaM and the isolated CaM-binding domain from
eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a
pair of Ca2+-binding sites, enfold the domain, as has been observed
in several other such CaM-peptide complexes
(7). Consistent with this
structure, investigations of CaM-dependent activation of the neuronal synthase
suggest that both CaM lobes must participate
(8,
9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497,
Ser-617, Ser-635, and Ser-1179
(10–12).
There are equivalent phosphorylation sites in the human enzyme
(10–12).
Phosphorylation of the bovine enzyme at Thr-497, which is located in the
CaM-binding domain, blocks CaM binding and enzyme activation
(7,
11,
13,
14). Ser-116 can be basally
phosphorylated in cells (10,
11,
13,
15), and dephosphorylation of
this site has been correlated with increased NO production
(13,
15). However, it has also been
reported that a phosphomimetic substitution at this position has no effect on
enzyme activity measured in vitro
(13). Ser-1179 is
phosphorylated in response to a variety of stimuli, and this has been reliably
correlated with enhanced NO production in cells
(10,
11). Indeed, NO production is
elevated in transgenic endothelium expressing an eNOS mutant containing an
S1179D substitution, but not in tissue expressing an S1179A mutant
(16). Shear stress or insulin
treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in
endothelial cells, and this is correlated with increased NO production in the
absence of extracellular Ca2+
(17–19).
Akt-catalyzed phosphorylation or an S1179D substitution has also been
correlated with increased synthase activity in cell extracts at low
intracellular free [Ca2+]
(17). Increased NO production
has also been observed in cells expressing an eNOS mutant containing an S617D
substitution, and physiological stimuli such as shear-stress, bradykinin,
VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and
Ser-1179 (12,
13,
20). Although S617D eNOS has
been reported to have the same maximum activity in vitro as the wild
type enzyme (20), in our hands
an S617D substitution increases the maximal CaM-dependent synthase activity of
purified mutant enzyme ∼2-fold, partially disinhibits reductase activity,
and reduces the EC50(Ca2+) values for CaM binding and
enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp
substitution at Ser-1179 in eNOS on the Ca2+ dependence of CaM
binding and CaM-dependent activation of reductase and synthase activities. We
also describe the effects on these properties of combining this substitution
with one at Ser-617. Finally, we demonstrate that Akt-catalyzed
phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar
functional effects. Our results suggest that phosphorylation of eNOS at
Ser-617 and Ser-1179 can substantially increase synthase activity in cells at
a typical basal free Ca2+ concentration of 50–100
nm, while single phosphorylations at these sites produce smaller
activity increases, and can do so only at higher free Ca2+
concentrations. 相似文献
4.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
5.
6.
Rebecca M. Dixon Jack R. Mellor Jonathan G. Hanley 《The Journal of biological chemistry》2009,284(21):14230-14235
Oxygen and glucose deprivation (OGD) induces delayed cell death in
hippocampal CA1 neurons via Ca2+/Zn2+-permeable,
GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents
in hippocampal neurons show marked inward rectification and increased
sensitivity to channel blockers selective for GluR2-lacking AMPARs. This
occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression
and a rapid internalization of GluR2-containing AMPARs during the OGD insult,
which are replaced by GluR2-lacking receptors. The mechanisms that underlie
this rapid change in subunit composition are unknown. Here, we demonstrate
that this trafficking event shares features in common with events that mediate
long term depression and long term potentiation and is initiated by the
activation of N-methyl-d-aspartic acid receptors. Using
biochemical and electrophysiological approaches, we show that peptides that
interfere with PICK1 PDZ domain interactions block the OGD-induced switch in
subunit composition, implicating PICK1 in restricting GluR2 from synapses
during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at
synapses during OGD as a result of PICK1 PDZ interactions are involved in
OGD-induced delayed cell death. This work demonstrates that PICK1 plays a
crucial role in the response to OGD that results in altered synaptic
transmission and neuronal death and has implications for our understanding of
the molecular mechanisms that underlie cell death during stroke.Oxygen and glucose deprivation
(OGD)3 associated with
transient global ischemia induces delayed cell death, particularly in
hippocampal CA1 pyramidal cells
(1–3),
a phenomenon that involves Ca2+/Zn2+-permeable,
GluR2-lacking AMPARs (4).
AMPARs are heteromeric complexes of subunits GluR1–4
(5), and most AMPARs in the
hippocampus contain GluR2, which renders them calcium-impermeable and results
in a marked inward rectification in their current-voltage relationship
(6–8).
Ischemia induces a delayed down-regulation of GluR2 mRNA and protein
expression (4,
9–11),
resulting in enhanced AMPAR-mediated Ca2+ and Zn2+
influx into CA1 neurons (10,
12). In these neurons,
AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification
1–2 days following ischemia and increased sensitivity to 1-naphthyl
acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs
(13–16).
Blockade of these channels at 9–40 h following ischemia is
neuroprotective, indicating a crucial role for Ca2+-permeable
AMPARs in ischemic cell death
(16).In addition to delayed changes in AMPAR subunit composition as a result of
altered mRNA expression, it was recently reported that
Ca2+-permable, GluR2-lacking AMPARs are targeted to synaptic sites
via membrane trafficking at much earlier times during OGD
(17). This subunit
rearrangement involves endocytosis of AMPARs containing GluR2 complexed with
GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3
(17). However, the molecular
mechanisms behind this trafficking event are unknown, and furthermore, it is
not known whether these trafficking-mediated changes in AMPAR subunit
composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial
involvement in long term depression (LTD) and long term potentiation (LTP),
activity-dependent forms of synaptic plasticity thought to underlie learning
and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching
events (brought about by trafficking that involves endo/exocytosis) are
central to the necessary changes in synaptic receptor complement
(7,
18–20).
It is possible that similar mechanisms regulate AMPAR trafficking during
OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that
binds, via the PDZ domain, to a number of membrane proteins including AMPAR
subunits GluR2/3. This interaction is required for AMPAR internalization from
the synaptic plasma membrane in response to Ca2+ influx via NMDAR
activation in hippocampal neurons
(21–23).
This process is the major mechanism that underlies the reduction in synaptic
strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged
as a mechanism that regulates the GluR2 content of synaptic receptors, which
in turn determines their Ca2+ permeability
(7,
20). This is likely to be of
profound importance in both plasticity and pathological mechanisms.
Importantly, PICK1 overexpression has been shown to induce a shift in synaptic
AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly
rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1
(24). This suggests that PICK1
may mediate the rapid switch in subunit composition occurring during OGD
(17). Here, we demonstrate
that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1
PDZ interactions, and importantly, that this early trafficking event that
occurs during OGD contributes to the signaling that results in delayed
neuronal death. 相似文献
7.
Danielle M. Paul Edward P. Morris Robert W. Kensler John M. Squire 《The Journal of biological chemistry》2009,284(22):15007-15015
The troponin complex on the thin filament plays a crucial role in the
regulation of muscle contraction. However, the precise location of troponin
relative to actin and tropomyosin remains uncertain. We have developed a
method of reconstructing thin filaments using single particle analysis that
does not impose the helical symmetry of actin and is independent of a starting
model. We present a single particle three-dimensional reconstruction of the
thin filament. Atomic models of the F-actin filament were fitted into the
electron density maps and troponin and tropomyosin located. The structure
provides evidence that the globular head region of troponin labels the two
strands of actin with a 27.5-Å axial stagger. The density attributed to
troponin appears tapered with the widest point toward the barbed end. This
leads us to interpret the polarity of the troponin complex in the thin
filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process
with implications ranging from cell migration to muscle contraction. Skeletal
and cardiac muscle thin filaments consist of actin and the regulatory proteins
troponin and tropomyosin. Contraction is initiated by release of
Ca2+ into the sarcomere and the consequent binding of
Ca2+ to regulatory sites on troponin. Troponin is believed to
undergo a conformational change leading to an azimuthal movement of
tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP,
and generate force. The molecular basis by which troponin acts to regulate
muscle contraction is only partly understood. It is essential that the
structure of troponin in the thin filament at high and low Ca2+ is
determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972
(1). In this structure each
tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å
stagger between troponin molecules. The 7-Å tropomyosin structure
(2), the atomic model of
F-actin (3), and the troponin
“core domain” (4)
have recently been used to generate atomic models of the thin filament in low
and high Ca2+ states
(5). While the position of
troponin in these models was constrained by known distance measurements
between filament components, the exact arrangement of the complex on the
filament has not been determined a priori. Although recently
published crystal structures of partial troponin complexes
(4,
6) have provided valuable
insights into the arrangement of the globular head or core domain, the complex
in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended
tail (7). The globular core
contains the Ca2+-binding subunit
(TnC),2 the inhibitory
subunit (TnI), and the C-terminal part (residues 156–262) of the
tropomyosin-binding subunit (TnT). The extended tail consists of the
N-terminal part of TnT (residues 1–155). A structural rearrangement
associated with Ca2+ dissociation from the troponin core has been
observed (4) such that the
helix connecting the two domains of TnC collapses, releasing the TnI
inhibitory segment. It is postulated that the TnI inhibitory segment then
becomes able to bind actin, in so doing biasing tropomyosin
(8). To understand properly how
Ca2+ binding to TnC leads to movement of tropomyosin, it is
necessary to determine a high resolution structure of troponin attached to the
thin filament, allowing unambiguous docking of the available crystal
structures and direct observation of any changes at a molecular level caused
by Ca2+ binding.Direct visualization of the thin filament is possible using electron
microscopy. Tropomyosin strands have been resolved in the low and high
Ca2+ states confirming the movement of tropomyosin and the steric
blocking model (9,
10). Until recently the actin
helical repeat has been imposed in the majority of reconstructions of the thin
filament causing artifacts. Helical averaging using the actin repeat spreads
troponin density over every actin monomer, which prevents the detailed
position and shape of the troponin complex from being found
(11). It is possible to avoid
this effect by applying a single particle approach. Individual filament images
are divided into segments and each segment treated as a particle.
Three-dimensional reconstruction may then be carried out by single particle
techniques of alignment, classification
(12,
13), Euler angle assignment
(14–16)
and exact filter back-projection
(17,
18).Two forms of single particle analysis have emerged: helical single particle
analysis (19), where the
determined helical symmetry is applied to the final reconstruction, and
non-helical single particle analysis, which treats the complex as a truly
asymmetric particle. Helical single particle analysis has been used to
successfully reconstruct a myosin containing invertebrate thick filament to a
resolution of 25 Å (20),
and non-helical single particle analysis has been applied to the vertebrate
skeletal muscle thick filament allowing azimuthal perturbations of the myosin
heads to be observed (21).Model-based single particle image processing methods have recently been
applied to the structural analysis of the vertebrate
(5,
22,
23) and the insect thin
filament (24). We have
deliberately avoided starting with a model and any potential model bias by
using a reference-free alignment procedure. The adaptation of conventional
procedures and their application to the structural study of the muscle thin
filament has been documented
(25). 相似文献
8.
9.
Neeliyath A. Ramakrishnan Marian J. Drescher Dennis G. Drescher 《The Journal of biological chemistry》2009,284(3):1364-1372
The molecular mechanisms underlying synaptic exocytosis in the hair cell,
the auditory and vestibular receptor cell, are not well understood. Otoferlin,
a C2 domain-containing Ca2+-binding protein, has been implicated as
having a role in vesicular release. Mutations in the OTOF gene cause
nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In
the present study, we generated otoferlin fusion proteins containing two of
the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F
and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and
SNAP-25 in a Ca2+-dependent manner (with optimal 61
μm free Ca2+ required for binding). These
interactions were greatly diminished for C2F with the P1825A mutation,
possibly because of a reduction in tertiary structural change, induced by
Ca2+, for the mutated C2F compared with the native C2F. The
otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity
(Kd = 1.7 × 10–5 m) than
for the C2F interaction (Kd = 2.6 ×
10–9 m). In contrast, it was the otoferlin C2D
domain that bound the Cav1.3 II-III loop, in a
Ca2+-dependent manner. The L1011P mutation in C2D rendered this
binding insensitive to Ca2+ and considerably diminished. Overall,
we demonstrated that otoferlin interacts with two main target-SNARE proteins
of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the
calcium channel, with the otoferlin C2F and C2D domains of central importance
for binding. Because mutations in the otoferlin C2 domains that cause deafness
in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the
Cav1.3 calcium channel, it is these interactions that may mediate
regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear
hair cell function.Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref.
1). In mechanosensory hair
cells, calcium microdomains (2)
and possibly nanodomains (3)
are formed when voltage-gated calcium channels open upon depolarization.
Calcium at these sites is thought to activate protein interactions, leading to
vesicle fusion. Some of the key players in this process are the
target-SNARE2
proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin
(4). Vesicle-SNARE
synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal
synapse, modulating calcium channels and vesicle release by a
Ca2+-dependent interaction with other SNARE proteins in the
presence of lipid molecules
(4–6).
However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not
detected (7). Instead, fast
neurotransmitter release in auditory and vestibular hair cells, facilitated
largely by an L-type voltagegated calcium channel, Cav1.3
(8,
9), is thought to be modulated
by a newly discovered protein, otoferlin, acting as the Ca2+ sensor
and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic
deafness (10). Gene sequences
of different deaf families show that the OTOF gene can undergo
mutation at multiple locations
(11–13).
Recently, it has been demonstrated that otoferlin is necessary for synaptic
exocytosis from hair cells
(14). Further, an engineered
mutation in the C2B domain of otoferlin has been shown to cause deafness in
mice (15). However, the
precise function of otoferlin as a synaptic protein is not well
understood.Specific mutations in the otoferlin C2F (P1825A) or C2D (L1011P) domains in
humans have been documented to cause DFNB9 deafness
(11,
12). Previous studies
suggested that a region of otoferlin containing all three C2 domains, D, E,
and F, binds directly to the t-SNARE molecules syntaxin 1A and SNAP-25 in
response to an increase in Ca2+ concentration
(14). However, it is not
understood how a single amino acid substitution in one domain of otoferlin,
such as C2F (11) or C2D
(12), might independently lead
to deafness. Here, we examine the role of otoferlin as a Ca2+
sensor as well as a facilitator of vesicle fusion, as indicated by
protein-protein interactions and their [Ca2+] dependence. 相似文献
10.
Christian Rosker Gargi Meur Emily J. A. Taylor Colin W. Taylor 《The Journal of biological chemistry》2009,284(8):5186-5194
Ryanodine receptors (RyR) are Ca2+ channels that mediate
Ca2+ release from intracellular stores in response to diverse
intracellular signals. In RINm5F insulinoma cells, caffeine, and
4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca2+
entry that was independent of store-operated Ca2+ entry, and
blocked by prior incubation with a concentration of ryanodine that inactivates
RyR. Patch-clamp recording identified small numbers of large-conductance
(γK = 169 pS) cation channels that were activated by
caffeine, 4CmC or low concentrations of ryanodine. Similar channels were
detected in rat pancreatic β-cells. In RINm5F cells, the channels were
blocked by cytosolic, but not extracellular, ruthenium red. Subcellular
fractionation showed that type 3 IP3 receptors (IP3R3)
were expressed predominantly in endoplasmic reticulum, whereas RyR2 were
present also in plasma membrane fractions. Using RNAi selectively to reduce
expression of RyR1, RyR2, or IP3R3, we showed that RyR2 mediates
both the Ca2+ entry and the plasma membrane currents evoked by
agonists of RyR. We conclude that small numbers of RyR2 are selectively
expressed in the plasma membrane of RINm5F pancreatic β-cells, where they
mediate Ca2+ entry.Ryanodine receptors
(RyR)3 and inositol
1,4,5-trisphosphate receptors (IP3R)
(1,
2) are the archetypal
intracellular Ca2+ channels. Both are widely expressed, although
RyR are more restricted in their expression than IP3R
(3,
4). In common with many cells,
pancreatic β-cells and insulin-secreting cell lines express both
IP3R (predominantly IP3R3)
(5,
6) and RyR (predominantly RyR2)
(7). Both RyR and
IP3R are expressed mostly within membranes of the endoplasmic (ER),
where they mediate release of Ca2+. Functional RyR are also
expressed in the secretory vesicles
(8,
9) or, and perhaps more likely,
in the endosomes of β-cells
(10). Despite earlier
suggestions (11),
IP3R are probably not present in the secretory vesicles of
β-cells (8,
12,
13).All three subtypes of IP3R are stimulated by IP3 with
Ca2+ (1), and the
three subtypes of RyR are each directly regulated by Ca2+. However,
RyR differ in whether their most important physiological stimulus is
depolarization of the plasma membrane (RyR1), Ca2+ (RyR2) or
additional intracellular messengers like cyclic ADP-ribose. The latter
stimulates both Ca2+ release and insulin secretion in β-cells
(8,
14). The activities of both
families of intracellular Ca2+ channels are also modulated by many
additional signals that act directly or via phosphorylation
(15,
16). Although they commonly
mediate release of Ca2+ from the ER, both IP3R and RyR
select rather poorly between Ca2+ and other cations (permeability
ratio, PCa/PK ∼7)
(1,
17). This may allow
electrogenic Ca2+ release from the ER to be rapidly compensated by
uptake of K+ (18),
and where RyR or IP3R are expressed in other membranes it may allow
them to affect membrane potential.Both Ca2+ entry and release of Ca2+ from
intracellular stores contribute to the oscillatory increases in cytosolic
Ca2+ concentration ([Ca2+]i) that
stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells
(7). Glucose rapidly
equilibrates across the plasma membrane (PM) of β-cells and its oxidative
metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing
KATP channels to close
(19). This allows an
unidentified leak current to depolarize the PM
(20) and activate
voltage-gated Ca2+ channels, predominantly L-type Ca2+
channels (21). The resulting
Ca2+ entry is amplified by Ca2+-induced Ca2+
release from intracellular stores
(7), triggering exocytotic
release of insulin-containing dense-core vesicles
(22). The importance of this
sequence is clear from the widespread use of sulfonylurea drugs, which close
KATP channels, in the treatment of type 2 diabetes. Ca2+
uptake by mitochondria beneath the PM further stimulates ATP production,
amplifying the initial response to glucose and perhaps thereby contributing to
the sustained phase of insulin release
(23). However, neither the
increase in [Ca2+]i nor the insulin release
evoked by glucose or other nutrients is entirely dependent on Ca2+
entry (7,
24) or closure of
KATP channels (25).
This suggests that glucose metabolism may also more directly activate RyR
(7,
26) and/or IP3R
(27) to cause release of
Ca2+ from intracellular stores. A change in the ATP/ADP ratio is
one means whereby nutrient metabolism may be linked to opening of
intracellular Ca2+ channels because both RyR
(28) and IP3R
(1) are stimulated by ATP.The other major physiological regulators of insulin release are the
incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic
hormone (29). These hormones,
released by cells in the small intestine, stimulate synthesis of cAMP in
β-cells and thereby potentiate glucose-evoked insulin release
(30). These pathways are also
targets of drugs used successfully to treat type 2 diabetes
(29). The responses of
β-cells to cAMP involve both cAMP-dependent protein kinase and epacs
(exchange factors activated by cAMP)
(31,
32). The effects of the latter
are, at least partly, due to release of Ca2+ from intracellular
stores via RyR
(33–35)
and perhaps also via IP3R
(36). The interplays between
Ca2+ and cAMP signaling generate oscillatory changes in the
concentrations of both messengers
(37). RyR and IP3R
are thus implicated in mediating responses to each of the major physiological
regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores,
which probably include both the ER and secretory vesicles and/or endosomes,
functional RyR2 are also expressed in small numbers in the PM of RINm5F
insulinoma cells and rat pancreatic β-cells. 相似文献
11.
Sean R. Stowell Moonjae Cho Christa L. Feasley Connie M. Arthur Xuezheng Song Jennifer K. Colucci Sougata Karmakar Padmaja Mehta Marcelo Dias-Baruffi Rodger P. McEver Richard D. Cummings 《The Journal of biological chemistry》2009,284(8):4989-4999
Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell
surface exposure of phosphatidylserine (PS), a ligand that targets cells for
phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer
equilibrium appears to modulate Gal-1-induced PS exposure, although the
mechanism underlying this regulation remains unclear. Here we show that
monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant
form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired
dimerization and fails to induce cell surface PS exposure while retaining the
ability to recognize carbohydrates and signal Ca2+ flux in
leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas
ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1
dimerization. Continual incubation of leukocytes with Gal-1 resulted in
gradual oxidative inactivation with concomitant loss of cell surface PS,
whereas rapid oxidation prevented mGal-1 from inducing PS exposure.
Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and
mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to
signal sustained PS exposure in leukocytes and mGal-1 to signal both
Ca2+ flux and PS exposure. Taken together, these results
demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to
oxidative inactivation and provides a mechanism whereby ligand partially
protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated
leukocytes following an inflammatory episode. Several factors, including
members of the galectin and tumor necrosis factor families
(1,
2), regulate leukocyte turnover
by inducing apoptotic cell death. In contrast, several galectin family
members, in particular galectin-1
(Gal-1),2 uniquely
regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure,
which normally sensitizes apoptotic cells to phagocytic removal
(3,
4), independent of apoptosis, a
process recently termed preaparesis
(5).Previous studies suggested that dimerization may be required for
Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two
point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays
impaired Gal-1 dimerization and fails to induce PS exposure
(6). However, the manner in
which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear.
Previous studies suggest that dimerization may be required for efficient
cross-linking of functional receptors or the formation of signaling lattices
(7–9).
Consistent with this, monomeric mutants of several other galectins fail to
induce PS exposure or signal leukocytes
(4,
8). Gal-1 signaling of PS
exposure requires initial signaling events, such as mobilization of
intracellular Ca2+ followed by sustained receptor engagement
(10). Although mGal-1 fails to
induce PS exposure (6), whether
mGal-1 can induce these initial signaling events remains unknown
(10).In addition to directly regulating signaling, monomer-dimer equilibrium may
also regulate other aspects of Gal-1 function. Unlike many other proteins
involved in the regulation of immunity, Gal-1 displays unique sensitivity to
oxidative inactivation
(11–15).
Although engagement of ligand partially protects Gal-1 from oxidation
(15), the impact of Gal-1
oxidation on signaling remains enigmatic. During oxidation, Gal-1 forms three
distinct intramolecular disulfide bridges that facilitate profound
conformational changes that preclude ligand binding and Gal-1 dimerization
(12–14),
suggesting that monomerdimer equilibrium may also regulate Gal-1 sensitivity
to oxidative inactivation.Previous studies utilized dithiothreitol (DTT) in treatment conditions to
protect Gal-1 from oxidative inactivation
(16,
17). Indeed, failure to
include DTT precluded Gal-1-induced death in T cells
(3,
18), suggesting that Gal-1
undergoes rapid oxidation in vivo in the absence of reducing
conditions. However, DTT itself can induce apoptosis in leukocytes
(19), leaving questions
regarding the impact of Gal-1 oxidation on these signaling events. In
contrast, recent studies utilizing iodoacetamide-alkylated Gal-1 (iGal-1),
previously shown to protect Gal-1 from oxidative inactivation
(20–29),
demonstrated that DTT actually primes cells to become sensitive to
Gal-1-induced apoptosis regardless of Gal-1 sensitivity to oxidation
(5).As the engagement of leukocyte ligands requires glycan recognition and
oxidation precludes this binding
(11,
15), understanding the impact
of oxidation on Gal-1 signals will facilitate a greater appreciation of the
factors that govern Gal-1 oxidation and therefore function. Our results
demonstrate that Gal-1 monomer-dimer equilibrium provides a key regulatory
point controlling both Gal-1 sensitivity to oxidation and its ability to
signal PS exposure in leukocytes. These results provide novel insights into
Gal-1 function and explain at a biochemical level the mechanisms regulating
Gal-1 oxidative inactivation and signaling. 相似文献
12.
Hongjie Yuan Katie M. Vance Candice E. Junge Matthew T. Geballe James P. Snyder John R. Hepler Manuel Yepes Chian-Ming Low Stephen F. Traynelis 《The Journal of biological chemistry》2009,284(19):12862-12873
Zinc is hypothesized to be co-released with glutamate at synapses of the
central nervous system. Zinc binds to NR1/NR2A
N-methyl-d-aspartate (NMDA) receptors with high affinity
and inhibits NMDAR function in a voltage-independent manner. The serine
protease plasmin can cleave a number of substrates, including
protease-activated receptors, and may play an important role in several
disorders of the central nervous system, including ischemia and spinal cord
injury. Here, we demonstrate that plasmin can cleave the native NR2A
amino-terminal domain (NR2AATD), removing the functional high
affinity Zn2+ binding site. Plasmin also cleaves recombinant
NR2AATD at lysine 317 (Lys317), thereby producing a
∼40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments
observed in rat brain membrane preparations. A homology model of the
NR2AATD predicts that Lys317 is near the surface of the
protein and is accessible to plasmin. Recombinant expression of NR2A with an
amino-terminal deletion at Lys317 is functional and Zn2+
insensitive. Whole cell voltage-clamp recordings show that Zn2+
inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected
HEK 293 cells and cultured cortical neurons is significantly reduced by
plasmin treatment. Mutating the plasmin cleavage site Lys317 on
NR2A to alanine blocks the effect of plasmin on Zn2+ inhibition.
The relief of Zn2+ inhibition by plasmin occurs in
PAR1-/- cortical neurons and thus is independent of interaction
with protease-activated receptors. These results suggest that plasmin can
directly interact with NMDA receptors, and plasmin may increase NMDA receptor
responses through disruption or removal of the amino-terminal domain and
relief of Zn2+ inhibition.N-Methyl-d-aspartate
(NMDA)2 receptors are
one of three types of ionotropic glutamate receptors that play critical roles
in excitatory neurotransmission, synaptic plasticity, and neuronal death
(1–3).
NMDA receptors are comprised of glycine-binding NR1 subunits in combination
with at least one type of glutamate-binding NR2 subunit
(1,
4). Each subunit contains three
transmembrane domains, one cytoplasmic re-entrant membrane loop, one bi-lobed
domain that forms the ligand binding site, and one bi-lobed amino-terminal
domain (ATD), thought to share structural homology to periplasmic amino
acid-binding proteins
(4–6).
Activation of NMDA receptors requires combined stimulation by glutamate and
the co-agonist glycine in addition to membrane depolarization to overcome
voltage-dependent Mg2+ block of the ion channel
(7). The activity of NMDA
receptors is negatively modulated by a variety of extracellular ions,
including Mg2+, polyamines, protons, and Zn2+ ions,
which can exert tonic inhibition under physiological conditions
(1,
4). Several extracellular
modulators such as Zn2+ and ifenprodil are thought to act at the
ATD of the NMDA receptor
(8–14).Zinc is a transition metal that plays key roles in both catalytic and
structural capacities in all mammalian cells
(15). Zinc is required for
normal growth and survival of cells. In addition, neuronal death in
hypoxia-ischemia and epilepsy has been associated with Zn2+
(16–18).
Abnormal metabolism of zinc may contribute to induction of cytotoxicity in
neurodegenerative diseases, such as Alzheimer''s disease, Parkinson''s disease,
and amyotrophic lateral sclerosis
(19). Zinc is co-released with
glutamate at excitatory presynaptic terminals and inhibits native NMDA
receptor activation (20,
21). Zn2+ inhibits
NMDA receptor function through a dual mechanism, which includes
voltage-dependent block and voltage-independent inhibition
(22–24).
Voltage-independent Zn2+ inhibition at low nanomolar concentrations
(IC50, 20 nm) is observed for NR2A-containing NMDA
receptors
(25–28).
Evidence has accumulated that the amino-terminal domain of the NR2A subunit
controls high-affinity Zn2+ inhibition of NMDA receptors, and
several histidine residues in this region may constitute part of an
NR2A-specific Zn2+ binding site
(8,
9,
11,
12). For the NR2A subunit,
several lines of evidence suggest that Zn2+ acts by enhancing
proton inhibition (8,
11,
29,
30).Serine proteases present in the circulation, mast cells, and elsewhere
signal directly to cells by cleaving protease-activated receptors (PARs),
members of a subfamily of G-protein-coupled receptors. Cleavage exposes a
tethered ligand domain that binds to and activates the cleaved receptors
(31,
32). Protease receptor
activation has been studied extensively in relation to coagulation and
thrombolysis (33). In addition
to their circulation in the bloodstream, some serine proteases and PARs are
expressed in the central nervous system, and have been suggested to play roles
in physiological conditions (e.g. long-term potentiation or memory)
and pathophysiological states such as glial scarring, edema, seizure, and
neuronal death (31,
34–36).Functional interactions between proteases and NMDA receptors have
previously been suggested. Earlier studies reported that the blood-derived
serine protease thrombin potentiates NMDA receptor response more than 2-fold
through activation of PAR1
(37). Plasmin, another serine
protease, similarly potentiates NMDA receptor response
(38). Tissue-plasminogen
activator (tPA), which catalyzes the conversion of the zymogen precursor
plasminogen to plasmin and results in PAR1 activation, also interacts with and
cleaves the ATD of the NR1 subunit of the NMDA receptor
(39,
40). This raises the
possibility that plasmin may also interact directly with the NMDA receptor
subunits to modulate receptor response. We therefore investigated the ability
of plasmin to cleave the NR2A NMDA receptor subunit. We found that nanomolar
concentrations of plasmin can cleave within the ATD, a region that mediates
tonic voltage-independent Zn2+ inhibition of NR2A-containing NMDA
receptors. We hypothesized that plasmin cleavage reduces the
Zn2+-mediated inhibition of NMDA receptors by removing the
Zn2+ binding domain. In the present study, we have demonstrated
that Zn2+ inhibition of agonist-evoked NMDA currents is decreased
significantly by plasmin treatment in recombinant NR1/NR2A-transfected HEK 293
cells and cultured cortical neurons. These concentrations of plasmin may be
pathophysiologically relevant in situations in which the blood-brain barrier
is compromised, which could allow blood-derived plasmin to enter brain
parenchyma at concentrations in excess of these that can cleave NR2A. Thus,
ability of plasmin to potentiate NMDA function through the relief of the
Zn2+ inhibition could exacerbate the harmful actions of NMDA
receptor overactivation in pathological situations. In addition, if newly
cleaved NR2AATD enters the bloodstream during ischemic injury, it
could serve as a biomarker of central nervous system injury. 相似文献
13.
Richard L. Daniels Yoshio Takashima David D. McKemy 《The Journal of biological chemistry》2009,284(3):1570-1582
Cold temperatures robustly activate a small cohort of somatosensory nerves,
yet during a prolonged cold stimulus their activity will decrease, or adapt,
over time. This process allows for the discrimination of subtle changes in
temperature. At the molecular level, cold is detected by transient receptor
potential melastatin 8 (TRPM8), a nonselective cation channel expressed on a
subset of peripheral afferent fibers. We and others have reported that TRPM8
channels also adapt in a calcium-dependent manner when activated by the
cooling compound menthol. Additionally, TRPM8 activity is sensitive to the
phospholipid phosphoinositol 4,5-bisphosphate (PIP2), a substrate
for the enzyme phospholipase C (PLC). These results suggest an adaptation
model whereby TRPM8-mediated Ca2+ influx activates PLC, thereby
decreasing PIP2 levels and resulting in reduced TRPM8 activity.
Here we tested this model using pharmacological activation of PLC and by
manipulating PIP2 levels independent of both PLC and
Ca2+. PLC activation leads to adaptation-like reductions in cold-
or menthol-evoked TRPM8 currents in both heterologous and native cells.
Moreover, PLC-independent reductions in PIP2 had a similar effect
on cold- and menthol-evoked currents. Mechanistically, either form of
adaptation does not alter temperature sensitivity of TRPM8 but does lead to a
change in channel gating. Our results show that adaptation is a shift in
voltage dependence toward more positive potentials, reversing the trend toward
negative potentials caused by agonist. These data suggest that PLC activity
not only mediates adaptation to thermal stimuli, but likely underlies a more
general mechanism that establishes the temperature sensitivity of
somatosensory neurons.The detection of temperature is a fundamental task of the nervous system.
Temperature-sensing sensory afferent neurons reside in either the trigeminal
(TG)2 or dorsal root
(DRG) sensory ganglia and project peripherally, terminating as free nerve
endings that innervate areas of the head or trunk, respectively
(1,
2). Subpopulations of these
afferents respond to distinct sub-modalities of thermal stimuli, including
noxious heat, innocuous cooling and warmth, and painfully cold temperatures.
Each carries thermal information to the dorsal horn of the spinal cord,
synapsing with neurons that project centrally
(1,
3).The discovery of thermosensitive ion channels of the transient receptor
potential (TRP) family demonstrated an underlying molecular mechanism for
temperature detection (4). Cold
temperature sensation is largely mediated by TRPM8, a nonselective cation
channel expressed on a small subset of neurons
(5,
6). TRPM8 is activated by
cooling compounds, such as menthol, as well as cold temperatures below ∼28
°C, in vitro (7,
8). Recent reports on the
behavioral phenotype of TRPM8-null mice suggest that this lone channel is
required for the majority of cold sensing in vivo
(5,
9–11).
These and other data strongly implicate TRPM8 in not only the detection of
both innocuous cool and some aspects of noxious cold but also injury-induced
hypersensitivity to cold and, paradoxically, cooling-mediated analgesia
(11,
12). Thus, understanding
regulatory mechanisms that alter TRPM8 activity will provide keen insights
into temperature sensation, nociception, and analgesia.One fundamental property of cold-sensitive neurons is an intrinsic ability
to adapt to prolonged cold stimuli, a mechanism that is likely critical for
discrimination of changing environmental conditions
(13,
14). We and others have shown
that cold-sensitive neurons adapt to cold and menthol over time in
vitro (6,
15), a phenomenon also
observed with recombinant TRPM8 channels activated by menthol
(7). During sustained exposure
to menthol, TRPM8 currents adapt in a manner that is dependent upon the
presence of external calcium
(7). Interestingly, cold- and
menthol-evoked currents are highly sensitive to cellular manipulation. In
heterologous cells, TRPM8 currents quickly decrease or run down upon membrane
patch excision (16,
17). Moreover, in membrane
patches excised from cold- and menthol-sensitive DRG neurons, cold thresholds
for current activation exhibit a shift of ∼10 °C to colder
temperatures in comparison with thresholds recorded in intact cells
(18).Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane
phospholipid that accounts for ∼1% of all lipids in the inner leaflet of
the plasma membrane and is known to regulate a variety of ion channels,
including TRPM8 (16,
17). When applied to the
cytoplasmic face of excised membrane patches containing TRPM8 channels,
PIP2 can recover menthol-evoked currents to near pre-rundown levels
(16,
17). PIP2 is
proposed to interact with channels either through electrostatic interactions
or by binding to target proteins at specific phosphoinositide-binding sites
(19,
20). Membrane PIP2
levels are a product of enzymatic activity, such as phosphoinositide kinases
that synthesize PIP2 from membrane precursors and phospholipase C
(PLC) that hydrolyzes it, creating membrane-bound diacylglycerol (DAG) and
cytosolic inositol trisphosphate (IP3), both of which function as
second messengers. Of the three different PLC isotypes, PLCδ isoforms
are modulated by increases in intracellular calcium
(21).When taken in context with the sensitivity of TRPM8 currents to
PIP2 levels, a model has been proposed whereby adaption is a result
of channel-mediated Ca2+ influx activating one or more PLCδ
isoforms (16,
17). The subsequent reductions
in PIP2 levels then promote reduced or adapted TRPM8 currents.
However, this hypothesis has not been conclusively shown in intact
heterologous cells or in somatosensory neurons expressing TRPM8. Moreover,
other alternative hypotheses for TRPM8 adaptation have been proposed,
including Ca2+-dependent kinase activity mediated by protein kinase
C (22,
23). Thus, the cellular and
molecular mechanisms for Ca2+-mediated TRPM8 adaptation are
unclear.Here we show, in both heterologous cells and native TRPM8-expressing
neurons, that Ca2+-independent activation of PLC results in adapted
TRPM8 currents. Moreover, PLC- and Ca2+-independent PIP2
depletion in heterologous cells produces similar effects on TRPM8 activity,
again reducing both cold- and menthol-evoked currents. Mechanistically, we
find that all such manipulations do not alter the temperature sensitivity of
the channel but do lead to a shift in the voltage dependence of TRPM8 channel
gating. 相似文献
14.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
15.
Joey Lai Oliver K. Bernhard Stuart G. Turville Andrew N. Harman John Wilkinson Anthony L. Cunningham 《The Journal of biological chemistry》2009,284(17):11027-11038
C-type lectin receptors expressed on the surface of dendritic cells and
macrophages are able to bind glycoproteins of microbial pathogens via mannose,
fucose, and N-acetylglucosamine. Langerin on Langerhans cells,
dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin
on dendritic cells, and mannose receptor (MR) on dendritic cells and
macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120
principally via high mannose oligosaccharides. These C-type lectin receptors
can also oligomerize to facilitate enhanced ligand binding. This study
examined the effect of oligomerization of MR on its ability to bind to mannan,
monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry
analysis of cross-linked MR showed homodimerization on the surface of primary
monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric
MR were precipitated by mannan, but only the dimeric form was
co-immunoprecipitated by gp120. These results were confirmed independently by
flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a
cellular HIV virion capture assay. As expected, mannan bound to the
carbohydrate recognition domains of MR dimers mostly in a calcium-dependent
fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on
MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only
partially blocked by mannan, and was also partially inhibited by
N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding
occurs via the calcium-dependent, non-calcium-dependent carbohydrate
recognition domains and the cysteine-rich domain at the C terminus of MR
dimers, presenting a much broader target for potential inhibitors of gp120-MR
binding.The mannose receptor
(MR)2 is a C-type
lectin receptor that is expressed on the surface of a variety of cells,
including immature monocyte-derived dendritic cells (MDDC), dermal dendritic
cells, macrophages, and hepatic endothelial cells. It is a multifunctional
protein, involved in antigen recognition and internalization during the early
stages of the innate immune response
(1) as well as physiological
clearance of the endogenous pituitary hormones lutropin and thyrotropin
(2,
3). Recognition of foreign
antigens occurs via mannose, fucose, and GlcNAc residues
(4,
5), which are generally not
found as terminal residues on mammalian glycoproteins but are highly abundant
on surface proteins of pathogens such as the HIV-1 envelope gp120
(6,
7). Once bound, pathogens can
be internalized by endocytosis or phagocytosis, where they are targeted to
lysosomes for proteolytic degradation and presentation on major
histocompatibility complex class II
(8). In immature DCs, soluble
recombinant HIV envelope proteins are processed by this pathway, initially
binding to both dendritic cell-specific intracellular adhesion molecule 3
grabbing non-integrin (DC-SIGN) and MR and ultimately co-localizing with MR
but not DC-SIGN in lysosomes
(9). Furthermore, in immature
DCs and to a greater extent mature DCs, a proportion of intact HIV-1 enters a
unique vesicular compartment that co-localizes with tetraspanin proteins such
as CD81 (10,
11). Recently, this
compartment has been shown to be continuous with the plasma membrane
(11) and does not represent a
continuation of the endolysosomal network. Interestingly, this compartment can
translocate virus from DCs to CD4 T cells, upon the formation of a virological
synapse
(10–12).
Although viral uptake can occur in DCs independent of HIV env
(2), the efficiency of HIV
binding and uptake is greatly enhanced by the presence of C-type lectin-env
interactions. At least initial binding to DC-SIGN (and most likely also MR) is
required for T cell trans-infection
(13).Structurally, the extracellular domain of MR consists of an N-terminal
cysteine-rich domain (Cys-RD), followed by a fibronectin type II domain and
eight carbohydrate recognition domains (CRD) on a single polypeptide backbone
(1). Of the eight CRDs, CRD
4–8 have been shown to be required for high affinity binding of ligands
containing terminal mannose/fucose/GlcNAc residues, with CRD 4 having
demonstrable monosaccharide binding in isolation
(14). Binding and release of
ligand within the low pH environment of the endolysosomal compartment are also
Ca2+-dependent. Acid-induced removal of Ca2+ binding in
CRD 4 and 5 was shown to cause a conformational rearrangement of the domain,
resulting in a loss of carbohydrate binding activity
(15). In contrast, binding of
sulfated carbohydrates to the Cys-RD appears to be Ca2+-independent
as no Ca2+-binding sites were observed in its crystal structure
(2,
16).Oligomerization of CLRs such as DC-SIGN
(17), Langerin
(18), and mannose-binding
protein (19) has been reported
to be essential for binding of oligosaccharide-bearing ligands. Early studies
on MR suggested that it exists solely as a monomeric molecule and that
clustering of multiple CRDs within the single polypeptide backbone was
necessary for high affinity binding of oligosaccharide moieties
(20). However, more recent
studies have shown that dimerization is possible in the presence of
Ca2+ (21) and that
an equilibrium may exist between monomeric and dimeric forms on the cell
surface (22). It is currently
unclear what effect dimerization has on ligand binding to the CRDs; however,
there is evidence that dimerization of MR is required for high affinity
binding of ligands bearing terminal N-acetylgalactosamine 4-sulfate
(GalNAc-4-SO4) such as lutropin and thyrotropin
(22) to the Cys-RD.To date, studies on the oligomerization and ligand binding activity of MR
have used solubilized protein from cell lysates
(20) or purified recombinant
fragments (21). Because the
membrane microenvironment can influence protein associations, soluble forms of
MR may not necessarily be a true model of the quaternary structure and
function of the native protein. Here, we used a well established method of
cross-linking (23) on MDDCs,
monocyte-derived macrophages (MDMs), and MR-transfected Rat-6 cells to
preserve lateral protein-protein interactions between MR on the cell surface
prior to solubilization. Mass spectrometry analysis of affinity-purified
complexes showed they were homo-oligomers, and further resolution of the
complex on a low percentage polyacrylamide gel by SDS-PAGE strongly indicates
that they are dimers. Dimerization of MR was also found to be essential for
binding mannan, monomeric gp120, native trimeric gp140, and HIV-1 viral
particles. Persistence of monomeric gp120 and trimeric gp140 binding to
dimeric MR in the presence of EGTA and various CRD and other inhibitors,
however, suggested that gp120-mediated HIV-1 binding is not
Ca2+-dependent and that at least binding probably occurs to both
Ca2+-dependent and -independent CRDs and also the Cys-RD. 相似文献
16.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
17.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
18.
19.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献
20.
Andrés Norambuena Claudia Metz Lucas Vicu?a Antonia Silva Evelyn Pardo Claudia Oyanadel Loreto Massardo Alfonso González Andrea Soza 《The Journal of biological chemistry》2009,284(19):12670-12679
Galectins have been implicated in T cell homeostasis playing complementary
pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent
pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase
D/phosphatidic acid signaling pathway that has not been reported for any
galectin before. Gal-8 increases phosphatidic signaling, which enhances the
activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a
subsequent decrease in basal protein kinase A activity. Strikingly, rolipram
inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4
activation releases a negative influence of cAMP/protein kinase A on ERK1/2.
The resulting strong ERK1/2 activation leads to expression of the death factor
Fas ligand and caspase-mediated apoptosis. Several conditions that decrease
ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking
antibodies. In addition, experiments with freshly isolated human peripheral
blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28,
show that Gal-8 is pro-apoptotic on activated T cells, most likely on a
subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic
lupus erythematosus block the apoptotic effect of Gal-8. These results
implicate Gal-8 as a novel T cell suppressive factor, which can be
counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly
studied as regulators of the immune response and potential therapeutic agents
for autoimmune disorders (1).
To date, 15 galectins have been identified and classified according with the
structural organization of their distinctive monomeric or dimeric carbohydrate
recognition domain for β-galactosides
(2,
3). Galectins are secreted by
unconventional mechanisms and once outside the cells bind to and cross-link
multiple glycoconjugates both at the cell surface and at the extracellular
matrix, modulating processes as diverse as cell adhesion, migration,
proliferation, differentiation, and apoptosis
(4–10).
Several galectins have been involved in T cell homeostasis because of their
capability to kill thymocytes, activated T cells, and T cell lines
(11–16).
Pro-apoptotic galectins might contribute to shape the T cell repertoire in the
thymus by negative selection, restrict the immune response by eliminating
activated T cells at the periphery
(1), and help cancer cells to
escape the immune system by eliminating cancer-infiltrating T cells
(17). They have also a
promising therapeutic potential to eliminate abnormally activated T cells and
inflammatory cells (1). Studies
on the mostly explored galectins, Gal-1, -3, and -9
(14,
15,
18–20),
as well as in Gal-2 (13),
suggest immunosuppressive complementary roles inducing different pathways to
apoptosis. Galectin-8
(Gal-8)4 is one of the
most widely expressed galectins in human tissues
(21,
22) and cancerous cells
(23,
24). Depending on the cell
context and mode of presentation, either as soluble stimulus or extracellular
matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis
(6,
7,
9,
10,
22,
25). Its role has been mostly
studied in relation to tumor malignancy
(23,
24). However, there is some
evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or
inflammatory disorders. For instance, the intrathymic expression and
pro-apoptotic effect of Gal-8 upon CD4highCD8high
thymocytes suggest a role for Gal-8 in shaping the T cell repertoire
(16). Gal-8 could also
modulate the inflammatory function of neutrophils
(26), Moreover Gal-8-blocking
agents have been detected in chronic autoimmune disorders
(10,
27,
28). In rheumatoid arthritis,
Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid
cells, but can be counteracted by a specific rheumatoid version of CD44
(CD44vRA) (27). In systemic
lupus erythematosus (SLE), a prototypic autoimmune disease, we recently
described function-blocking autoantibodies against Gal-8
(10,
28). Thus it is important to
define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in
immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with
specific integrins, such as α1β1, α3β1, and
α5β1 but not α4β1, and as a matrix protein promotes cell
adhesion and asymmetric spreading through activation of the extracellular
signal-regulated kinases 1 and 2 (ERK1/2)
(10). These early effects
occur within 5–30 min. However, ERK1/2 signaling supports long term
processes such as T cell survival or death, depending on the moment of the
immune response. During T cell activation, ERK1/2 contributes to enhance the
expression of interleukin-2 (IL-2) required for T cell clonal expansion
(29). It also supports T cell
survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by
other previously activated T cells
(30,
31). Later on, ERK1/2 is
required for activation-induced cell death, which controls the extension of
the immune response by eliminating recently activated and restimulated T cells
(32,
33). In activation-induced
cell death, ERK1/2 signaling contributes to enhance the expression of FasL and
its receptor Fas/CD95 (32,
33), which constitute a
preponderant pro-apoptotic system in T cells
(34). Here, we ask whether
Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to
participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling
(35) deserves special
attention. cAMP/PKA signaling plays an immunosuppressive role in T cells
(36) and is altered in SLE
(37). Phosphodiesterases
(PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA
during T cell activation (38,
39). PKA has been described to
control the activity of ERK1/2 either positively or negatively in different
cells and processes (35). A
little explored integration among ERK1/2 and PKA occurs via phosphatidic acid
(PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that
hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA
plays roles in signaling interacting with a variety of targeting proteins that
bear PA-binding domains (40).
In this way PA recruits Raf-1 to the plasma membrane
(41). It is also converted by
phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG),
which among other functions, recruits and activates the GTPase Ras
(42). Both Ras and Raf-1 are
upstream elements of the ERK1/2 activation pathway
(43). In addition, PA binds to
and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP
levels and PKA down-regulation
(44). The regulation and role
of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell
homeostasis, because it is also unknown whether galectins stimulate the PLD/PA
pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering
cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our
results for the first time show that a galectin increases the PA levels,
down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE
activity, and induces an ERK1/2-dependent expression of the pro-apoptotic
factor FasL. The enhanced PDE activity induced by Gal-8 is required for the
activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces
apoptosis in human peripheral blood mononuclear cells (PBMC), especially after
activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other
galectins the property of killing activated T cells contributing to the T cell
homeostasis. The pathway involves a particularly integrated signaling context,
engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for
galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its
susceptibility to inhibition by anti-Gal-8 autoantibodies. 相似文献