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1.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
2.
Jenny Erales Sabrina Lignon Brigitte Gontero 《The Journal of biological chemistry》2009,284(19):12735-12744
A new role is reported for CP12, a highly unfolded and flexible protein,
mainly known for its redox function with A4
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized
CP12 can prevent the in vitro thermal inactivation and aggregation of
GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not
redox-dependent. The protection is specific to CP12, because other proteins,
such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do
not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific
chaperone, since it does not protect other proteins, such as catalase, alcohol
dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is
necessary to prevent the aggregation and inactivation, since the mutant C66S
that does not form any complex with GAPDH cannot accomplish this protection.
Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is
partially able to protect and to slow down the inactivation and aggregation.
Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of
GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox
function but also behaves as a specific “chaperone-like protein”
for GAPDH, although a stable and not transitory interaction is observed. This
new function of CP12 may explain why it is also present in complexes involving
A2B2 GAPDHs that possess a regulatory C-terminal
extension (GapB subunit) and therefore do not require CP12 to be
redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most
photosynthetic organisms, including cyanobacteria
(1,
2), higher plants
(3), the diatom
Asterionella formosa
(4,
5), and green
(1) and red algae
(6). It allows the formation of
a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH),3 two key
enzymes of the Calvin cycle pathway, and was recently shown to interact with
fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway
(7). The
phosphoribulokinase·GAPDH·CP12 complex has been extensively
studied in Chlamydomonas reinhardtii
(8,
9) and in Arabidopsis
thaliana (10,
11). In the green alga C.
reinhardtii, the interaction between CP12 and GAPDH is strong
(8). GAPDH may exist as a
homotetramer composed of four GapA subunits (A4) in higher plants,
cyanobacteria, and green and red algae
(6,
12), but in higher plants, it
can also exist as a heterotetramer (A2B2), composed of
two subunits, GapA and GapB
(13,
14). GapB, up to now, has
exclusively been found in Streptophyta, but recently two
prasinophycean green algae, Ostreococcus tauri and Ostreococcus
lucimarinus, were also shown to possess a GapB gene, whereas
CP12 is missing (15).
The GapB subunit is similar to the GapA subunit but has a C-terminal extension
containing two redox-regulated cysteine residues
(16). Thus, although the
A4 GAPDHs lack these regulatory cysteine residues
(13,
14,
17–20),
they are also redox-regulated through its interaction with CP12, since the C
terminus of this small protein resembles the C-terminal extension of the GapB
subunit. The regulatory cysteine residues for GapA are thus supplied by CP12,
as is well documented in the literature
(1,
8,
11,
16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs)
(21–26).
The amino acid composition of these proteins causes them to have no or few
secondary structures. Their total or partial lack of structure and their high
flexibility allow them to be molecular adaptors
(27,
28). They are often able to
bind to several partners and are involved in most cellular functions
(29,
30). Recently, some IUPs have
been described in photosynthetic organisms
(31,
32).There are many functional categories of IUPs
(22,
33). They can be, for
instance, involved in permanent binding and have (i) a scavenger role,
neutralizing or storing small ligands; (ii) an assembler role by forming
complexes; and (iii) an effector role by modulating the activity of a partner
molecule (33). These functions
are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating
its redox properties (8,
34,
35), and can also bind a metal
ion (36,
37). IUPs can also bind
transiently to partners, and some of them have been found to possess a
chaperone activity (31,
38). This chaperone function
was first shown for α-synuclein
(39) and for α-casein
(40), which are fully
disordered. The amino acid composition of IUPs is less hydrophobic than those
of soluble proteins; hence, they lack hydrophobic cores and do not become
insoluble when heated. Since CP12 belongs to this family, we tested if it was
resistant to heat treatment and finally, since it is tightly bound to GAPDH,
if it could prevent aggregation of its partner, GAPDH, an enzyme well known
for its tendency to aggregate
(41–44)
and consequently a substrate commonly used in chaperone studies
(45,
46).Unlike chaperones, which form transient, dynamic complexes with their
protein substrates through hydrophobic interactions
(47,
48), CP12 forms a stable
complex with GAPDH. The interaction involves the C-terminal part of the
protein and the presence of negatively charged residues on CP12
(35). However, only a
site-directed mutagenesis has been performed to characterize the interaction
site on GAPDH. Although the mutation could have an indirect effect, the
residue Arg-197 was shown to be a good candidate for the interaction site
(49).In this report, we accordingly used proteolysis experiments coupled with
mass spectrometry to detect which regions of GAPDH are protected by its
association with CP12. To conclude, the aim of this report was to characterize
a chaperone function of CP12 that had never been described before and to map
the interaction site on GAPDH using an approach that does not involve
site-directed mutagenesis. 相似文献
3.
Xiaojing Wang Snezana Levic Michael Anne Gratton Karen Jo Doyle Ebenezer N. Yamoah Anthony E. Pegg 《The Journal of biological chemistry》2009,284(2):930-937
Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the
SpmS and Phex genes, were found to be profoundly hearing
impaired. This defect was due to alteration in polyamine content due to the
absence of spermine synthase, the product of the SpmS gene. It was
reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that
ubiquitously expresses spermine synthase under the control of a composite
cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost
complete loss of the endocochlear potential in the Gy mice, which parallels
the hearing deficiency, and this was also reversed by the production of
spermine from the spermine synthase transgene. Gy mice showed a striking toxic
response to treatment with the ornithine decarboxylase inhibitor
α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to
DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor
function resulting in death within 5 days. This effect was due to an inability
to maintain normal balance and was also prevented by the transgenic expression
of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no
effect on balance. The loss of balance in Gy mice treated with DFMO was due to
inhibition of polyamine synthesis because it was prevented by administration
of putrescine. Our results are consistent with a critical role for polyamines
in regulation of Kir channels that maintain the endocochlear potential and
emphasize the importance of normal spermidine:spermine ratio in the hearing
and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes
for ornithine decarboxylase and S-adenosylmethionine decarboxylase,
which are enzymes needed for the synthesis of putrescine, spermidine, and
spermine, are lethal at early stages of embryonic development
(1,
2). There is convincing
evidence that the formation of hypusine in eIF5A, which requires spermidine as
a precursor, is essential for eukaryotes
(3). However, the function(s)
of spermine is not so well established. Yeast mutants with inactivated
spermine synthase grow at a normal rate
(4). Mammalian cells in culture
also grow normally in the presence of inhibitors of spermine synthase
(5) or after inactivation of
the spermine synthase gene (SpmS)
(6–8).
Inactivation of both of the genes that were originally described as encoding
spermine synthases in plants leads to profound developmental defects
(9–11),
but recently it was discovered that one of these genes actually encodes a
thermospermine synthase, and it appears that the lack of thermospermine may be
responsible for these defects
(12).In contrast, spermine is clearly required for normal development in
mammals. The rare human Snyder-Robinson syndrome is caused by mutations in
SpmS located in the X chromosome that drastically reduces the amount
of spermine synthase (13,
14). This leads to mental
retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry,
thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X
chromosomal deletion that includes SpmS and have no detectable
spermine synthase activity, do survive but are only viable on the B6C3H
background
(15–17).
This mouse strain having an X-linked dominant mutation was isolated from a
female offspring of an irradiated mouse and was termed gyro
(Gy)2 based on a
circling behavior pattern in affected males
(18). Subsequent studies have
shown that the Gy mice have a deletion of part of the X chromosome that
inactivates both Phex, a gene that regulates phosphate metabolism,
and SpmS (16,
19). The lack of SpmS
causes a total absence of spermine
(6,
7,
15,
16). Such Gy mice suffer from
hypophosphatemia, have a greatly reduced size, sterility, and neurological
abnormalities, and have a short life span
(6,
16,
18). All of these changes
except the hypophosphatemia are reversed when spermine synthase activity is
restored (20).The original characterization of Gy mice also reported preliminary
indications that these mice had hearing defects lacking the Preyer reflex
(21,
22). This is of particular
interest in the context of polyamine metabolism because a drug,
α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine
decarboxylase has been shown to cause occasional hearing loss in some patients
(23–26).
Although DFMO was ineffective for cancer treatment, it is an extremely
promising agent for cancer chemoprevention
(27,
28). When combined with
sulindac, DFMO treatment produced a substantial reduction in the recurrence of
colorectal adenomas in a large clinical trial
(27). DFMO is a major drug for
the treatment of African sleeping sickness caused by Trypanosoma
brucei (29,
30). It is also used as a
topically applied cream for treatment of unwanted facial hair in women
(31,
32). DFMO is generally well
tolerated even at high doses, but reversible hearing loss has been reported in
multiple clinical trials (25,
33), and a rarer irreversible
defect has also been reported
(34). These side effects are
not observed at lower doses of DFMO
(26,
27).Ototoxicity has been demonstrated to occur in experimental animals treated
with DFMO including rats (35),
guinea pigs (36), gerbils
(37), and mice
(38). Using
immunohistochemistry, a high level of ornithine decarboxylase was observed in
the inner ear of the rat, with the highest in the organ of Corti and lateral
wall followed by the cochlear nerve
(39). Measurements of
polyamines in the relevant structures are very difficult due to the small
amount of tissue available, but as expected, DFMO treatment reduced polyamine
levels and ornithine decarboxylase activity in the inner ear of the guinea pig
(36). A plausible explanation
for the importance of polyamines in auditory physiology is based on their well
documented role as regulators of potassium channels
(38). The inward rectification
of Kir channels is caused by blockage of the outward current by polyamines
(40–42).
Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel
showed that inward rectification was reduced and that there was a marked
reduction in endocochlear potential (EP). It was proposed that DFMO treatment
increases the outward Kir4.1 current, resulting in a drop in EP
(38).In the experiments reported here, we have studied in more detail the role
of polyamines in auditory physiology using Gy mice and crosses of these mice
with transgenic CAG/SpmS mice
(43). These mice express
spermine synthase under the control of a composite cytomegalovirus-IE
enhancer/chicken β-actin promoter, which was designed to provide
ubiquitous expression
(44–46).
Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that
there was a high level of expression of the transgene in many different organs
and that this level was maintained for at least 1 year
(43). Our studies confirm that
Gy mice are totally deaf and that this condition is reversed by the expression
of the SpmS gene. These changes are due to a virtually complete loss
of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy
mice. Unexpectedly, it was found that these mice show a rapid and profound
toxicity to this drug, leading to death within a few days. Within 5 days of
exposure to DFMO in the drinking water, the DFMO-treated mice suffered a
catastrophic loss of balance due to inner ear effects. This toxicity was also
prevented by the transgenic expression of spermine synthase in the Gy
background. 相似文献
4.
5.
6.
7.
Jason D. Hoffert Chung-Lin Chou Mark A. Knepper 《The Journal of biological chemistry》2009,284(22):14683-14687
Vasopressin controls renal water excretion largely through actions to
regulate the water channel aquaporin-2 in collecting duct principal cells. Our
knowledge of the mechanisms involved has increased markedly in recent years
with the advent of methods for large-scale systems-level profiling such as
protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide
microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible
aspects of everyday physiology. An outdoor tennis game on a hot summer day can
result in substantial water losses by sweating, and the kidneys respond by
reducing water excretion. In contrast, excessive intake of water, a frequent
occurrence in everyday life, results in excretion of copious amounts of clear
urine. These responses serve to exact tight control on the tonicity of body
fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg
of H2O through the regulated return of water from the pro-urine in
the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails.
For example, polyuria (rapid uncontrolled excretion of water) is a sometimes
devastating consequence of lithium therapy for bipolar disorder. On the other
side of the coin are water balance disorders that result from excessive renal
water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia
due to excessive water retention can be seen with severe congestive heart
failure, hepatic cirrhosis, and the syndrome of inappropriate
antidiuresis.The chief regulator of water excretion is the peptide hormone
AVP,2 whereas the
chief molecular target for regulation is the water channel AQP2. In this
minireview, we describe new progress in the understanding of the molecular
mechanisms involved in regulation of AQP2 by AVP in collecting duct cells,
with emphasis on new information derived from “systems-level”
approaches involving large-scale profiling and screening techniques such as
oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid
analysis. Most of the progress with these techniques is in the identification
of individual molecules involved in AVP signaling and binding interactions
with AQP2. Additional related issues are addressed in several recent reviews
(1–4). 相似文献
8.
9.
10.
11.
12.
13.
14.
15.
Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
16.
Mammalian defensins are cationic antimicrobial peptides that play a central
role in host innate immunity and as regulators of acquired immunity. In
animals, three structural defensin subfamilies, designated as α, β,
and θ, have been characterized, each possessing a distinctive
tridisulfide motif. Mature α- and β-defensins are produced by
simple proteolytic processing of their prepropeptide precursors. In contrast,
the macrocyclic θ-defensins are formed by the head-to-tail splicing of
nonapeptides excised from a pair of prepropeptide precursors. Thus,
elucidation of the θ-defensin biosynthetic pathway provides an
opportunity to identify novel factors involved in this unique process. We
incorporated the θ-defensin precursor, proRTD1a, into a bait construct
for a yeast two-hybrid screen that identified rhesus macaque stromal
cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a
component of the endoplasmic reticulum (ER) chaperone complex, which we found
to also interact with α- and β-defensins. However, analysis of the
SDF2L1 domain requirements for binding of representative α-, β-,
and θ-defensins revealed that α- and β-defensins bind SDF2L1
similarly, but differently from the interactions that mediate binding of
SDF2L1 to pro-θ-defensins. Thus, SDF2L1 is a factor involved in
processing and/or sorting of all three defensin subfamilies.Mammalian defensins are tridisulfide-containing antimicrobial peptides that
contribute to innate immunity in all species studied to date. Defensins are
comprised of three structural subfamilies: the α-, β-, and
θ-defensins (1). α-
and β-Defensins are peptides of about 29–45-amino acid residues
with similar three-dimensional structures. Despite their similar tertiary
conformations, the disulfide motifs of α- and β-defensins differ.
Expression of human α-defensins is tissue-specific. Four myeloid
α-defensins (HNP1–4) are expressed predominantly by neutrophils
and monocytes wherein they are packaged in granules, while two enteric
α-defensins (HD-5 and HD-6) are expressed at high levels in Paneth cells
of the small intestine. Myeloid α-defensins constitute about 5% of the
protein mass of human neutrophils. HNPs are discharged into the phagosome
during phagocytic ingestion of microbial particles. HD-5 and HD-6 are produced
and stored as propeptides in Paneth cell granules and are processed
extracellularly by intestinal trypsin
(2). β-Defensins are
produced primarily by various epithelia (e.g. skin, urogenital tract,
airway) and are secreted by the producing cells in their mature forms. In
contrast to pro-α-defensins, which contain a conserved prosegment of
∼40 amino acids, the prosegments in β-defensins vary in length and
sequence. θ-Defensins are found only in Old World monkeys and orangutans
and are the only known circular peptides in animals. These 18-residue
macrocyclic peptides are formed by ligation of two nonamer sequences excised
from two precursor polypeptides, which are truncated versions of ancestral
α-defensins. Like myeloid α-defensins, θ-defensins are
stored primarily in neutrophil and monocyte granules
(3).Numerous laboratories have demonstrated that the antimicrobial properties
of defensins derive from their ability to bind and disrupt target cell
membranes (4), and studies have
shown defensins to be active against Gram-positive and -negative bacteria
(5), viruses
(6–9),
fungi (10,
11), and parasites such as
Giardia lamblia (12).
Defensins also play a regulatory role in acquired immunity as they are known
to chemoattract T lymphocytes, monocytes, and immature dendritic cells
(13,
14), act as adjuvants,
stimulate B cell responses, and up-regulate proliferation and cytokine
production by spleen cells and T helper cells
(15,
16).Defensins are produced as pre-propeptides and undergo post-translational
processing to form the mature peptides. While much has been learned about
regulation of defensin expression, little is known about the factors involved
in their biosynthesis. Valore and Ganz
(17) investigated the
processing of defensins in cultured cells and demonstrated that maturation of
HNPs occurs through two proteolytic steps that lead to formation of mature
α-defensins, but the proteases involved have yet to be identified.
Moreover, there are virtually no published data regarding endoplasmic
reticulum (ER)2
factors that are responsible for the folding, processing, and sorting steps
necessary for defensin maturation and secretion or trafficking to the proper
subcellular compartment. It is likely that several chaperones, proteases, and
protein-disulfide isomerase (PDI) family proteins are involved. Consistent
with this possibility, Gruber et al.
(18) recently demonstrated the
role of a PDI in biosynthesis of cyclotides, small ∼30-residue macrocyclic
peptides produced by plants.The primary structures of α- and θ-defensin precursors are
closely related. We therefore undertook studies to identify proteins that
interact with representative propeptides of each defensin subfamily with the
goal of determining common and unique processes that regulate biosynthesis of
α- and θ-defensins. We used two-hybrid analysis to first identify
interactors of the θ-defensin precursor, proRTD1a. As described, we
identified SDF2L1, a component of the ER-chaperone complex as an interactor,
and showed that it also specifically interacts with α- and
β-defensins. This suggests that SDF2L1 is involved in the
maturation/trafficking of defensins at a step common to all three subfamilies
of mammalian defensins. 相似文献
17.
Karen Vanhoorelbeke Simon F. De Meyer Inge Pareyn Chantal Melchior Sebastien Plan?on Christiane Margue Olivier Pradier Pierre Fondu Nelly Kieffer Timothy A. Springer Hans Deckmyn 《The Journal of biological chemistry》2009,284(22):14914-14920
Three heterozygous mutations were identified in the genes encoding platelet
integrin receptor αIIbβ3 in a patient with an ill defined platelet
disorder: one in the β3 gene (S527F) and two in the αIIb gene
(R512W and L841M). Five stable Chinese hamster ovary cell lines were
constructed expressing recombinant αIIbβ3 receptors bearing the
individual R512W, L841M, or S527F mutation; both the R512W and L841M
mutations; or all three mutations. All receptors were expressed on the cell
surface, and mutations R512W and L841M had no effect on integrin function.
Interestingly, the β3 S527F mutation produced a constitutively active
receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound
to non-activated αIIbβ3 receptors carrying the S527F mutation,
indicating that the conformation of this receptor was altered and corresponded
to the high affinity ligand binding state. In addition, the conformational
change induced by S527F was evident from basal anti-ligand-induced binding
site antibody binding to the receptor. A molecular model bearing this mutation
was constructed based on the crystal structure of αIIbβ3 and
revealed that the S527F mutation, situated in the third integrin epidermal
growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor
from adopting a wild type-like bent conformation. Movement of I-EGF3 into a
cleft in the bent conformation may be hampered both by steric hindrance
between Phe527 in β3 and the calf-1 domain in αIIb and
by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin
receptors that consist of noncovalently linked α/β-heterodimers.
They are cell-surface receptors that play a role in cell-cell and cell-matrix
interactions. Under resting conditions, integrin receptors adopt the low
affinity conformation and do not interact with their ligands. Inside-out
signaling turns the receptor into a high affinity conformation capable of
ligand binding. Ligand binding itself induces additional conformational
changes resulting in exposure of neoantigenic sites called ligand-induced
binding sites (LIBS)3
and generates in turn outside-in signaling, which triggers a range of
downstream signals (1,
2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In
flowing blood under resting conditions, αIIbβ3 does not interact
with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere
at sites of vascular injury and become activated. As a consequence,
αIIbβ3 adopts the high affinity conformation and binds fibrinogen.
This results in platelet aggregation and thrombus formation, which eventually
will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal
ligand-binding head domain (the β-propeller domain in the αIIb
chain and the βI domain in the β3 chain) standing on two long legs
or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb
chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial
growth factor-like (I-EGF), and β-tail domains in the β3 chain),
followed by transmembrane and cytoplasmic domains
(1,
2). X-ray crystal structures of
the extracellular domain of non-activated αVβ3 revealed that the
legs are severely bent, putting the head domain next to the membrane-proximal
portions of the legs (4,
5). The bending occurs between
I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1
domains in the α-subunit. This bent conformation represents the low
affinity state of the receptor. The high affinity state of the receptor is
induced by activation and is associated with a large-scale conformational
rearrangement in which the integrin extends with a switchblade-like motion
(2). Recently, the crystal
structure of the entire extracellular domain of αIIbβ3 in its low
affinity conformation was resolved and revealed that this integrin also adopts
the bent conformation under resting conditions
(6). Structural rearrangements
in αIIbβ3 between the bent and extended conformations are similar
to what has been reported for other integrins
(7).We report here that the S527F mutation in the I-EGF3 region of the β3
polypeptide chain of the αIIbβ3 receptor induces a constitutively
active receptor adopting an extended high affinity conformation. This was
evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding.
These data were further corroborated by modeling the replacement of
Ser527 with Phe in the crystal structure of the extracellular
domain of αIIbβ3. In this model, the S527F mutation decreases the
flexibility of I-EGF3 and appears to prevent movement of the lower β-leg
into the cleft between the upper β-leg and the lower α-leg. As a
consequence, formation of the bent conformation of the non-activated receptor
is hampered. 相似文献
18.