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1.
Madepalli K. Lakshmana Il-Sang Yoon Eunice Chen Elizabetta Bianchi Edward H. Koo David E. Kang 《The Journal of biological chemistry》2009,284(18):11863-11872
Accumulation of the amyloid β (Aβ) peptide derived from the
proteolytic processing of amyloid precursor protein (APP) is the defining
pathological hallmark of Alzheimer disease. We previously demonstrated that
the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP)
robustly promoted Aβ generation independent of FE65 and specifically
interacted with Ran-binding protein 9 (RanBP9). In this study we found that
RanBP9 strongly increased BACE1 cleavage of APP and Aβ generation. This
pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the
Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell
surface APP and accelerated APP internalization, consistent with enhanced
β-secretase processing in the endocytic pathway. The N-terminal half of
RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with
APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP
interactions with LRP and BACE1 and increased lipid raft association of APP.
Importantly, knockdown of endogenous RanBP9 significantly reduced Aβ
generation in Chinese hamster ovary cells and in primary neurons,
demonstrating its physiological role in BACE1 cleavage of APP. These findings
not only implicate RanBP9 as a novel and potent regulator of APP processing
but also as a potential therapeutic target for Alzheimer disease.The major defining pathological hallmark of Alzheimer disease
(AD)2 is the
accumulation of amyloid β protein (Aβ), a neurotoxic peptide derived
from β- and γ-secretase cleavages of the amyloid precursor protein
(APP). The vast majority of APP is constitutively cleaved in the middle of the
Aβ sequence by α-secretase (ADAM10/TACE/ADAM17) in the
non-amyloidogenic pathway, thereby abrogating the generation of an intact
Aβ peptide. Alternatively, a small proportion of APP is cleaved in the
amyloidogenic pathway, leading to the secretion of Aβ peptides
(37–42 amino acids) via two proteolytic enzymes, β- and
γ-secretase, known as BACE1 and presenilin, respectively
(1).The proteolytic processing of APP to generate Aβ requires the
trafficking of APP such that APP and BACE1 are brought together in close
proximity for β-secretase cleavage to occur. We and others have shown
that the low density lipoprotein receptor-related protein (LRP), a
multifunctional endocytosis receptor
(2), binds to APP and alters
its trafficking to promote Aβ generation. The loss of LRP substantially
reduces Aβ release, a phenotype that is reversed when full-length
(LRP-FL) or truncated LRP is transfected in LRP-deficient cells
(3,
4). Specifically, LRP-CT
lacking the extracellular ligand binding regions but containing the
transmembrane domain and the cytoplasmic tail is capable of rescuing
amyloidogenic processing of APP and Aβ release in LRP deficient cells
(3). Moreover, the LRP soluble
tail (LRP-ST) lacking the transmembrane domain and only containing the
cytoplasmic tail of LRP is sufficient to enhance Aβ secretion
(5). This activity of LRP-ST is
achieved by promoting APP/BACE1 interaction
(6), although the precise
mechanism is unknown. Although we had hypothesized that one or more
NPXY domains in LRP-ST might underlie the pro-amyloidogenic
processing of APP, we recently found that the 37 C-terminal residues of LRP
(LRP-C37) lacking the NPXY motif was sufficient to robustly promote
Aβ production independent of FE65
(7). Because LRP-C37 likely
acts by recruiting other proteins, we used the LRP-C37 region as bait in a
yeast two-hybrid screen, resulting in the identification of 4 new LRP-binding
proteins (7). Among these, we
focused on Ran-binding protein 9 (RanBP9) in this study, which we found to
play a critical role in the trafficking and processing of APP. RanBP9, also
known as RanBPM, acts as a multi-modular scaffolding protein, bridging
interactions between the cytoplasmic domains of a variety of membrane
receptors and intracellular signaling targets. These include Axl and Sky
(8), MET receptor
protein-tyrosine kinase (9),
and β2-integrin LFA-1
(10). Similarly, RanBP9
interacts with Plexin-A receptors to strongly inhibit axonal outgrowth
(11) and functions to regulate
cell morphology and adhesion
(12,
13). Here we show that RanBP9
robustly promotes BACE1 processing of APP and Aβ generation. 相似文献
2.
3.
Hyaluronan Activates Cell Motility of v-Src-transformed Cells via
Ras-Mitogen–activated Protein Kinase and Phosphoinositide
3-Kinase-Akt in a Tumor-specific Manner 下载免费PDF全文
Yasuyoshi Sohara Naoki Ishiguro Kazuya Machida Hisashi Kurata Aye Aye Thant Takeshi Senga Satoru Matsuda Koji Kimata Hisashi Iwata Michinari Hamaguchi 《Molecular biology of the cell》2001,12(6):1859-1868
We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src-transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44-neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src-transformed cells. 相似文献
4.
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6.
Yongmei Pu Susan H. Garfield Noemi Kedei Peter M. Blumberg 《The Journal of biological chemistry》2009,284(2):1302-1312
Classic and novel protein kinase C (PKC) isozymes contain two zinc finger
motifs, designated “C1a” and “C1b” domains, which
constitute the recognition modules for the second messenger diacylglycerol
(DAG) or the phorbol esters. However, the individual contributions of these
tandem C1 domains to PKC function and, reciprocally, the influence of protein
context on their function remain uncertain. In the present study, we prepared
PKCδ constructs in which the individual C1a and C1b domains were
deleted, swapped, or substituted for one another to explore these issues. As
isolated fragments, both the δC1a and δC1b domains potently bound
phorbol esters, but the binding of [3H]phorbol 12,13-dibutyrate
([3H]PDBu) by the δC1a domain depended much more on the
presence of phosphatidylserine than did that of the δC1b domain. In
intact PKCδ, the δC1b domain played the dominant role in
[3H]PDBu binding, membrane translocation, and down-regulation. A
contribution from the δC1a domain was nonetheless evident, as shown by
retention of [3H]PDBu binding at reduced affinity, by increased
[3H]PDBu affinity upon expression of a second δC1a domain
substituting for the δC1b domain, and by loss of persistent plasma
membrane translocation for PKCδ expressing only the δC1b domain,
but its contribution was less than predicted from the activity of the isolated
domain. Switching the position of the δC1b domain to the normal position
of the δC1a domain (or vice versa) had no apparent effect on the
response to phorbol esters, suggesting that the specific position of the C1
domain within PKCδ was not the primary determinant of its activity.One of the essential steps for protein kinase C
(PKC)2 activation is
its translocation from the cytosol to the membranes. For conventional
(α, βI, βII, and γ) and novel (δ, ε, η,
and θ) PKCs, this translocation is driven by interaction with the
lipophilic second messenger sn-1,2-diacylglycerol (DAG), generated
from phosphatidylinositol 4,5-bisphosphate upon the activation of
receptor-coupled phospholipase C or indirectly from phosphatidylcholine via
phospholipase D (1). A pair of
zinc finger structures in the regulatory domain of the PKCs, the
“C1” domains, are responsible for the recognition of the DAG
signal. The DAG-C1 domain-membrane interaction is coupled to a conformational
change in PKC, both causing the release of the pseudosubstrate domain from the
catalytic site to activate the enzyme and triggering the translocation to the
membrane (2). By regulating
access to substrates, PKC translocation complements the intrinsic enzymatic
specificity of PKC to determine its substrate profile.The C1 domain is a highly conserved cysteine-rich motif (∼50 amino
acids), which was first identified in PKC as the interaction site for DAG or
phorbol esters (3). It
possesses a globular structure with a hydrophilic binding cleft at one end
surrounded by hydrophobic residues. Binding of DAG or phorbol esters to the C1
domain caps the hydrophilic cleft and forms a continuous hydrophobic surface
favoring the interaction or penetration of the C1 domain into the membrane
(4). In addition to the novel
and classic PKCs, six other families of proteins have also been identified,
some of whose members possess DAG/phorbol ester-responsive C1 domains. These
are the protein kinase D (5),
the chimaerin (6), the munc-13
(7), the RasGRP (guanyl
nucleotide exchange factors for Ras and Rap1)
(8), the DAG kinase
(9), and the recently
characterized MRCK (myotonic dystrophy kinase-related
Cdc42-binding kinase) families
(10). Of these C1
domain-containing proteins, the PKCs have been studied most extensively and
are important therapeutic targets
(11). Among the drug
candidates in clinical trials that target PKC, a number such as bryostatin 1
and PEP005 are directed at the C1 domains of PKC rather than at its catalytic
site.Both the classic and novel PKCs contain in their N-terminal regulatory
region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester
(12). Multiple studies have
sought to define the respective roles of these two C1 domains in PKC
regulation, but the issue remains unclear. Initial in vitro binding
measurements with conventional PKCs suggested that 1 mol of phorbol ester
bound per mole of PKC
(13-15).
On the other hand, Stubbs et al., using a fluorescent phorbol ester
analog, reported that PKCα bound two ligands per PKC
(16). Further, site-directed
mutagenesis of the C1a and C1b domains of intact PKCα indicated that the
C1a and C1b domains played equivalent roles for membrane translocation in
response to phorbol 12-myristate 13-acetate (PMA) and (-)octylindolactam V
(17). Likewise, deletion
studies indicated that the C1a and C1b domains of PKCγ bound PDBu
equally with high potency (3,
18). Using a functional assay
with PKCα expression in yeast, Shieh et al.
(19) deleted individual C1
domains and reported that C1a and C1b were both functional and equivalent upon
stimulation by PMA, with either deletion causing a similar reduction in
potency of response, whereas for mezerein the response depended essentially on
the C1a domain, with much weaker response if only the C1b domain was present.
Using isolated C1 domains, Irie et al.
(20) suggested that the C1a
domain of PKCα but not those of PKCβ or PKCγ bound
[3H]PDBu preferentially; different ligands showed a generally
similar pattern but with different extents of selectivity. Using synthesized
dimeric bisphorbols, Newton''s group reported
(21) that, although both C1
domains of PKCβII are oriented for potential membrane interaction, only
one C1 domain bound ligand in a physiological context.In the case of novel PKCs, many studies have been performed on PKCδ
to study the equivalency of the twin C1 domains. The P11G point mutation of
the C1a domain, which caused a 300-fold loss of binding potency in the
isolated domain (22), had
little effect on the phorbol ester-dependent translocation of PKCδ in
NIH3T3 cells, whereas the same mutation of the C1b caused a 20-fold shift in
phorbol ester potency for inducing translocation, suggesting a major role of
the C1b domain for phorbol ester binding
(23). A secondary role for the
C1a domain was suggested, however, because mutation in the C1a domain as well
as the C1b domain caused a further 7-fold shift in potency. Using the same
mutations in the C1a and C1b domains, Bögi et al.
(24) found that the binding
selectivity for the C1a and C1b domains of PKCδ appeared to be
ligand-dependent. Whereas PMA and the indole alkaloids indolactam and
octylindolactam were selectively dependent on the C1b domain, selectivity was
not observed for mezerein, the 12-deoxyphorbol 13-monoesters prostratin and
12-deoxyphorbol 13-phenylacetate, and the macrocyclic lactone bryostatin 1
(24). In in vitro
studies using isolated C1a and C1b domains of PKCδ, Cho''s group
(25) described that the two C1
domains had opposite affinities for DAG and phorbol ester; i.e. the
C1a domain showed high affinity for DAG and the C1b domain showed high
affinity for phorbol ester. No such difference in selectivity was observed by
Irie et al. (20).PKC has emerged as a promising therapeutic target both for cancer and for
other conditions, such as diabetic retinopathy or macular degeneration
(26-30).
Kinase inhibitors represent one promising approach for targeting PKC, and
enzastaurin, an inhibitor with moderate selectivity for PKCβ relative to
other PKC isoforms (but still with activity on some other non-PKC kinases) is
currently in multiple clinical trials. An alternative strategy for drug
development has been to target the regulatory C1 domains of PKC. Strong proof
of principle for this approach is provided by multiple natural products,
e.g. bryostatin 1 and PEP005, which are likewise in clinical trials
and which are directed at the C1 domains. A potential advantage of this
approach is the lesser number of homologous targets, <30 DAG-sensitive C1
domains compared with over 500 kinases, as well as further opportunities for
specificity provided by the diversity of lipid environments, which form a
half-site for ligand binding to the C1 domain. Because different PKC isoforms
may induce antagonistic activities, inhibition of one isoform may be
functionally equivalent to activation of an antagonistic isoform
(31).Along with the benzolactams
(20,
32), the DAG lactones have
provided a powerful synthetic platform for manipulating ligand: C1 domain
interactions (31). For
example, the DAG lactone derivative 130C037 displayed marked selectivity among
the recombinant C1a and C1b domains of PKCα and PKCδ as well as
substantial selectivity for RasGRP relative to PKCα
(33). Likewise, we have shown
that a modified DAG lactone (dioxolanones) can afford an additional point of
contact in ligand binding to the C1b domain of PKCδ
(34). Such studies provide
clear examples that ligand-C1 domain interactions can be manipulated to yield
novel patterns of recognition. Further selectivity might be gained with
bivalent compounds, exploiting the spacing and individual characteristics of
the C1a and C1b domains (35).
A better understanding of the differential roles of the two C1 domains in PKC
regulation is critical for the rational development of such compounds. In this
study, by molecularly manipulating the C1a or C1b domains in intact
PKCδ, we find that both the C1a and C1b domains play important roles in
PKCδ regulation. The C1b domain is predominant for ligand binding and
for membrane translocation of the whole PKCδ molecule. The C1a domain of
intact PKCδ plays only a secondary role in ligand binding but stabilizes
the PKCδ molecule at the plasma membrane for downstream signaling. In
addition, we show that the effect of the individual C1 domains of PKCδ
does not critically depend on their position within the regulatory domain. 相似文献
7.
Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
8.
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
9.
10.
11.
Saumitri Bhattacharyya Jeremy Keirsey Beatriz Russell Juraj Kavecansky Kate Lillard-Wetherell Kambiz Tahmaseb John J. Turchi Joanna Groden 《The Journal of biological chemistry》2009,284(22):14966-14977
The BLM helicase associates with the telomere structural proteins TRF1 and
TRF2 in immortalized cells using the alternative
lengthening of telomere (ALT) pathways. This work
focuses on identifying protein partners of BLM in cells using ALT. Mass
spectrometry and immunoprecipitation techniques have identified three proteins
that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein
1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα
(TOPOIIα). BLM predominantly co-localizes with these proteins in foci
actively synthesizing DNA during late S and G2/M phases of the cell
cycle when ALT is thought to occur. Immunoprecipitation studies also indicate
that only HSP90 and TOPOIIα are components of a specific complex
containing BLM, TRF1, and TRF2 but that this complex does not include TEP1.
TEP1, TOPOIIα, and HSP90 interact directly with BLM in vitro
and modulate its helicase activity on telomere-like DNA substrates but not on
non-telomeric substrates. Initial studies suggest that knockdown of
BLM in ALT cells reduces average telomere length but does not do so
in cells using telomerase.Bloom syndrome
(BS)4 is a genetic
disease caused by mutation of both copies of the human BLM gene. It
is characterized by sun sensitivity, small stature, immunodeficiency, male
infertility, and an increased susceptibility to cancer of all sites and types.
The high incidence of spontaneous chromosome breakage and other unique
chromosomal anomalies in cells from BS patients indicate an increase in
homologous recombination in somatic cells
(1). Another notable feature of
non-immortalized and immortalized cells from BS individuals is the presence of
telomeric associations (TAs) between homologous chromosomes
(2). Work from our group and
others have suggested a role for BLM in recombination-mediated mechanisms of
telomere elongation or ALT (alternative lengthening of telomeres), processes
that maintain/elongate telomeres in the absence of telomerase
(3–5).
However, the exact mechanism by which BLM contributes to telomere stability is
unknown.Several proteins interact with and regulate BLM helicase activity,
including two telomere-specific proteins, TRF1 and TRF2
(6,
7). Although TRF2 stimulates
BLM unwinding of telomeric and non-telomeric 3′-overhang substrates,
TRF1 inhibits BLM unwinding of telomeric substrates. TRF2-mediated stimulation
of BLM helicase activity on a telomeric substrate is observed when TRF2 is
present in excess or with equimolar amount of TRF1 but not when TRF1 is
present in molar excess. Both proteins associate with BLM specifically in ALT
cells in vivo, suggesting their involvement in the ALT pathways. In
addition to TRF1 and TRF2, the telomere single-strand DNA-binding protein POT1
strongly stimulates BLM helicase activity on long telomeric forked duplexes
and D-loop structures (8).
Other proteins also play an important role in telomere maintenance in
telomerase-negative cells, including RAD50, NBS1, and MRE11, which co-localize
with TRF1 and TRF2 in specialized ALT-associated promyelocytic leukemia (PML)
nuclear bodies (APBs)
(9–11).
Thus, we hypothesize that BLM complex formation may be essential for the ALT
mechanism, and its modification may occur dynamically during the specific
nucleic acid transactions required to protect the telomere in cells using the
ALT pathways.This study has identified previously unknown protein partners of BLM and
TRF2 in ALT cells using double immunoprecipitation and mass spectrometry (MS).
These include telomerase-associated protein 1 (TEP1), heat shock protein 90
(HSP90), and topoisomerase IIα (TOPOIIα). These proteins associate
with BLM and TRF2 in cells using ALT but not in cells using telomerase and
directly interact with BLM in vitro. This complex of proteins
localizes to sites of new DNA synthesis in vivo in ALT cells,
suggesting a role in telomere maintenance. We also identified HSP90 and
TOPOIIα in another ALT-specific complex consisting of BLM, TRF1, and
TRF2 but not TEP1. In vitro analyses demonstrate that HSP90 inhibits
BLM helicase activity using both telomeric and non-telomeric substrates,
whereas TEP1 and TOPOIIα initially slow the kinetics of BLM unwinding
only using telomeric substrates. These findings suggest the presence of
dynamic BLM-associated ALT complexes that include previously unidentified
interacting proteins. The function of TEP1 in the BLM·TRF2 complex
remains unclear, although its previously described interaction with the RNA
subunit of telomerase (12)
suggests an interesting hypothesis of cross-talk between mechanisms of
telomere elongation. 相似文献
12.
Michael K?mpf Birgit Absmanner Markus Schwarz Ludwig Lehle 《The Journal of biological chemistry》2009,284(18):11900-11912
N-Linked glycosylation involves the ordered, stepwise synthesis of
the unique lipid-linked oligosaccharide precursor
Glc3Man9 GlcNAc2-PP-Dol on the endoplasmic
reticulum (ER), catalyzed by a series of glycosyltransferases. Here we
characterize Alg2 as a bifunctional enzyme that is required for both the
transfer of the α1,3- and the α1,6-mannose-linked residue from
GDP-mannose to Man1GlcNAc2-PP-Dol forming the
Man3GlcNAc2-PP-Dol intermediate on the cytosolic side of
the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four
transmembrane-spanning helices, two at the N terminus and two at the C
terminus. Contradictory to topology predictions, we prove that only the two
N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic
sequences contribute to ER localization in a nontransmembrane manner.
Surprisingly, none of the four domains is essential for transferase activity
because truncated Alg2 variants can exert their function as long as Alg2 is
associated with the ER by either its N- or C-terminal hydrophobic regions. By
site-directed mutagenesis we demonstrate that an EX7E
motif, conserved in a variety of glycosyltransferases, is not important for
Alg2 function in vivo and in vitro. Instead, we identify a
conserved lysine residue, Lys230, as being essential for activity,
which could be involved in the binding of the phosphate of the glycosyl
donor.Asparagine-linked glycosylation is an essential protein modification highly
conserved in eukaryotes
(1–4),
and several features of this pathway even occur in prokaryotes
(5–7).
In eukaryotes, biosynthesis of N-glycans starts with the assembly of
the common core oligosaccharide precursor Glc3Man9
GlcNAc2-PP-Dol, the glycan moiety of which is subsequently
transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent
polypeptide chain by the oligosaccharyl-transferase complex
(8–10).
The initial steps of the dolichol pathway up to
Man5GlcNAc2-PP-Dol take place on the cytosolic site of
the endoplasmic reticulum
(ER),2 using sugar
nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to
the luminal site, which is facilitated by Rft1
(11) and another not yet
identified protein (12), it is
extended by four mannose and three glucose residues deriving from Man-P-Dol
and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially
in an ordered fashion based on differences in the substrate specificity of the
various glycosyltransferases
(13). In the yeast
Saccharomyces cerevisiae, alg mutants (for
asparagine-linked glycosylation) have been
isolated, defective in lipid-linked oligosaccharide (LLO) assembly
(14–17),
and shown to be invaluable to define the pathway as well as to isolate the
genes encoding the respective glycosyltransferases by complementing a
particular phenotype characteristic of the respective mutant. Likewise various
mutant cell lines from mammalian origin have been described that produce
truncated lipid-linked oligosaccharides
(18–20).One of the temperature-sensitive alg mutants, alg2, was
shown to accumulate lipid-linked Man2GlcNAc2 at the
restrictive temperature (15),
indicating that alg2 might have a defect in the glycosyltransferase
catalyzing the transfer of the third, α1,6-linked mannose, i.e.
in the formation of the branched pentasaccharide
Man3GlcNAc2-PP-Dol (see
Fig. 8). On the other hand,
biochemical studies in human fibroblasts from a patient with a defect in the
human ALG2 ortholog, causing congenital disorder of glycosylation
type CDG1i, pointed to a role in the transfer of the second, α1,3-linked
mannose residue, because no elongation of Man(1,6)ManGlcNAc2-PP-Dol
occurred (21). In contrast,
control fibroblasts were able to do so, albeit with reduced efficiency when
compared with Man(1,3)ManGlcNAc2-PP-Dol as glycosyl acceptor.
Because a bioinformatic approach of the yeast data base did not reveal an
unknown open reading frame that might encode an additional putative
mannosyltransferase being involved in LLO synthesis, we reasoned that
ALG2 may have a dual function, i.e. synthesizing both
Man2GlcNAc2-PP-Dol and
Man3GlcNAc2-PP-Dol. While the current study was in
progress, evidence was presented that a membrane fraction from Escherichia
coli, expressing ALG2 from yeast, is able to carry out an
α1,3- and α1,6-mannosylation to form the branched pentasaccharide
intermediate (22). However,
the contribution of native E. coli enzymes could not entirely be
ruled out. So far Alg2 has not been studied biochemically in yeast. Here, we
confirm and extent this finding by investigating Alg2 in yeast. We first
established a radioactive in vitro assay and demonstrate that Alg2,
immunoprecipitated from detergent extracts of yeast microsomal membranes, is
indeed sufficient to catalyze both elongation of
Man1GlcNAc2-PP-Dol to
Man2GlcNAc2-PP-Dol and subsequently to
Man3GlcNAc2-PP-Dol. Furthermore we investigated the
membrane topology of Alg2 mannosyltransferase. Evidence will be presented that
Alg2 is composed only of the two N-terminal of four predicted transmembrane
domains (TMDs), whereas the C-terminal hydrophobic sequences contribute to ER
localization merely in a nontransmembrane manner. Surprisingly, none of the
four domains is essential for Alg2 activity because deletion of either the two
N-terminal or C-terminal domains gives rise to an active transferase. Finally,
we perform a mutational analysis of Alg2 and identify amino acids required for
its activity.Open in a separate windowFIGURE 8.Early steps of lipid-linked oligosaccharide formation on the cytosolic
side of the ER membrane. Biosynthesis starts with the transfer of a
GlcNAc-phosphate to dolichol phosphate with formation of the pyrophosphate
bond, catalyzed by Alg7. The second step is catalyzed be the dimeric
Alg14/Alg13 complex, whereby membrane-bound Alg14 recruits cytosolic Alg13 to
the membrane with formation of the active GlcNAc transferase. Following the
addition of the β1,4-linked mannose by Alg1, Alg2 catalyzes, as
demonstrated here, both the transfer of the α1,3- and α1,6-linked
mannose. The two final α1,2-mannose residues are transferred by Alg11,
before the Man5GlcNAc2-PP heptasaccharide is
translocated across the ER membrane to the lumen, where further elongation
takes place to the full-length core saccharide. All of the sugar residues are
donated by sugar nucleotides. 相似文献
13.
14.
15.
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
16.
Thanh H. Pham Xiaofei Gao Gyanendra Singh Philip R. Hardwidge 《The Journal of biological chemistry》2013,288(48):34567-34574
Enterohemorrhagic Escherichia coli and other attaching/effacing bacterial pathogens cause diarrhea in humans. These pathogens use a type III secretion system to inject virulence proteins (effectors) into host cells, some of which inhibit the innate immune system. The enterohemorrhagic E. coli NleH1 effector prevents the nuclear translocation of RPS3 (ribosomal protein S3) to inhibit its participation as a nuclear “specifier” of NF-κB binding to target gene promoters. NleH1 binds to RPS3 and inhibits its phosphorylation on Ser-209 by IκB kinase-β (IKKβ). However, the precise mechanism of this inhibition is unclear. NleH1 possesses a Ser/Thr protein kinase activity that is essential both for its ability to inhibit the RPS3/NF-κB pathway and for full virulence of the attaching/effacing mouse pathogen Citrobacter rodentium. However, neither RPS3 nor IKKβ is a substrate of NleH1 kinase activity. We therefore screened ∼9,000 human proteins to identify NleH1 kinase substrates and identified CRKL (v-Crk sarcoma virus CT10 oncogene-like protein), a substrate of the BCR/ABL kinase. Knockdown of CRKL abundance prevented NleH1 from inhibiting RPS3 nuclear translocation and NF-κB activity. CRKL residues Tyr-198 and Tyr-207 were required for interaction with NleH1. Lys-159, the kinase-active site of NleH1, was necessary for its interaction with CRKL. We also identified CRKL as an IKKβ interaction partner, mediated by CRKL Tyr-198. We propose that the CRKL interaction with IKKβ recruits NleH1 to the IKKβ complex, where NleH1 then inhibits the RPS3/NF-κB pathway. 相似文献
17.
Hanbang Zhang Gretchen M. Ehrenkaufer Justine M. Pompey Jason A. Hackney Upinder Singh 《PLoS pathogens》2008,4(11)
Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5′-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5′-polyphosphate siRNAs extends to single-celled eukaryotic organisms. 相似文献
18.
19.