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Yuya Sato Toshihiko Uemura Keisuke Morimitsu Ryoko Sato-Nishiuchi Ri-ichiroh Manabe Junichi Takagi Masashi Yamada Kiyotoshi Sekiguchi 《The Journal of biological chemistry》2009,284(21):14524-14536
Integrin α8β1 interacts with a variety of Arg-Gly-Asp
(RGD)-containing ligands in the extracellular matrix. Here, we examined the
binding activities of α8β1 integrin toward a panel of
RGD-containing ligands. Integrin α8β1 bound specifically to
nephronectin with an apparent dissociation constant of 0.28 ± 0.01
nm, but showed only marginal affinities for fibronectin and other
RGD-containing ligands. The high-affinity binding to α8β1 integrin
was fully reproduced with a recombinant nephronectin fragment derived from the
RGD-containing central “linker” segment. A series of deletion
mutants of the recombinant fragment identified the LFEIFEIER sequence on the
C-terminal side of the RGD motif as an auxiliary site required for
high-affinity binding to α8β1 integrin. Alanine scanning
mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a
critical motif ensuring the high-affinity integrin-ligand interaction.
Although a synthetic LFEIFEIER peptide failed to inhibit the binding of
α8β1 integrin to nephronectin, a longer peptide containing both the
RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was
∼2,000-fold more potent than a peptide containing only the RGD motif.
Furthermore, trans-complementation assays using recombinant fragments
containing either the RGD motif or LFEIFEIER sequence revealed a clear
synergism in the binding to α8β1 integrin. Taken together, these
results indicate that the specific high-affinity binding of nephronectin to
α8β1 integrin is achieved by bipartite interaction of the integrin
with the RGD motif and LFEIFEIER sequence, with the latter serving as a
synergy site that greatly potentiates the RGD-driven integrin-ligand
interaction but has only marginal activity to secure the interaction by
itself.Integrins are a family of adhesion receptors that interact with a variety
of extracellular ligands, typically cell-adhesive proteins in the
extracellular matrix
(ECM).2 They play
mandatory roles in embryonic development and the maintenance of tissue
architectures by providing essential links between cells and the ECM
(1). Integrins are composed of
two non-covalently associated subunits, termed α and β. In mammals,
18 α and 8 β subunits have been identified, and combinations of
these subunits give rise to at least 24 distinct integrin heterodimers. Based
on their ligand-binding specificities, ECM-binding integrins are classified
into three groups, namely laminin-, collagen- and RGD-binding integrins
(2,
3), of which the RGD-binding
integrins have been most extensively investigated. The RGD-binding integrins
include α5β1, α8β1, αIIbβ3, and
αV-containing integrins, and have been shown to interact with a variety
of ECM ligands, such as fibronectin and vitronectin, with distinct binding
specificities.The α8 integrin subunit was originally identified in chick nerves
(4). Integrin α8β1
is expressed in the metanephric mesenchyme and plays a crucial role in
epithelial-mesenchymal interactions during the early stages of kidney
morphogenesis. Disruption of the α8 gene in mice was found to be
associated with severe defects in kidney morphogenesis
(5) and stereocilia development
(6). To date, α8β1
integrin has been shown to bind to fibronectin, vitronectin, osteopontin,
latency-associated peptide of transforming growth factor-β1, tenascin-W,
and nephronectin (also named POEM)
(7–13),
among which nephronectin is believed to be an α8β1 integrin ligand
involved in kidney development
(10).Nephronectin is one of the basement membrane proteins whose expression and
localization patterns are restricted in a tissue-specific and developmentally
regulated manner (10,
11). Nephronectin consists of
five epidermal growth factor-like repeats, a linker segment containing the RGD
cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor
protein-tyrosine phosphatase μ (MAM) domain (see
Fig. 3A). Although the
physiological functions of nephronectin remain only poorly understood, it is
thought to play a role in epithelial-mesenchymal interactions through binding
to α8β1 integrin, thereby transmitting signals from the epithelium
to the mesenchyme across the basement membrane
(10). Recently, mice deficient
in nephronectin expression were produced by homologous recombination
(14). These
nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype
reminiscent of α8 integrin knock-out mice
(14), despite the fact that
other RGD-containing ligands, including fibronectin and osteopontin, were
expressed in the embryonic kidneys
(9,
15). The failure of the other
RGD-containing ligands to compensate for the deficiency of nephronectin in the
developing kidneys suggests that nephronectin is an indispensable
α8β1 ligand that plays a mandatory role in epithelial-mesenchymal
interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its
fragments. A, schematic diagrams of full-length nephronectin
(NN) and its fragments. RGD-linker and RGD-linker
(GST), the central RGD-containing linker segments expressed in
mammalian and bacterial expression systems, respectively; PRGDV, a
short RGD-containing peptide modeled after nephronectin and expressed as a GST
fusion protein (see Fig.
4A for the peptide sequence). The arrowheads
indicate the positions of the RGD motif. B, purified recombinant
proteins were analyzed by SDS-PAGE in 7–15% gradient (left and
center panels) and 12% (right panels) gels, followed by
Coomassie Brilliant Blue (CBB) staining, immunoblotting with an
anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded
were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for
blotting with anti-FLAG and PNA) in the left and center
panels;1 μg in the right panel. C, recombinant proteins (10
nm) were coated on microtiter plates and assessed for their binding
activities toward α8β1 integrin (10 nm) in the presence
of 1 mm Mn2+. The backgrounds were subtracted as
described in the legend to Fig.
2. The results represent the mean ± S.D. of triplicate
determinations. D, titration curves of α8β1 integrin bound
to full-length nephronectin (NN, closed squares), the RGD-linker
segments expressed in 293F cells (RGD-linker, closed triangles) and
E. coli (RGD-linker (GST), open
triangles), the MAM domain (MAM, closed diamonds), and the PRGDV
peptide expressed as a GST fusion protein in E. coli (PRGDV
(GST), open circles). The assays were performed as described
in the legend to Fig.
2B. The results represent the means of duplicate
determinations.Although ligand recognition by RGD-binding integrins is primarily
determined by the RGD motif in the ligands, it is the residues outside the RGD
motif that define the binding specificities and affinities toward individual
integrins (16,
17). For example,
α5β1 integrin specifically binds to fibronectin among the many
RGD-containing ligands, and requires not only the RGD motif in the 10th type
III repeat but also the so-called “synergy site” within the
preceding 9th type III repeat for fibronectin recognition
(18). Recently, DiCara et
al. (19) demonstrated
that the high-affinity binding of αVβ6 integrin to its natural
ligands, e.g. foot-and-mouth disease virus, requires the RGD motif
immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to
align the two conserved hydrophobic residues along the length of the helix.
Given the presence of many naturally occurring RGD-containing ligands, it is
conceivable that the specificities of the RGD-binding integrins are dictated
by the sequences flanking the RGD motif or those in neighboring domains that
come into close proximity with the RGD motif in the intact ligand proteins.
However, the preferences of α8β1 integrin for RGD-containing
ligands and how it secures its high-affinity binding toward its preferred
ligands remain unknown.In the present study, we investigated the binding specificities of
α8β1 integrin toward a panel of RGD-containing cell-adhesive
proteins. Our data reveal that nephronectin is a preferred ligand for
α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side
of its RGD motif serves as a synergy site to ensure the specific high-affinity
binding of nephronectin to α8β1 integrin. 相似文献
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Parth Patwari William A. Chutkow Kiersten Cummings Valerie L. R. M. Verstraeten Jan Lammerding Eric R. Schreiter Richard T. Lee 《The Journal of biological chemistry》2009,284(37):24996-25003
Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the α-arrestin protein family; the α-arrestins are related to the classical β-arrestins and visual arrestins. Txnip is the only α-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved α-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related α-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast α-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of α-arrestin proteins.Thioredoxin-interacting protein (Txnip),3 an inhibitor of thioredoxin disulfide reductase activity in vitro (1–3), is robustly induced by glucose (4–6) and a critical regulator of metabolism in vivo (7–10). In humans, Txnip expression is suppressed by insulin and strongly up-regulated in diabetes (7). Txnip-deficient mice have fasting hypoglycemia and ketosis (8, 9, 11, 12) with a striking enhancement of glucose uptake by peripheral tissues (8, 9). We have proposed that Txnip inhibits thioredoxin by forming a mixed disulfide with thioredoxin at its catalytic active site cysteines in a disulfide exchange reaction (13). However, it is not known how Txnip metabolic functions relate to its ability to bind thioredoxin.Structurally, Txnip belongs to the arrestin superfamily of proteins (14). The prototypical arrestins (the visual arrestins and the β-arrestins) are key regulators of receptor signaling. The β-arrestins, named for their interaction with the β-adrenergic receptor, are now known to control signaling through the multiple families of receptors (15). These arrestin proteins have two wing-like arrestin domains arranged around a central core that detects and binds selectively to the charged phosphates of activated receptors (16). The arrestin domains then act as multifunctional scaffolds that cannot only quench receptor signals by recruiting endocytotic machinery and ubiquitin ligases, but also start new signal cascades (15). Recently, arrestin-β2 has also been shown to play a key role in metabolism as a controller of insulin receptor signaling that is deficient in diabetes (17).In addition to the classical visual/β-arrestins, a large number of arrestins more closely related to Txnip are present throughout multicellular evolution. These proteins have been termed the “α-arrestins,” as they are of more ancient origin than the visual/β family (14). Although no structures are known of the α-arrestins to date, they appear highly likely to share the overall fold: two β-sheet sandwich arrestin domains connected by a short linker sequence (14, 18). Confidence in this prediction has been enhanced by the surprising finding that the vps26 family of proteins, even more distantly related to the classical arrestins than Txnip, also share the arrestin fold (19). The vps26 proteins are a component of the retromer complex that controls retrograde transport of recycling endosomes to the trans-Golgi network. This functional overlap with visual/β-arrestin regulation of endocytosis suggests that control of endosome formation and transport may be a conserved function of the arrestin superfamily fold.The functions of the mammalian α-arrestins remain unclear. Humans have six α-arrestins: Txnip and five other proteins, which have been assigned the names Arrdc1–5 (arrestin domain-containing 1–5) (13). Very little is known about these other α-arrestins; thioredoxin binding is not conserved beyond Txnip (13, 20). More is known in yeast: recent reports suggest that α-arrestins function in regulation of endocytosis and protein ubiquitination through PXXY motifs in their C-terminal tails (21–25). However, as all the vertebrate α-arrestins have diverged from the ancestral α-arrestins (14), their structure-function relationships may differ from yeast α-arrestins.Given that other α-arrestins are not thioredoxin-binding proteins, we hypothesized that Txnip metabolic functions may be conserved in mammalian α-arrestins and independent of its interaction with thioredoxin. Overexpression of Txnip in vitro can decrease levels of available thioredoxin and increase levels of reactive oxygen species (1, 3, 26). However, in vivo studies of two different Txnip-deficient mouse models found no change in available thioredoxin levels (8, 27). Txnip reportedly binds to other proteins including Jab1 (28) and Dnajb5 (29), but it is not clear to what extent these interactions are themselves independent of a Txnip-thioredoxin complex (30).Using overexpression of a mutant Txnip that does not bind thioredoxin, we show here that a major metabolic function of Txnip, its inhibition of glucose uptake, does not require interaction with thioredoxin. Instead, we show that inhibition of glucose uptake is a conserved function of another human α-arrestin, Arrdc4. Studies of Txnip mutants and chimeric α-arrestins suggest that the metabolic functions of Txnip and Arrdc4 are intrinsic to the arrestin domains. 相似文献
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Nik A. B. N. Mahmood Esther Biemans-Oldehinkel Bert Poolman 《The Journal of biological chemistry》2009,284(21):14368-14376
We have previously shown that the C-terminal cystathionine β-synthase
(CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA,
in conjunction with an anionic membrane surface function, act as sensor of
internal ionic strength (Iin). Here, we show that a
surface-exposed cationic region in the CBS module domain is critical for ion
sensing. The consecutive substitution of up to five cationic residues led to a
gradual decrease of the ionic strength dependence of transport. In fact, a
5-fold mutant was essentially independent of salt in the range from 0 to 250
mm KCl (or NaCl), supplemented to medium of 30 mm
potassium phosphate. Importantly, the threshold temperature for transport was
lowered by 5–7 °C and the temperature coefficient
Q10 was lowered from 8 to ∼1.5 in the 5-fold mutant,
indicating that large conformational changes are accompanying the CBS-mediated
regulation of transport. Furthermore, by replacing the anionic C-terminal tail
residues that extend the CBS module with histidines, the transport of OpuA
became pH-dependent, presumably by additional charge interactions of the
histidine residues with the membrane. The pH dependence was not observed at
high ionic strength. Altogether the analyses of the CBS mutants support the
notion that the osmotic regulation of OpuA involves a simple biophysical
switching mechanism, in which nonspecific electrostatic interactions of a
protein module with the membrane are sufficient to lock the transporter in the
inactive state.In their natural habitats microorganisms are often exposed to changes in
the concentration of solutes in the environment
(1). A sudden increase in the
medium osmolality results in loss of water from the cell, loss of turgor, a
decrease in cell volume, and an increase in intracellular osmolyte
concentration. Osmoregulatory transporters such as OpuA in Lactococcus
lactis, ProP in Escherichia coli, and BetP in
Corynebacterium glutamicum diminish the consequences of the osmotic
stress by mediating the uptake of compatible solutes upon an increase in
extracellular osmolality
(2–4).
For the ATP-binding cassette
(ABC)5 transporter
OpuA, it has been shown that the system, reconstituted in proteoliposomes, is
activated by increased concentrations of lumenal ions (increased internal
ionic strength) (2,
5,
6). This activation is
instantaneous both in vivo and in vitro and only requires
threshold levels of ionic osmolytes. Moreover, the ionic threshold for
activation is highly dependent of the ionic lipid content (charge density) of
the membrane and requires the presence of so-called cystathionine
β-synthase (CBS) domains, suggesting that the ionic signal is transduced
to the transporter via critical interactions of the protein with membrane
lipids.The ABC transporter OpuA consists of two identical nucleotide-binding
domains (NBD) fused to CBS domains and two identical substrate-binding domains
fused to transmembrane domains. The NBD-CBS and substrate-binding
domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively.
Two tandem CBS domains are linked to the C-terminal end of the NBD; each
domain (CBS1 and CBS2) has a β-α-β-β-α secondary
structure (5)
(Fig. 1A). The CBS
domains are widely distributed in most if not all species of life but their
function is largely unknown. Most of the CBS domains are found as tandem
repeats but data base searches have also revealed tetra-repeat units
(5). The crystal structures of
several tandem CBS domains have been elucidated
(7–9,
32), and in a number of cases
it has been shown that two tandem CBS domains form dimeric structures with a
total of four CBS domains per structural module (hereafter referred to as CBS
module). The crystal structures of the full-length MgtE Mg2+
transporter confirm the dimeric configuration and show that the CBS domains
undergo large conformational changes upon Mg2+ binding or release
(10,
11). In general, ABC
transporters are functional as dimers, which implies that two tandem CBS
domains are present in the OpuA complex. Preliminary experiments with
disulfides engineered at the interface of two tandem CBS domains in OpuA
suggest that large structural rearrangements (association-dissociation of the
interfaces) play a determining role in the ionic strength-regulated transport.
Finally, a subset of CBS-containing proteins has a C-terminal extension, which
in OpuA is highly anionic (sequence: ADIPDEDEVEEIEKEEENK) and modulates the
ion sensing activity (6).Open in a separate windowFIGURE 1.Domain structure of CBS module of OpuA. A, sequence of
tandem CBS domains. The predicted secondary structure is indicated
above the sequence. The residues modified in this study are
underlined. The amino acid sequence end-points of OpuAΔ61 and
OpuAΔ119 are indicated by vertical arrows. B, homology
model of tandem CBS domain of OpuA. The CBS domains were individually modeled
on the crystal structure of the tandem CBS protein Ta0289 from T.
acidophilum (PDB entry 1PVM), using Phyre. Ta0289 was used for the
initial modeling, because its primary sequence was more similar to the CBS
domains of OpuA than those of the other crystallized CBS proteins. The
individual domain models were then assembled with reference to the atomic
coordinates of the tandem CBS domains of IMPDH from Streptococcus
pyogenes (PDB entry 1ZFJ) to form the tandem CBS pair, using PyMOL
(DeLano). The positions of the (substituted) cationic residues are
indicated.In this study, we have engineered the surface-exposed cationic residues of
the CBS module and the C-terminal anionic tail of OpuA
(Fig. 1B). The ionic
strength and lipid dependence of the OpuA mutants were determined in
vivo and in vitro. We show that substitution of five cationic
residues for neutral amino acids is sufficient to inactivate the ionic
strength sensor and convert OpuA into a constitutively active transporter.
Moreover, by substituting six anionic plus four neutral residues of the
C-terminal anionic tail for histidines, the transport reaction becomes
strongly pH-dependent. 相似文献
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Giovanni Maga Barbara van Loon Emmanuele Crespan Giuseppe Villani Ulrich H��bscher 《The Journal of biological chemistry》2009,284(21):14267-14275
Abasic (AP) sites are very frequent and dangerous DNA lesions. Their
ability to block the advancement of a replication fork has been always viewed
as a consequence of their inhibitory effect on the DNA synthetic activity of
replicative DNA polymerases (DNA pols). Here we show that AP sites can also
affect the strand displacement activity of the lagging strand DNA pol δ,
thus preventing proper Okazaki fragment maturation. This block can be overcome
through a polymerase switch, involving the combined physical and functional
interaction of DNA pol β and Flap endonuclease 1. Our data identify a
previously unnoticed deleterious effect of the AP site lesion on normal cell
metabolism and suggest the existence of a novel repair pathway that might be
important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage
in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic)
lesions occur spontaneously in DNA; in eukaryotes it has been estimated that
about 104 depurination and 102 depyrimidation events
occur per genome per day. An equally important source of abasic DNA lesions
results from the action of DNA glycosylases, such as uracil glycosylase, which
excises uracil arising primarily from spontaneous deamination of cytosines
(1). Although most AP sites are
removed by the base excision repair
(BER)5 pathway, a
small fraction of lesions persists, and DNA with AP lesions presents a strong
block to DNA synthesis by replicative DNA polymerases (DNA pols)
(2,
3). Several studies have been
performed to address the effects of AP sites on the template DNA strand on the
synthetic activity of a variety of DNA pols. The major replicative enzyme of
eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP
lesion, but only in the presence of the auxiliary factor proliferating cell
nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared
with an undamaged DNA template
(4). On the other hand, the
family X DNA pols β and λ were shown to bypass an AP site but in a
very mutagenic way (5). Recent
genetic evidence in Saccharomyces cerevisiae cells showed that DNA
pol δ is the enzyme replicating the lagging strand
(6). According to the current
model for Okazaki fragment synthesis
(7–9),
the action of DNA pol δ is not only critical for the extension of the
newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA
segment of about 30 nucleotides on the pre-existing downstream Okazaki
fragment to create an intermediate Flap structure that is the target for the
subsequent action of the Dna2 endonuclease and the Flap endonuclease 1
(Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid
fragment synthesized by the DNA pol α/primase, potentially containing
nucleotide misincorporations caused by the lack of a proofreading exonuclease
activity of DNA pol α/primase. This results in a more accurate copy
synthesized by DNA pol δ. The intrinsic strand displacement activity of
DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A
(RP-A), has been also proposed to be essential for the S phase-specific long
patch BER pathway (10,
11). Although it is clear that
an AP site on the template strand is a strong block for DNA pol
δ-dependent synthesis on single-stranded DNA, the functional
consequences of such a lesion on the ability of DNA pol δ to carry on
strand displacement synthesis have never been investigated so far. Given the
high frequency of spontaneous hydrolysis and/or cytidine deamination events,
any detrimental effect of an AP site on the strand displacement activity of
DNA pol δ might have important consequences both for lagging strand DNA
synthesis and for long patch BER. In this work, we addressed this issue by
constructing a series of synthetic gapped DNA templates with a single AP site
at different positions with respect to the downstream primer to be displaced
by DNA pol δ (see Fig.
1A). We show that an AP site immediately upstream of a
single- to double-strand DNA junction constitutes a strong block to the strand
displacement activity of DNA pol δ, even in the presence of RP-A and
PCNA. Such a block could be resolved only through a “polymerase
switch” involving the concerted physical and functional interaction of
DNA pol β and Fen-1. The closely related DNA pol λ could only
partially substitute for DNA pol β. Based on our data, we propose that
stalling of a replication fork by an AP site not only is a consequence of its
ability to inhibit nucleotide incorporation by the replicative DNA pols but
can also stem from its effects on strand displacement during Okazaki fragment
maturation. In summary, our data suggest the existence of a novel repair
pathway that might be important in preventing replication fork stalling and
identify a previously unnoticed deleterious effect of the AP site lesion on
normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region
inhibits the strand displacement activity of DNA polymerase δ. The
reactions were performed as described under “Experimental
Procedures.” A, schematic representation of the various DNA
templates used. The size of the resulting gaps is indicated in nt. The
position of the AP site on the 100-mer template strand is indicated relative
to the 3′ end. Base pairs in the vicinity of the lesion are indicated by
dashes. The size of the gaps (35–38 nt) is consistent with the
size of ssDNA covered by a single RP-A molecule, which has to be released
during Okazaki fragment synthesis when the DNA pol is approaching the
5′-end of the downstream fragment. When the AP site is covered by the
downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the
nucleotide placed on the opposite strand is C to mimic the situation generated
by spontaneous loss of a guanine or excision of an oxidized guanine, whereas
when the AP site is covered by the primer (nicked AP template), the nucleotide
placed on the opposite strand is A to mimic the most frequent incorporation
event occurring opposite an AP site. B, human PCNA was titrated in
the presence of 15 nm (lanes 2–4 and
10–12) or 30 nm (lanes 6–8 and
14–16) recombinant human four subunit DNA pol δ, on a
linear control (lanes 1–8) or a 38-nt gap control (lanes
9–16) template. Lanes 1, 5, 9, and 13, control
reactions in the absence of PCNA. C, human PCNA was titrated in the
presence of 60 nm DNA pol δ, on a linear AP (lanes
2–4) or 38-nt gap AP (lanes 6–9) template. Lanes
1 and 5, control reactions in the absence of PCNA. 相似文献
12.
13.
Control of TANK-binding Kinase 1-mediated Signaling by the
��134.5 Protein of Herpes Simplex Virus
1
Dustin Verpooten Yijie Ma Songwang Hou Zhipeng Yan Bin He 《The Journal of biological chemistry》2009,284(2):1097-1105
TANK-binding kinase 1 (TBK1) is a key component of Toll-like
receptor-dependent and -independent signaling pathways. In response to
microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and
cytokine expression. Here we show that TBK1 is a novel target of the
γ134.5 protein, a virulence factor whose expression is
regulated in a temporal fashion. Remarkably, the γ134.5
protein is required to inhibit IRF3 phosphorylation, nuclear translocation,
and the induction of antiviral genes in infected cells. When expressed in
mammalian cells, the γ134.5 protein forms complexes with TBK1
and disrupts the interaction of TBK1 and IRF3, which prevents the induction of
interferon and interferon-stimulated gene promoters. Down-regulation of TBK1
requires the amino-terminal domain. In addition, unlike wild type virus, a
herpes simplex virus mutant lacking γ134.5 replicates
efficiently in TBK1-/- cells but not in TBK1+/+ cells.
Addition of exogenous interferon restores the antiviral activity in both
TBK1-/- and TBK+/+ cells. Hence, control of
TBK1-mediated cell signaling by the γ134.5 protein
contributes to herpes simplex virus infection. These results reveal that TBK1
plays a pivotal role in limiting replication of a DNA virus.Herpes simplex virus 1
(HSV-1)3 is a large
DNA virus that establishes latent or lytic infection, in which the virus
triggers innate immune responses. In HSV-infected cells, a number of antiviral
mechanisms operate in a cell type- and time-dependent manner
(1). In response to
double-stranded RNA (dsRNA), Toll-like receptor 3 (TLR3) recruits an adaptor
TIR domain-containing adaptor inducing IFN-β and stimulates cytokine
expression (2,
3). In the cytoplasm, RNA
helicases, RIG-I (retinoid acid-inducible gene-I), and MDA5 (melanoma
differentiation associated gene 5) recognize intracellular viral
5′-triphosphate RNA or dsRNA
(2,
4). Furthermore, a
DNA-dependent activator of IFN-regulatory factor (DAI) senses double-stranded
DNA in the cytoplasm and induces cytokine expression
(5). There is also evidence
that viral entry induces antiviral programs independent of TLR and RIG-I
pathways (6). While recognizing
distinct viral components, these innate immune pathways relay signals to the
two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB
kinase (IKKi) (2).The IKK-related kinases function as essential components that phosphorylate
IRF3 (interferon regulatory factor 3), as well as the closely related IRF7,
which translocates to the nucleus and induces antiviral genes, such as
interferon-α/β and ISG56 (interferon-stimulated gene 56)
(7,
8). TBK1 is constitutively
expressed, whereas IKKi is engaged as an inducible gene product of innate
immune signaling (9,
10). IRF3 activation is
attenuated in TBK1-deficient but not in IKKi-deficient cells
(11,
12). Its activation is
completely abolished in double-deficient cells
(12), suggesting a partially
redundant function of TBK1 and IKKi. Indeed, IKKi also negatively regulates
the STAT-signaling pathway
(13). TBK1/IKKi interacts with
several proteins, such as TRAF family member-associated NF-κB activator
(TANK), NAP1 (NAK-associated protein 1), similar to NAP1TBK1 adaptor
(SINTBAD), DNA-dependent activator of IFN-regulatory factors (DAI), and
secretory protein 5 (Sec5) in host cells
(5,
14–18).
These interactions are thought to regulate TBK1/IKKi, which delineates innate
as well as adaptive immune responses.Upon viral infection, expression of HSV proteins interferes with the
induction of antiviral immunity. When treated with UV or cycloheximide, HSV
induces an array of antiviral genes in human lung fibroblasts
(19,
20). Furthermore, an HSV
mutant, with deletion in immediate early protein ICP0, induces ISG56
expression (21). Accordingly,
expression of ICP0 inhibits the induction of antiviral programs mediated by
IRF3 or IRF7
(21–23).
However, although ICP0 negatively regulates IFN-β expression, it is not
essential for this effect
(24). In HSV-infected human
macrophages or dendritic cells, an immediate early protein ICP27 is required
to suppress cytokine induction involving IRF3
(25). In this context, it is
notable that an HSV mutant, lacking a leaky late gene γ134.5,
replicates efficiently in cells devoid of IFN-α/β genes
(26). Additionally, the
γ134.5 null mutant induces differential cytokine expression
as compared with wild type virus
(27). Thus, HSV modulation of
cytokine expression is a complex process that involves multiple viral
components. Currently, the molecular mechanism governing this event is
unclear. In this study, we show that HSV γ134.5 targets TBK1
and inhibits antiviral signaling. The data herein reveal a previously
unrecognized mechanism by which γ134.5 facilitates HSV
replication. 相似文献
14.
Yvette R. Pittman Kimberly Kandl Marcus Lewis Louis Valente Terri Goss Kinzy 《The Journal of biological chemistry》2009,284(7):4739-4747
Eukaryotic translation elongation factor 1A (eEF1A) both shuttles
aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single
domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide
exchange factor eEF1Bα. We show that eEF1Bα has the ability to
disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα
F163, which binds in this domain, demonstrates effects on growth, eEF1A
binding, nucleotide exchange activity, and cell morphology. These phenotypes
can be partially restored by an intragenic W130A mutation. Furthermore, the
combination of F163A with the lethal K205A mutation restores viability by
drastically reducing eEF1Bα affinity for eEF1A. This also results in a
consistent increase in actin bundling and partially corrected morphology. The
consequences of the overlapping functions in this eEF1A domain and its unique
differences from the bacterial homologs provide a novel function for
eEF1Bα to balance the dual roles in actin bundling and protein
synthesis.The final step of gene expression takes place at the ribosome as mRNA is
translated into protein. In the yeast Saccharomyces cerevisiae,
elongation of the polypeptide chain requires the orchestrated action of three
soluble factors. The eukaryotic elongation factor 1
(eEF1)2 complex
delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating
ribosome (1). The eEF1A subunit
is a classic G-protein that acts as a “molecular switch” for the
active and inactive states based on whether GTP or GDP is bound, respectively
(2). Once an anticodon-codon
match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP
hydrolysis resulting in the release of inactive GDP-bound eEF1A from the
ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely
slow (3,
4), a guanine nucleotide
exchange factor (GEF) complex, eEF1B, is required
(5,
6). The yeast S.
cerevisiae eEF1B complex contains two subunits, the essential catalytic
subunit eEF1Bα (5) and
the non-essential subunit eEF1Bγ
(7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP:
Mg2+ and eEF1A:eEF1Bα C terminus:GDPNP
(8,
9) demonstrated a surprising
structural divergence from the bacterial EF-Tu-EF-Ts
(10) and mammalian
mitochondrial EF-Tumt-EF-Tsmt
(11). While the G-proteins
have a similar topology and consist of three well-defined domains, a striking
difference was observed in binding sites for their GEFs. The C terminus of
eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A,
creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts
and mammalian mitochondrial EF-Tsmt, make extensive contacts with
domain I and III of EF-Tu and EF-Tumt, respectively. The altered
binding interface of eEF1Bα to domain II of eEF1A is particularly
unexpected given the functions associated with domain II of eEF1A and EF-Tu.
The crystal structure of the EF-Tu:GDPNP:Phe-tRNAPhe complex
reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II
and tRNA to sustain stable contacts
(12). That eEF1A employs the
same aa-tRNA binding site is supported by genetic and biochemical data
(13-15).
Interestingly, eEF1Bα contacts many domain II eEF1A residues in the
region hypothesized to be involved in the binding of the aa-tRNA CCA end
(8). Because, the shared
binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is
significantly different between the eukaryotic and bacterial/mitochondrial
systems, eEF1Bα may play a unique function aside from guanine nucleotide
release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This
noncanonical function of eEF1A was initially observed in Dictyostelium
amoebae (16). It is
estimated that greater than 60% of Dictyostelium eEF1A is associated
with the actin cytoskeleton
(17). The eEF1A-actin
interaction is conserved among species from yeast to mammals, suggesting the
importance of eEF1A for cytoskeleton integrity. Using a unique genetic
approach, multiple eEF1A mutations were identified that altered cell growth
and morphology, and are deficient in bundling actin in vitro
(18,
19). Intriguingly, most
mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding
site. Previous studies have demonstrated that actin bundling by eEF1A is
significantly reduced in the presence of aa-tRNA while eEF1A bound to actin
filaments is not in complex with aa-tRNA
(20). Therefore, actin and
aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of
yeast eEF1A or actin-bundling deficient mutants do not affect translation
elongation (18,
19,
21), suggesting
eEF1A-dependent cytoskeletal organization is independent of its translation
elongation function (18,
20). Thus, while aa-tRNA
binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling
function associated with eEF1A domain II places greater importance on its
relationship with the “novel” binding interface between eEF1A
domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα
binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates
the equilibrium between actin and translation functions of eEF1A and is
perhaps the result of evolutionary selective pressure to balance the
eukaryotic-specific role of eEF1A in actin organization. Here, we present
kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the
importance of the interactions between domain II of eEF1A and eEF1Bα to
prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide
exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A
support that this complex formation is a determining factor for eEF1A-induced
actin organization. Interestingly, the F163A that reduces eEF1A affinity is an
intragenic suppressor of the lethal K205A eEF1Bα mutant that displays
increased affinity for eEF1A. This, along with a consistent change in the
actin bundling correlated with the affinity of eEF1Bα for eEF1A,
indicates that eEF1Bα is a balancer, directing eEF1A to translation
elongation and away from actin, and alterations in this balance result in
detrimental effects on cell growth and eEF1A function. 相似文献
15.
Wei Lu Csaba F. L��szl�� Zhixin Miao Hao Chen Shiyong Wu 《The Journal of biological chemistry》2009,284(36):24281-24288
UV light induces phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2α in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2α phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2α phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2α after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2α phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor NG-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2α phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2α phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.UV irradiation inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α).2 Two eIF2α kinases, double strand RNA-dependent protein kinase-like ER kinase (PERK) and general control of amino acid biosynthesis kinase (GCN2), are known to phosphorylate the serine 51 of eIF2α in response to UV irradiation (1–4). However, the one or more upstream pathways that activate eIF2α kinase(s) upon UV irradiation are not known. In this report, we provide evidence that UV-induced nitric-oxide synthase (NOS) activation and nitric oxide (NO•) production regulate both PERK and GCN2 activation upon UVB irradiation.Expression of inducible nitric-oxide synthase in a mouse macrophage cell line leads to the phosphorylation of eIF2α and inhibition of translation (5). In cultured neuronal and pancreatic cell lines, production of NO• and peroxynitrite (ONOO−) induces endoplasmic reticulum (ER) stress, which activates PERK and results in cell dysfunction and apoptosis (6–9). Cytokine-stimulated inducible nitric-oxide synthase activation in astrocytes depletes l-arginine and activates GCN2, which phosphorylates eIF2α (10). UV irradiation also activates NOS and elevates cellular NO• (11–13). However, the UV-induced NOS activation and NO• production have never been shown to be related to the activation of eIF2α kinase(s). Now we demonstrate that UV-induced activation of NOS mediates the activation of both PERK and GCN2, which coordinately regulate the phosphorylation of eIF2α. 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.
18.
Tomoya Isaji Yuya Sato Tomohiko Fukuda Jianguo Gu 《The Journal of biological chemistry》2009,284(18):12207-12216
N-Glycosylation of integrin α5β1 plays a crucial role
in cell spreading, cell migration, ligand binding, and dimer formation, but
the detailed mechanisms by which N-glycosylation mediates these
functions remain unclear. In a previous study, we showed that three potential
N-glycosylation sites (α5S3–5) on the β-propeller of
the α5 subunit are essential to the functional expression of the
subunit. In particular, site 5 (α5S5) is the most important for its
expression on the cell surface. In this study, the function of the
N-glycans on the integrin β1 subunit was investigated using
sequential site-directed mutagenesis to remove the combined putative
N-glycosylation sites. Removal of the N-glycosylation sites
on the I-like domain of the β1 subunit (i.e. the Δ4-6
mutant) decreased both the level of expression and heterodimeric formation,
resulting in inhibition of cell spreading. Interestingly, cell spreading was
observed only when the β1 subunit possessed these three
N-glycosylation sites (i.e. the S4-6 mutant). Furthermore,
the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5
mutant of the α5 subunit. Taken together, the results of the present
study reveal for the first time that N-glycosylation of the I-like
domain of the β1 subunit is essential to both the heterodimer formation
and biological function of the subunit. Moreover, because the
α5S3-5/β1S4-6 mutant represents the minimal
N-glycosylation required for functional expression of the β1
subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α
and a β subunit (1). The
interaction between integrin and the extracellular matrix is essential to both
physiologic and pathologic events, such as cell migration, development, cell
viability, immune homeostasis, and tumorigenesis
(2,
3). Among the integrin
superfamily, β1 integrin can combine with 12 distinct α subunits
(α1–11, αv) to form heterodimers, thereby acquiring a wide
variety of ligand specificity
(1,
4). Integrins are thought to be
regulated by inside-out signaling mechanisms that provoke conformational
changes, which modulate the affinity of integrin for the ligand
(5). However, an increasing
body of evidence suggests that cell-surface carbohydrates mediate a variety of
interactions between integrin and its extracellular environment, thereby
affecting integrin activity and possibly tumor metastasis as well
(6–8).Guo et al. (9)
reported that an increase in β1–6-GlcNAc sugar chains on the
integrin β1 subunit stimulated cell migration. In addition, elevated
sialylation of the β1 subunit, because of Ras-induced STGal-I transferase
activity, also induced cell migration
(10,
11). Conversely, cell
migration and spreading were reduced by the addition of a bisecting GlcNAc,
which is a product of N-acetylglucosaminyltransferase III
(GnT-III),2 to the
α5β1 and α3β1 integrins
(12,
13). Alterations of
N-glycans on integrins might also regulate their cis interactions
with membrane-associated proteins, including the epidermal growth factor
receptor, the galectin family, and the tetraspanin family of proteins
(14–19).In addition to the positive and negative regulatory effects of
N-glycan, several research groups have reported that
N-glycans must be present on integrin α5β1 for the
αβ heterodimer formation and proper integrin-matrix interactions.
Consistent with this hypothesis, in the presence of the glycosylation
inhibitor, tunicamycin, normal integrin-substrate binding and transport to the
cell surface are inhibited
(20). Moreover, treatment of
purified integrin with N-glycosidase F blocked both the inherent
association of the subunits and the interaction between integrin and
fibronectin (FN) (21). These
results suggest that N-glycosylation is essential to the functional
expression of α5β1. However, because integrin α5β1
contains 26 potential N-linked glycosylation sites, 14 in the α
subunit and 12 in the β subunit, identification of the sites that are
essential to its biological functions is key to understanding the molecular
mechanisms by which N-glycans alter integrin function. Recently, our
group determined that N-glycosylation of the β-propeller domain
on the α5 subunit is essential to both heterodimerization and biological
functions of the subunit. Furthermore, we determined that sites 3–5 are
the most important sites for α5 subunit-mediated cell spreading and
migration on FN (22). The
purpose of this study was to clarify the roles of N-glycosylation of
the β1 subunit. Therefore, we performed combined substitutions in the
putative N-glycosylation sites by replacement of asparagine residues
with glutamine residues. We subsequently introduced these mutated genes into
β1-deficient epithelial cells (GE11). The results of these mutation
experiments revealed that the N-glycosylation sites on the I-like
domain of the β1 subunit, sites number 4–6 (S4-6), are essential to
both heterodimer formation and biological functions, such as cell
spreading. 相似文献
19.
20.
Yun Liu Yun-wu Zhang Xin Wang Han Zhang Xiaoqing You Francesca-Fang Liao Huaxi Xu 《The Journal of biological chemistry》2009,284(18):12145-12152
Excessive accumulation of β-amyloid peptides in the brain is a major
cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived
from β-amyloid precursor protein (APP) through sequential cleavages by
β- and γ-secretases, whose enzymatic activities are tightly
controlled by subcellular localization. Delineation of how intracellular
trafficking of these secretases and APP is regulated is important for
understanding Alzheimer disease pathogenesis. Although APP trafficking is
regulated by multiple factors including presenilin 1 (PS1), a major component
of the γ-secretase complex, and phospholipase D1 (PLD1), a
phospholipid-modifying enzyme, regulation of intracellular trafficking of
PS1/γ-secretase and β-secretase is less clear. Here we demonstrate
that APP can reciprocally regulate PS1 trafficking; APP deficiency results in
faster transport of PS1 from the trans-Golgi network to the cell
surface and increased steady state levels of PS1 at the cell surface, which
can be reversed by restoring APP levels. Restoration of APP in APP-deficient
cells also reduces steady state levels of other γ-secretase components
(nicastrin, APH-1, and PEN-2) and the cleavage of Notch by
PS1/γ-secretase that is more highly correlated with cell surface levels
of PS1 than with APP overexpression levels, supporting the notion that Notch
is mainly cleaved at the cell surface. In contrast, intracellular trafficking
of β-secretase (BACE1) is not regulated by APP. Moreover, we find that
PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell
surface accumulation of PS1 in an APP-independent manner. Our results clearly
elucidate a physiological function of APP in regulating protein trafficking
and suggest that intracellular trafficking of PS1/γ-secretase is
regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease
(AD)4 is the formation
of senile plaques in the brains of patients. The major components of those
plaques are β-amyloid peptides (Aβ), whose accumulation triggers a
cascade of neurodegenerative steps ending in formation of senile plaques and
intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible
brain regions (1,
2). Aβ is proteolytically
derived from the β-amyloid precursor protein (APP) through sequential
cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl
protease (3,
4), and by γ-secretase, a
high molecular weight complex consisting of at least four components:
presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and
presenilin enhancer-2 (PEN-2)
(5,
6). APP is a type I
transmembrane protein belonging to a protein family that includes APP-like
protein 1 (APLP1) and 2 (APLP2) in mammals
(7,
8). Full-length APP is
synthesized in the endoplasmic reticulum (ER) and transported through the
Golgi apparatus. Most secreted Aβ peptides are generated within the
trans-Golgi network (TGN), also the major site of steady state APP in
neurons
(9–11).
APP can be transported to the cell surface in TGN-derived secretory vesicles
if not proteolyzed to Aβ or an intermediate metabolite. At the cell
surface APP is either cleaved by α-secretase to produce soluble
sAPPα (12) or
reinternalized for endosomal/lysosomal degradation
(13,
14). Aβ may also be
generated in endosomal/lysosomal compartments
(15,
16). In contrast to neurotoxic
Aβ peptides, sAPPα possesses neuroprotective potential
(17,
18). Thus, the subcellular
distribution of APP and proteases that process it directly affect the ratio of
sAPPα to Aβ, making delineation of the mechanisms responsible for
regulating trafficking of all of these proteins relevant to AD
pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the
two mammalian PS gene homologues, PS1 and PS2, PS1
encodes the major form (PS1) in active γ-secretase
(19,
20). Nascent PSs undergo
endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a
carboxyl-terminal fragment (CTF) to form a functional PS heterodimer
(21). Based on observations
that PSs possess two highly conserved aspartate residues indispensable for
γ-secretase activity and that specific transition state analogue
γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers
(5,
22), PSs are believed to be
the catalytic component of the γ-secretase complex. PS assembles with
three other components, NCT, APH-1, and PEN-2, to form the functional
γ-secretase (5,
6). Strong evidence suggests
that PS1/γ-secretase resides principally in the ER, early Golgi, TGN,
endocytic and intermediate compartments, most of which (except the TGN) are
not major subcellular sites for APP
(23,
24). In addition to generating
Aβ and cleaving APP to release the APP intracellular domain,
PS1/γ-secretase cleaves other substrates such as Notch
(25), cadherin
(26), ErbB4
(27), and CD44
(28), releasing their
respective intracellular domains. Interestingly, PS1/γ-secretase
cleavage of different substrates seems to occur at different subcellular
compartments; APP is mainly cleaved at the TGN and early endosome domains,
whereas Notch is predominantly cleaved at the cell surface
(9,
11,
29). Thus, perturbing
intracellular trafficking of PS1/γ-secretase may alter interactions
between PS1/γ-secretase and APP, contributing to either abnormal Aβ
generation and AD pathogenesis or decreased access of PS1/γ-secretase to
APP such that Aβ production is reduced. However, mechanisms regulating
PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates
intracellular trafficking of several membrane proteins, including other
γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate
APP (reviewed in Ref. 30).
Intracellular APP trafficking is highly regulated and requires other factors
such as mint family members and SorLA
(2). Moreover, we recently
found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that
regulates membrane trafficking events, can interact with PS1, and can regulate
budding of APP-containing vesicles from the TGN and delivery of APP to the
cell surface (31,
32). Interestingly, Kamal
et al. (33)
identified an axonal membrane compartment that contains APP, BACE1, and PS1
and showed that fast anterograde axonal transport of this compartment is
mediated by APP and kinesin-I, implying a traffic-regulating role for APP.
Increased APP expression is also shown to decrease retrograde axonal transport
of nerve growth factor (34).
However, whether APP indeed regulates intracellular trafficking of proteins
including BACE1 and PS1/γ-secretase requires further validation. In the
present study we demonstrate that intracellular trafficking of PS1, as well as
that of other γ-secretase components, but not BACE1, is regulated by
APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase
complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In
addition, we find that PLD1 also regulates intracellular trafficking of PS1
through a different mechanism and more potently than APP. 相似文献