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《Journal of receptor and signal transduction research》2013,33(1):75-85
AbstractThe β2-adrenergic receptor (β2AR) couples to Gs, activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. β2AR also couple to Gi after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of β2AR-Gi coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express β2AR or β2AR and Giα2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted β2AR desensitization. Membrane AC activities showed that Giα2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing Giα2. in the absence of such overexpression, β2AR desensitization was 23 ± 7%, while with 5-fold Giα2 overexpression desensitization was 58 ± 5% (p<0.01, n=4). the effect of Gi on desensitization was receptor-specific, in that forskolin responses were not altered by Giα2 overexpression. Thus, acquired β2AR coupling to Gi is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase Gi levels contribute to β2AR dysfunction. 相似文献
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Daniel Beltr��n-Valero de Bernab�� Kei-ichiro Inamori Takako Yoshida-Moriguchi Christine J. Weydert Hollie A. Harper Tobias Willer Michael D. Henry Kevin P. Campbell 《The Journal of biological chemistry》2009,284(17):11279-11284
The interaction between epithelial cells and the extracellular matrix is
crucial for tissue architecture and function and is compromised during cancer
progression. Dystroglycan is a membrane receptor that mediates interactions
between cells and basement membranes in various epithelia. In many
epithelium-derived cancers, β-dystroglycan is expressed, but
α-dystroglycan is not detected. Here we report that α-dystroglycan
is correctly expressed and trafficked to the cell membrane but lacks laminin
binding as a result of the silencing of the like-acetylglucosaminyltransferase
(LARGE) gene in a cohort of highly metastatic epithelial cell lines
derived from breast, cervical, and lung cancers. Exogenous expression of LARGE
in these cancer cells restores the normal glycosylation and laminin binding of
α-dystroglycan, leading to enhanced cell adhesion and reduced cell
migration in vitro. Our findings demonstrate that LARGE repression is
responsible for the defects in dystroglycan-mediated cell adhesion that are
observed in epithelium-derived cancer cells and point to a defect of
dystroglycan glycosylation as a factor in cancer progression.Normal epithelial cells are tightly associated with one another and with
the underlying basement membrane to maintain tissue architecture and function.
During cancer progression, primitive cancer cells escape from this control by
modifying the binding affinities of their cell membrane receptors. Several
receptors have been described as important for this process. Of these, the
integrins are the best studied
(1). The receptor dystroglycan
has been reported to be required for the development and maintenance of
epithelial tissues (2,
3). A direct requirement for
dystroglycan in epithelia is further demonstrated by the profound effect that
loss of dystroglycan expression has on cell polarity and laminin binding in
cultured mammary epithelial cells
(4,
5). However, dystroglycan is
not only important in the establishment and maintenance of epithelial
structure. Associations have also been made between the loss of
α-dystroglycan immunoreactivity and cancer progression in tumors of
epithelial origin, including breast, colon, cervix, and prostate cancers
(4,
6–9).
The dystroglycan loss of function could thus serve as an effective means by
which cancerous cells modify their adhesion to the extracellular matrix
(ECM).2Dystroglycan is a ubiquitously expressed cell membrane protein that plays a
key function in cellular integrity, linking the intracellular cytoskeleton to
the extracellular matrix. The dystroglycan gene encodes a preprotein that is
cleaved into two peptides
(10). The C-terminal
component, known as β-dystroglycan, is embedded within the cell membrane,
whereas the N-terminal component, α-dystroglycan, is present within the
extracellular periphery but remains associated with β-dystroglycan
through non-covalent bonds. β-Dystroglycan binds to actin
(11), dystrophin
(11), utrophin
(11), and Grb2
(12) through its C-terminal
intracellular domain. α-Dystroglycan, on the other hand, binds to ECM
proteins that contain laminin globular domains including laminins
(13,
14), agrin
(15), and perlecan
(16), as well as to the
transmembrane protein neurexin
(17). α-Dystroglycan is
extensively decorated by three different types of glycan modifications: mucin
type O-glycosylation, O-mannosylation, and
N-glycosylation. The state of α-dystroglycan glycosylation has
been shown to be critical for the ability of the protein to bind to laminin
globular domain-containing proteins of the ECM
(18).Previous studies of epithelium-derived cancers
(4,
9) demonstrated that the loss
of immunoreactivity of α-dystroglycan antibodies correlates with tumor
grade and poor prognosis. This reduced detection of α-dystroglycan,
however, is based on a loss of α-dystroglycan reactivity to antibodies
(known as IIH6 and VIA4-1) that recognize the laminin-binding glyco-epitope of
α-dystroglycan, i.e. the protein is only functional when it is
glycosylated in such a way (henceforth, referred to as functional
glycosylation). However, in most of the cancer samples that have been studied
to date, β-dystroglycan is expressed at normal levels at the cell
membrane. Thus, the aforementioned cancer-associated loss of
α-dystroglycan expression may reflect a failure in the
post-translational processing of dystroglycan rather than in the synthesis of
α-dystroglycan itself.A similar defect in dystroglycan has been reported in a group of congenital
muscular dystrophies (19).
This spectrum of human developmental syndromes involves the brain, eye, and
skeletal muscle and shows a dramatic gradient of phenotypic severity that
ranges from the most devastating in Walker-Warburg syndrome to the least
severe in limb-girdle muscular dystrophy. Six distinct known and putative
glycosyltransferases have been shown to underlie these syndromes: protein
O-mannosyltransferase 1 (POMT1), protein
O-mannosyltransferase 2 (POMT2), protein O-mannose
β-1,2-acetylglucosaminyltransferase 1 (POMGnT1), like
acetylglucosaminyltransferase (LARGE), Fukutin, and Fukutin-related protein
(FKRP)
(20–25).
Indeed, all muscular dystrophy patients with mutations in any of these genes
fail to express the functionally glycosylated α-dystroglycan epitope
that is recognized by the IIH6 and VIA4-1 antibodies.To investigate the molecular mechanism responsible for the loss of
α-dystroglycan in epithelium-derived cancers and its role in metastatic
progression, we examined the expression and glycosylation status of
α-dystroglycan in a group of breast, cervical, and lung cancer cell
lines. Here we report that although α-dystroglycan is expressed in the
metastatic cell lines MDA-MB-231, HeLa, H1299, and H2030, it is not
functionally glycosylated. In screening these cell lines for expression of the
six known α-dystroglycan-modifying proteins, we observed that only one,
LARGE, was extensively down-regulated. We also report that the ectopic
restoration of LARGE expression in these cell lines led not only to the
production of a functional dystroglycan but also to the reversion of certain
characteristics associated with invasiveness, namely cell attachment to ECM
proteins and cell migration. 相似文献
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Tatsuya Hoshino Takushi Namba Masaya Takehara Tadashi Nakaya Yukihiko Sugimoto Wataru Araki Shuh Narumiya Toshiharu Suzuki Tohru Mizushima 《The Journal of biological chemistry》2009,284(27):18493-18502
Amyloid-β (Aβ) peptides, generated by the proteolysis of β-amyloid precursor protein by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E2 (PGE2), a strong inducer of inflammation, stimulates the production of Aβ through EP2 and EP4 receptors, and here we have examined the molecular mechanism. Activation of EP2 and EP4 receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP2, but not EP4, receptor-mediated stimulation of the Aβ production. In contrast, inhibitors of endocytosis suppressed EP4, but not EP2, receptor-mediated stimulation. Activation of γ-secretase was observed with the activation of EP4 receptors but not EP2 receptors. PGE2-dependent internalization of the EP4 receptor was observed, and cells expressing a mutant EP4 receptor lacking the internalization activity did not exhibit PGE2-stimulated production of Aβ. A physical interaction between the EP4 receptor and PS-1, a catalytic subunit of γ-secretases, was revealed by immunoprecipitation assays. PGE2-induced internalization of PS-1 and co-localization of EP4, PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP4 receptor null mice. These results suggest that PGE2-stimulated production of Aβ involves EP4 receptor-mediated endocytosis of PS-1 followed by activation of the γ-secretase, as well as EP2 receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.Alzheimer disease (AD)2 is the most common neurodegenerative disorder of the central nervous system and the leading cause of adult onset dementia. AD is characterized pathologically by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-β (Aβ) peptides Aβ40 and Aβ42 (1, 2). To generate Aβ, β-amyloid precursor protein (APP) is first cleaved by β-secretase and then by γ-secretase. Cleavage of APP by α-secretase produces non-amyloidogenic peptides (3, 4). The γ-secretase is an aspartyl protease complex composed of four core components, including presenilin (PS) 1 and PS2 (5). Early onset familial AD is linked to three genes, APP, PS1, and PS2 (5, 6), strongly suggesting that γ-secretase-dependent production of Aβ is a key factor in the pathogenesis of AD. Therefore, cellular factors that affect the γ-secretase-dependent production of Aβ may be good targets for the development of drugs to prevent and treat AD.Both APP and PS-1 are transmembrane proteins, and their intracellular localization is controlled by secretory and endocytic pathways. These proteins are modified in the endoplasmic reticulum and trafficked to the cell surface through the trans-Golgi network (TGN). Then, they are internalized again and trafficked to early endosomes. Next, they are trafficked to late endosomes and lysosomes (LEL), which are recycling endosomes that are targeted to the cell surface or the TGN (7–11). The production of Aβ seems to occur in a broad range of cellular compartments including the cell surface, TGN, and endosomes (12). Abnormalities of secretory and endocytic pathways have been observed in the brains of AD patients (9, 13). Importantly, factors that control these vesicle transport systems affect the production of Aβ. For example, overproduction of Rab5, a factor essential for traffic of vesicles to early endosomes, has been shown to stimulate the production of Aβ (14), and SorL1 has been shown to reduce the production of Aβ by stimulating the traffic of APP in early endosomes to the TGN (15, 16).It has been suggested that inflammation is important in the pathogenesis of AD; chronic inflammation has been observed in the brains of AD patients, and trauma to the brain and ischemia, both of which can activate inflammation, are major risk factors for AD (17–19). Cyclooxygenase (COX) is essential for the synthesis of prostaglandin E2 (PGE2), a potent inducer of inflammation and has two subtypes, COX-1 and COX-2. COX-1 is expressed constitutively, whereas expression of COX-2 is induced under inflammatory conditions and is responsible for the progression of inflammation (20–22). The following evidences of the involvement of PGE2 (and COX-2) in the progression of AD suggest that they are good targets for the development of AD drugs: (i) Elevated levels of PGE2 and overexpression of COX-2 have been observed in the brains of AD patients (23–25); (ii) the extent of COX-2 expression correlates with the amount of Aβ and the degree of progression of AD pathogenesis (26); (iii) transgenic mice constitutively overexpressing COX-2 show aging-dependent neural apoptosis and memory dysfunction (27); (iv) prolonged use of nonsteroidal anti-inflammatory drugs, inhibitors of COX, delays the onset and reduces the risk of AD (28); (v) PGE2 stimulates the production of reactive oxygen species in microglia cells, resulting in activation of β-secretase (29).We recently reported that PGE2 stimulates the production of Aβ in human embryonic kidney (HEK) 293 and human neuroblastoma (SH-SY5Y) cells that stably express a form of APP with two mutations (K651N/M652L) (APPsw) that elevate cellular and secreted levels of Aβ (30). Similar results were reported by another group (31). Using agonists and antagonists specific for each of the four PGE2 receptors (EP1, EP2, EP3, and EP4), we found that EP4 receptors alone and also both EP2 and EP4 receptors are involved in PGE2-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively (30). Furthermore, experiments with transgenic mice suggest that EP2 and EP4 receptors are involved in the production of Aβ in vivo (30). Based on these results, we propose that antagonists of the EP2 and/or EP4 receptors may be therapeutically beneficial for the treatment of AD. Understanding the mechanism governing EP2 and EP4 receptor-mediated stimulation of production of Aβ by PGE2 will be important for such drug development.Activation of EP2 and EP4 receptors causes activation of adenylate cyclase and an increase in the cellular level of cAMP (32). We have shown that an EP4 receptor agonist or both EP2 and EP4 receptor agonists increase the cellular level of cAMP in HEK293 or SH-SY5Y cells, respectively, and that a cAMP analogue, 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP), increases the level of Aβ in HEK293 cells (30). These findings suggest that the cellular level of cAMP is important for PGE2-stimulated production of Aβ. An increase in the cellular level of cAMP is known to activate protein kinase A (PKA), which is important for cAMP-regulated intracellular signal transduction (33). However, an inhibitor of PKA, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), does not block PGE2-stimulated production of Aβ in HEK293 cells (30). Other cAMP-regulated signal transduction factors, such as phosphatidylinositol 3-kinase and Epac (exchange protein directly activated by cAMP), were also shown not to be involved in PGE2-stimulated production of Aβ in HEK293 cells (30). Thus, the mechanism whereby the activation of EP2 and EP4 receptors stimulates the production of Aβ has remained unknown. In this study, by using inhibitors of adenylate cyclase and PKA, we found that activation of the EP2 receptor stimulates production of Aβ through activation of adenylate cyclase and PKA. We also propose that activation of the EP4 receptor causes its co-internalization with PS-1 (γ-secretase) into endosomes and that this co-internalization is important for EP4 receptor-mediated stimulation of Aβ production by PGE2 through the activation of γ-secretase. 相似文献
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Peter J. McCormick Marta Segarra Paola Gasperini A. Virginia Gulino Giovanna Tosato 《PloS one》2009,4(12)
WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is a rare immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. Leukocytes from patients with WHIM syndrome display impaired CXCR4 internalization and enhanced chemotaxis in response to its unique ligand SDF-1/CXCL12, which likely contribute to the clinical manifestations. Here, we investigated the biochemical mechanisms underlying CXCR4 deficiency in WHIM syndrome. We report that after ligand activation, WHIM-associated mutant CXCR4 receptors lacking the carboxy-terminal 19 residues internalize and activate Erk 1/2 slower than wild-type (WT) receptors, while utilizing the same trafficking endocytic pathway. Recruitment of β-Arrestin 2, but not β-Arrestin 1, to the active WHIM-mutant receptor is delayed compared to the WT CXCR4 receptor. In addition, while both kinases Grk3 and Grk6 bind to WT CXCR4 and are critical to its trafficking to the lysosomes, Grk6 fails to associate with the WHIM-mutant receptor whereas Grk3 associates normally. Since β-Arrestins and Grks play critical roles in phosphorylation and internalization of agonist-activated G protein-coupled receptors, these results provide a molecular basis for CXCR4 dysfunction in WHIM syndrome. 相似文献
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Hardeep Kaur Chitranshu Kumar Christophe Junot Michel B. Toledano Anand K. Bachhawat 《The Journal of biological chemistry》2009,284(21):14493-14502
GSH metabolism in yeast is carried out by the γ-glutamyl cycle as
well as by the DUG complex. One of the last steps in the
γ-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the
constitutent amino acids. Saccharomyces cerevisiae extracts carry
Cys-Gly dipeptidase activity, but the corresponding gene has not yet been
identified. We describe the isolation and characterization of a novel Cys-Gly
dipeptidase, encoded by the DUG1 gene. Dug1p had previously been
identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an
alternate GSH degradation pathway and has also been suggested to function as a
possible di- or tripeptidase based on genetic studies. We show here that Dug1p
is a homodimer that can also function in a Dug2-Dug3-independent manner as a
dipeptidase with high specificity for Cys-Gly and no activity toward tri- or
tetrapeptides in vitro. This activity requires zinc or manganese
ions. Yeast cells lacking Dug1p (dug1Δ) accumulate Cys-Gly.
Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase
M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We
also show that the Dug1p Schizosaccharomyces pombe orthologue
functions as the exclusive Cys-Gly peptidase in this organism. The human
orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by
complementation of the dug1Δ mutant and by biochemical
characterization, which revealed a high substrate specificity and affinity for
Cys-Gly. The results indicate that the Dug1p family represents a novel class
of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide
(l-γ-glutamyl-l-cysteinyl-glycine) present in
almost all eukaryotes (barring a few protozoa) and in a few prokaryotes
(1). In the cell, glutathione
exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the
millimolar range), a relatively low redox potential (-240 mV), and a high
stability conferred by the unusual peptidase-resistant γ-glutamyl bond
are three of the properties endowing GSH with the attribute of an important
cellular redox buffer. GSH also contributes to the scavenging of free radicals
and peroxides, the chelation of heavy metals, such as cadmium, the
detoxification of xenobiotics, the transport of amino acids, and the
regulation of enzyme activities through glutathionylation and serves as a
source of sulfur and nitrogen under starvation conditions
(2,
3). GSH metabolism is carried
out by the γ-glutamyl cycle, which coordinates its biosynthesis,
transport, and degradation. The six-step cycle is schematically depicted in
Fig. 1
(2).Open in a separate windowFIGURE 1.γ-Glutamyl cycle of glutathione metabolism.
γ-Glutamylcysteine synthetase and GSH synthetase carry out the first two
steps in glutathione biosynthesis. γ-glutamyltranspeptidase,
γ-glutamylcyclotransferase, 5-oxoprolinase, and Cys-Gly dipeptidase are
involved in glutathione catabolism. Activities responsible for
γ-glutamylcyclotransferase and 5-oxoprolinase have not been detected in
S. cerevisiae.In Saccharomyces cerevisiae, γ-glutamyl cyclotransferase and
5-oxoprolinase activities have not been detected, which has led to the
suggestion of the presence of an incomplete, truncated form of the
γ-glutamyl cycle (4) made
of γ-glutamyl transpeptidase
(γGT)4 and
Cys-Gly dipeptidase and only serving a GSH catabolic function. Although
γGT and Cys-Gly dipeptidase activities were detected in S.
cerevisiae cell extracts, only the γGT gene (ECM38) has
been identified so far. Cys-Gly dipeptidase activity has been identified in
humans (5,
6), rats
(7–10),
pigs (11,
12), Escherichia coli
(13,
14), and other organisms
(15,
16), and most of them belong
to the M17 or the M1 and M19 metallopeptidases gene families
(17).S. cerevisiae has an alternative γGT-independent GSH
degradation pathway (18) made
of the Dug1p, Dug2p, and Dug3p proteins that function together as a complex.
Dug1p also seem to carry nonspecific di- and tripeptidase activity, based on
genetic studies (19).We show here that Dug1p is a highly specific Cys-Gly dipeptidase, as is its
Schizosaccharomyces pombe homologue. We also show that the mammalian
orthologue of DUG1, CNDP2, can complement the defective utilization
of Cys-Gly as sulfur source of an S. cerevisiae strain lacking
DUG1 (dug1Δ). Moreover, CNDP2 has Cys-Gly dipeptidase
activity in vitro, with a strong preference for Cys-Gly over all
other dipeptides tested. CNDP2 and its homologue CNDP1 are members of the
metallopeptidases M20A family and have been known to carry carnosine
(β-alanyl-histidine) and carnosine-like (homocarnosine and anserine)
peptidase activity (20,
21). This study thus reveals
that the metallopeptidase M20A family represents a novel Cys-Gly peptidase
family, since only members of the M19, M1, and M17 family were known to carry
this function. 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
14.
Eri Takeuchi Yamato Sato Eriko Miura Hiroshi Yamaura Michisuke Yuzaki Dai Yanagihara 《PloS one》2012,7(10)
The cerebellum plays a fundamental, but as yet poorly understood, role in the control of locomotion. Recently, mice with gene mutations or knockouts have been used to investigate various aspects of cerebellar function with regard to locomotion. Although many of the mutant mice exhibit severe gait ataxia, kinematic analyses of limb movements have been performed in only a few cases. Here, we investigated locomotion in ho15J mice that have a mutation of the δ2 glutamate receptor. The cerebellum of ho15J mice shows a severe reduction in the number of parallel fiber-Purkinje synapses compared with wild-type mice. Analysis of hindlimb kinematics during treadmill locomotion showed abnormal hindlimb movements characterized by excessive toe elevation during the swing phase, and by severe hyperflexion of the ankles in ho15J mice. The great trochanter heights in ho15J mice were lower than in wild-type mice throughout the step cycle. However, there were no significant differences in various temporal parameters between ho15J and wild-type mice. We suggest that dysfunction of the cerebellar neuronal circuits underlies the observed characteristic kinematic abnormality of hindlimb movements during locomotion of ho15J mice. 相似文献
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20.
Kulandaivelu S. Vetrivel Xavier Meckler Ying Chen Phuong D. Nguyen Nabil G. Seidah Robert Vassar Philip C. Wong Masaki Fukata Maria Z. Kounnas Gopal Thinakaran 《The Journal of biological chemistry》2009,284(6):3793-3803
Alzheimer disease β-amyloid (Aβ) peptides are generated via
sequential proteolysis of amyloid precursor protein (APP) by BACE1 and
γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane
microdomains, termed lipid rafts. BACE1 processing in raft microdomains of
cultured cells and neurons was characterized in previous studies by disrupting
the integrity of lipid rafts by cholesterol depletion. These studies found
either inhibition or elevation of Aβ production depending on the extent
of cholesterol depletion, generating controversy. The intricate interplay
between cholesterol levels, APP trafficking, and BACE1 processing is not
clearly understood because cholesterol depletion has pleiotropic effects on
Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this
study, we used an alternate strategy to explore the function of BACE1 in
membrane microdomains without altering the cellular cholesterol level. We
demonstrate that BACE1 undergoes S-palmitoylation at four Cys
residues at the junction of transmembrane and cytosolic domains, and Ala
substitution at these four residues is sufficient to displace BACE1 from lipid
rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null
fibroblasts and neuroblastoma cells revealed that S-palmitoylation
neither contributes to protein stability nor subcellular localization of
BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1
did not have discernible influence on BACE1 processing of APP or secretion of
Aβ. These results indicate that post-translational
S-palmitoylation of BACE1 is not required for APP processing, and
that BACE1 can efficiently cleave APP in both raft and non-raft
microdomains.Alzheimer disease-associated β-amyloid
(Aβ)3 peptides
are derived from the sequential proteolysis of β-amyloid precursor
protein (APP) by β- and γ-secretases. The major β-secretase is
an aspartyl protease, termed BACE1 (β-site
APP-cleaving enzyme 1)
(1–4).
BACE1 cleaves APP within the extracellular domain of APP, generating the N
terminus of Aβ. In addition, BACE1 also cleaves to a lesser extent within
the Aβ domain between Tyr10 and Glu11
(β′-cleavage site). Processing of APP at these sites results in the
shedding/secretion of the large ectodomain (sAPPβ) and generating
membrane-tethered C-terminal fragments +1 and +11 (β-CTF)
(5). The multimeric
γ-secretase cleaves at multiple sites within the transmembrane domain of
β-CTF, generating C-terminal heterogeneous Aβ peptides (ranging in
length between 38 and 43 residues) that are secreted, as well as cytosolic APP
intracellular domains (6). In
addition to BACE1, APP can be cleaved by α-secretase within the Aβ
domain between Lys16 and Leu17, releasing sAPPα
and generating α-CTF. γ-Secretase cleavage of α-CTF
generates N-terminal truncated Aβ, termed p3.Genetic ablation of BACE1 completely abolishes Aβ production,
establishing BACE1 as the major neuronal enzyme responsible for initiating
amyloidogenic processing of APP
(4,
7). Interestingly, both the
expression and activity of BACE1 is specifically elevated in neurons adjacent
to senile plaques in brains of individuals with Alzheimer disease
(8). In the past few years
additional substrates of BACE1 have been identified that include APP
homologues APLP1 and APLP2 (9),
P-selectin glycoprotein ligand-1
(10), β-galactoside
α2,6-sialyltransferase
(11), low-density lipoprotein
receptor-related protein (12),
β-subunits of voltage-gated sodium channels
(13), and neuregulin-1
(14,
15), thus extending the
physiological function of BACE1 beyond Alzheimer disease pathogenesis.BACE1 is a type I transmembrane protein with a long extracellular domain
harboring a catalytic domain and a short cytoplasmic tail. BACE1 is
synthesized as a proenzyme, which undergoes post-translational modifications
that include removal of a pro-domain by a furin-like protease,
N-glycosylation, phosphorylation, S-palmitoylation, and
acetylation, during the transit in the secretory pathway
(16–20).
In non-neuronal cells the majority of BACE1 localizes to late Golgi/TGN and
endosomes at steady-state and a fraction of BACE1 also cycles between the cell
surface and endosomes (21).
The steady-state localization of BACE1 is consistent with the acidic pH
optimum of BACE1 in vitro, and BACE1 cleavage of APP is observed in
the Golgi apparatus, TGN, and endosomes
(22–25).
BACE1 endocytosis and recycling are mediated by the GGA family of adaptors
binding to a dileucine motif (496DISLL) in its cytoplasmic tail
(21,
26–31).
Phosphorylation at Ser498 within this motif modulates GGA-dependent
retrograde transport of BACE1 from endosomes to TGN
(21,
26–31).Over the years, a functional relationship between cellular cholesterol
level and Aβ production has been uncovered, raising the intriguing
possibility that cholesterol levels may determine the balance between
amyloidogenic and non-amyloidogenic processing of APP
(32–34).
Furthermore, several lines of evidence from in vitro and in
vivo studies indicate that cholesterol- and sphingolipid-rich membrane
microdomains, termed lipid rafts, might be the critical link between
cholesterol levels and amyloidogenic processing of APP. Lipid rafts function
in the trafficking of proteins in the secretory and endocytic pathways in
epithelial cells and neurons, and participate in a number of important
biological functions (35).
BACE1 undergoes S-palmitoylation
(19), a reversible
post-translational modification responsible for targeting a variety of
peripheral and integral membrane proteins to lipid rafts
(36). Indeed, a significant
fraction of BACE1 is localized in lipid raft microdomains in a
cholesterol-dependent manner, and addition of glycosylphosphatidylinositol
(GPI) anchor to target BACE1 exclusively to lipid rafts increases APP
processing at the β-cleavage site
(37,
38). Antibody-mediated
co-patching of cell surface APP and BACE1 has provided further evidence for
BACE1 processing of APP in raft microdomains
(33,
39). Components of the
γ-secretase complex also associate with detergent-resistant membrane
(DRM) fractions enriched in raft markers such as caveolin, flotillin, PrP, and
ganglioside GM1 (40). The
above findings suggest a model whereby APP is sequentially processed by BACE1
and γ-secretase in lipid rafts.Despite the accumulating evidence, cleavage of APP by BACE1 in non-raft
membrane regions cannot be unambiguously ruled out because of the paucity of
full-length APP (APP FL) and BACE1 in DRM isolated from adult brain and
cultured cells (41). Moreover,
it was recently reported that moderate reduction of cholesterol (<25%)
displaces BACE1 from raft domains, and increases BACE1 processing by promoting
the membrane proximity of BACE1 and APP in non-raft domains
(34). Nevertheless, this study
also found that BACE1 processing of APP is inhibited with further loss of
cholesterol (>35%), consistent with earlier studies
(32,
33). Nevertheless, given the
pleiotropic effects of cholesterol depletion on membrane properties and
vesicular trafficking of secretory and endocytic proteins
(42–47),
unequivocal conclusions regarding BACE1 processing of APP in lipid rafts
cannot be reached based on cholesterol depletion studies.In this study, we explored the function of BACE1 in lipid raft microdomains
without manipulating cellular cholesterol levels. In addition to the
previously reported S-palmitoylation sites
(Cys478/Cys482/Cys485) within the cytosolic
tail of BACE1 (19), we have
identified a fourth site (Cys474) within the transmembrane domain
of BACE1 that undergoes S-palmitoylation. A BACE1 mutant with Ala
substitution of all four Cys residues (BACE1-4C/A) fails to associate with DRM
in cultured cells, but is not otherwise different from wtBACE1 in terms of
protein stability, maturation, or subcellular localization. Surprisingly, APP
processing and Aβ generation were unaffected in cells stably expressing
the BACE1-4C/A mutant. Finally, we observed an increase in the levels of APP
CTFs in detergent-soluble fractions of BACE1-4C/A as compared with wtBACE1
cells. Thus, our data collectively indicate a non-obligatory role of
S-palmitoylation and lipid raft localization of BACE1 in
amyloidogenic processing of APP. 相似文献