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1.
Warren Sun Dea Shahinas Julie Bonvin Wenjuan Hou Matthew S. Kimber Joanne Turnbull Dinesh Christendat 《The Journal of biological chemistry》2009,284(19):13223-13232
TyrA proteins belong to a family of dehydrogenases that are dedicated to
l-tyrosine biosynthesis. The three TyrA subclasses are
distinguished by their substrate specificities, namely the prephenate
dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl
dehydrogenases, which utilize prephenate, l-arogenate, or both
substrates, respectively. The molecular mechanism responsible for TyrA
substrate selectivity and regulation is unknown. To further our understanding
of TyrA-catalyzed reactions, we have determined the crystal structures of
Aquifex aeolicus prephenate dehydrogenase bound with NAD+
plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or
l-tyrosine and have used these structures as guides to target
active site residues for site-directed mutagenesis. From a combination of
mutational and structural analyses, we have demonstrated that His-147 and
Arg-250 are key catalytic and binding groups, respectively, and Ser-126
participates in both catalysis and substrate binding through the ligand
4-hydroxyl group. The crystal structure revealed that tyrosine, a known
inhibitor, binds directly to the active site of the enzyme and not to an
allosteric site. The most interesting finding though, is that mutating His-217
relieved the inhibitory effect of tyrosine on A. aeolicus prephenate
dehydrogenase. The identification of a tyrosine-insensitive mutant provides a
novel avenue for designing an unregulated enzyme for application in metabolic
engineering.Tyrosine serves as a precursor for the synthesis of proteins and secondary
metabolites such as quinones
(1-3),
alkaloids (4), flavonoids
(5), and phenolic compounds
(5,
6). In prokaryotes and plants,
these compounds are important for viability and normal development
(7).The TyrA protein family consists of dehydrogenase homologues that are
dedicated to the biosynthesis of l-tyrosine. These enzymes
participate in two independent metabolic branches that result in the
conversion of prephenate to l-tyrosine, namely the arogenate route
and the 4-hydroxyphenylpyruvate
(HPP)3 routes.
Although both of these pathways utilize a common precursor and converge to
produce a common end-product, they differ in the sequential order of enzymatic
steps. Through the HPP route, prephenate is first decarboxylated by prephenate
dehydrogenase (PD) to yield HPP, which is subsequently transaminated to
l-tyrosine via a TyrB homologue
(8). Alternatively, through the
arogenate route, prephenate is first transaminated to l-arogenate
by prephenate aminotransferase and then decarboxylated by arogenate
dehydrogenase (AD) to yield l-tyrosine
(9-11)
(see Fig. 1A).Open in a separate windowFIGURE 1.A, metabolic routes from chorismate leading to the synthesis of
l-tyrosine and l-phenylalanine. In the arogenate,
4-hydroxyphenylpyruvate, or phenylpyruvate route, prephenate and arogenate are
branch point intermediates in both l-tyrosine and
l-phenylalanine biosynthesis. Prephenate dehydrogenase catalyzes
the oxidative decarboxylation of prephenate with NAD+ to produce
hydroxyphenylpyruvate, NADH, and CO2
(40). B, a comparison
of the chemical structure of the three ligands, HPP, HPpropionate, and
tyrosine, used in the crystallization of A. aeolicus prephenate
dehydrogenase. These ligands all have an -OH at the C4 position and a
propionyl side chain at the C1 position of the ring.There are three classes of TyrA enzymes that catalyze the oxidative
decarboxylation reactions in these two pathways. The enzymes are distinguished
by the affinity for cyclohexadienyl substrates. PD and AD accept prephenate or
l-arogenate, respectively, whereas the cyclohexadienyl
dehydrogenases can catalyze the reaction using either substrate
(12).To ensure efficient metabolite distribution of the pathway intermediates,
TyrA enzymes are highly regulated by various control mechanisms, including
feedback inhibition, and genetic regulation by the Tyr operon
(13-16).
In some cases, l-tyrosine competes directly with substrate, be it
prephenate or l-arogenate for the active site of arogenate or
cyclohexadienyl dehydrogenases
(14,
17-19).
The product HPP can also serve as an efficient competitive inhibitor with
respect to prephenate (20).
Additionally, at the protein level PDs are only shown to be regulated at
distinct allosteric sites or domains to modulate their activity. For example,
the results of kinetic studies on the bifunctional Escherichia coli
chorismate mutase-prephenate dehydrogenase (CM-PD) have indicated that this
enzyme likely possesses a distinct allosteric site for binding tyrosine
(21). In contrast, the
Bacillus subtilis PD is the only enzyme reported to be competitively
inhibited by HPP and l-tyrosine but is also noncompetitively
inhibited by l-phenylalanine and l-tryptophan
(12,
22). Additional regulatory
control is thought to originate through a C-terminal aspartate kinase-CM-TyrA
domain of the B. subtilis PD
(23).Biochemical analyses of PD from E. coli CM-PD have provided a
framework for understanding the molecular mechanism of the TyrA enzymes. The
E. coli PD-catalyzed reaction proceeds though a rapid equilibrium,
random kinetic mechanism with catalysis as the rate-limiting step
(24). Additionally, studies of
the pH dependence of the kinetic parameters V and
V/K indicate that a deprotonated group facilitates hydride
transfer from prephenate to NAD+ by polarizing the 4-hydroxyl group
of prephenate, whereas a protonated residue is required for binding prephenate
to the enzyme·NAD+ complex
(25). The conserved residues
His-197 and Arg-294 have been identified through extensive mutagenesis studies
to fulfill these two roles
(26,
27). Further analyses of the
activities of wild-type protein and site-directed variants in the presence of
a series of inhibitory substrate analogues support the idea that Arg-294 binds
prephenate through the ring carboxylate
(26).The structures of AD from Synechocystis sp. and PD from
Aquifex aeolicus (both in complex with NAD+) have been
reported by Legrand et al.
(28) and by our group
(29), respectively. Analyses
of these structures have provided structural information on the conserved
histidine and arginine residues. The structure A. aeolicus PD has
also led to the identification of other active site residues that may play a
role in enzyme catalysis, most notably Ser-126, which we propose facilitates
catalysis by orienting the catalytic histidine and the nicotinamide moiety of
NAD+ into their catalytically efficient conformations. Ambiguities
can arise from examination of the binary complexes, because prephenate has
only been modeled in the active site. For example, analysis of the AD
structure by Legrand et al.
(28) places Arg-217
(equivalent to Arg-294 in E. coli and Arg-250 in A.
aeolicus) too far from the active site to play a role in prephenate
binding. Thus, the full complement of interactions between prephenate and TyrA
proteins are still largely unknown, as are the interactions of the enzymes
with l-tyrosine.To further investigate the importance of residues involved in ligand
binding, specificity, and catalysis, we have carried out co-crystallization
studies of A. aeolicus PD with NAD+ and prephenate, with
NAD+ and 4-hydroxyphenylpropionate (HPpropionate), a product
analogue, and with NAD+ and l-tyrosine. Accordingly,
this study provides the first direct evidence that l-tyrosine binds
to the active site of a prephenate dehydrogenase. We have investigated the
role of Ser-126, His-147, His-217, and Arg-250 through the kinetic analysis of
site-directed mutants and structural analysis of the co-crystal complexes. To
understand the role of active site residues in substrate selectivity,
comparative structural analysis of AD and PD was also conducted. The current
study provides a basis for understanding the mechanism of substrate
selectivity between the different classes of TyrA enzymes and details how
A. aeolicus PD can accept prephenate as substrate and
l-tyrosine as a competitive inhibitor. 相似文献
2.
Gunthard Stübs Volker Fingerle Bettina Wilske Ulf B. G?bel Ulrich Z?hringer Ralf R. Schumann Nicolas W. J. Schr?der 《The Journal of biological chemistry》2009,284(20):13326-13334
Borrelia burgdorferi sensu lato is the causative agent of Lyme
disease (LD), an infectious disease occurring in North America, Europe, and
Asia in different clinical stages. B. burgdorferi sensu lato
encompasses at least 12 species, with B. burgdorferi sensu stricto,
B. garinii, and B. afzelii being of highest clinical
importance. Immunologic testing for LD as well as recent vaccination
strategies exclusively refer to proteinaceous antigens. However, B.
burgdorferi sensu stricto exhibits glycolipid antigens, including
6-O-acylated cholesteryl β-d-galactopyranoside
(ACGal), and first the data indicated that this compound may act as an
immunogen. Here we investigated whether B. garinii and B.
afzelii also possess this antigen, and whether antibodies directed
against these compounds are abundant among patients suffering from different
stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy
showed that both B. garinii and B. afzelii exhibit ACGal in
high quantities. In contrast, B. hermsii causing relapsing fever
features 6-O-acylated cholesteryl β-d-glucopyranoside
(ACGlc). Sera derived from patients diagnosed for LD contained antibodies
against ACGal, with 80% of patients suffering from late stage disease
exhibiting this feature. Antibodies reacted with ACGal from all three B.
burgdorferi species tested, but not with ACGlc from B. hermsii.
These data show that ACGal is present in all clinically important B.
burgdorferi species, and that specific antibodies against this compound
are frequently found during LD. ACGal may thus be an interesting tool for
improving diagnostics as well as for novel vaccination strategies.Lyme disease (LD)2
is caused by B. burgdorferi sensu lato (s.l.) and is transmitted by
ticks of the genus Ixodes
(1,
2). It is the most common
tick-borne disease in the U.S. with an incidence of 6 per 100,000, with
endemic areas such as Connecticut reaching 111 cases per 100,000
(3). LD is also frequent in
Asia and Europe, particularly in Germany, Austria, Slovenia, and Sweden
(2,
4). B. burgdorferi
s.l. comprises at least 12 species with B. burgdorferi sensu stricto
(Bbu), B. garinii (Bga), and B. afzelii (Baf) being of
highest clinical importance
(2). In the U.S., LD is
exclusively caused by Bbu, whereas in Europe all human pathogenic species are
found, with Bga and Baf being predominant
(2,
5,
6). LD is an infectious disease
occurring in different clinical stages: early localized infection is indicated
by erythema migrans (EM) in ∼70–90% of the patients
(7–9),
and early disseminated infection often causes neurological manifestations,
such as facial palsy, meningitis, meningoradiculitits, or meningoencephalitis
(early neuroborreliosis (NB))
(2,8,9).
The cardinal manifestation of late stage LD in the U.S. is Lyme arthritis
(LA), with ∼70% of the untreated EM cases developing this syndrome
(10,
11). In Europe, next to
arthritis, acrodermatitis chronica atrophicans (ACA) is a frequent late
manifestation, and has been associated with Baf
(11).Currently, diagnosis of LD is generally based on assessment of clinical
features in combination with immunologic serum testing, where both ELISA and a
confirming immunoblot are required
(12,
13). However, because in
Europe and Asia at least three species are causing LD, there is a substantial
variation of immunodominant antigens, which requires the combination of
various homologous antigens for effective serodiagnosis
(14–16).
Immunologic evaluation in these areas is therefore complicated, and no
consensus has been established yet
(12). In comparison to
diagnostic procedures, vaccination strategies directed against LD so far have
also been based on proteinaceous antigens: in the 1990s, recombinant vaccines
based on OspA were found to be effective
(17), but the production was
discontinued, one reason being the high production costs in comparison to
early treatment (2). Another
concern raised against this approach was a potential triggering of autoimmune
diseases by vaccination with Osps due to a similarity between an
immunodominant epitope in OspA and human leukocyte function-associated
antigen-1 (18).In contrast to proteins, information on membrane glycolipids in
Borrelia available today is rather scarce. In 1978, a preliminary
compositional analysis of lipid extracts of B. hermsii causing
relapsing fever (RF) indicated the presence of monoglucosyldiacylglycerol and
acylated as well as non-acylated cholesteryl glucosides
(19). Later, studies on Bbu
indicated the presence of complex glycolipids as well, but no chemical
analysis was performed (20,
21). A more recent study
identified mono-α-d-galactosyldiacylglycerol (MGalD) in Bbu,
and first data indicated that antibodies present in sera obtained from LD
patients detected this antigen
(22). We and others were
recently able to show that Bbu furthermore exhibits cholesteryl
6-O-acyl-β-d-galactopyranoside (ACGal) as well as its
non-acylated counterpart, cholesteryl β-d-galactopyranoside
(βCGal) (23,
24). Patient sera reacted with
ACGal more frequently as compared with MGalD
(23), and antibodies could be
raised in mice by intraperitoneal injection
(24), indicating that this
compound is a strong immunogen.The aim of this study was to elucidate whether ACGal is a common structure
present in the most relevant B. burgdorferi species of clinical
importance, and whether it is a specific feature of Borrelia causing
LD. Furthermore, we aimed at defining the frequency of the occurrence of
antibodies against this antigen in patients suffering from LD. To this end, we
performed a comparative structural analysis of glycolipid fractions of Bbu as
well as the two other B. burgdorferi s.l. species of clinical
importance, Baf and Bga, in comparison with B. hermsii (Bhe), the
causative agent of relapsing fever. We found ACGal to be present in all B.
burgdorferi species tested, whereas Bhe exhibited cholesteryl
6-O-acyl-β-d-glucopyranoside (ACGlc) instead.
Antibodies against ACGal could be detected in the majority of patients
diagnosed for arthritis or acrodermatitis, and these failed to cross-react
with ACGlc. These data demonstrate that ACGal is an abundant, but still highly
specific antigen in B. burgdorferi and thus a promising candidate for
vaccine development and improvement of serologic methods. 相似文献
3.
J. Shawn Goodwin Gaynor A. Larson Jarod Swant Namita Sen Jonathan A. Javitch Nancy R. Zahniser Louis J. De Felice Habibeh Khoshbouei 《The Journal of biological chemistry》2009,284(5):2978-2989
The psychostimulants d-amphetamine (AMPH) and methamphetamine
(METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic
neurons. Abnormal DA release is thought to occur by reverse transport through
the DA transporter (DAT), and it is believed to underlie the severe behavioral
effects of these drugs. Here we compare structurally similar AMPH and METH on
DAT function in a heterologous expression system and in an animal model. In
the in vitro expression system, DAT-mediated whole-cell currents were
greater for METH stimulation than for AMPH. At the same voltage and
concentration, METH released five times more DA than AMPH and did so at
physiological membrane potentials. At maximally effective concentrations, METH
released twice as much [Ca2+]i from internal
stores compared with AMPH. [Ca2+]i responses to
both drugs were independent of membrane voltage but inhibited by DAT
antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were
required for the AMPH- and METH-induced increase in
[Ca2+]i and for the enhanced effects of METH on
[Ca2+]i elevation. Calmodulin-dependent protein
kinase II and protein kinase C inhibitors alone or in combination also blocked
AMPH- or METH-induced Ca2+ responses. Finally, in the rat nucleus
accumbens, in vivo voltammetry showed that systemic application of
METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting
in excess external DA. Together these data demonstrate that METH has a
stronger effect on DAT-mediated cell physiology than AMPH, which may
contribute to the euphoric and addictive properties of METH compared with
AMPH.The dopamine transporter
(DAT)3 is a main
target for psychostimulants, such as d-amphetamine (AMPH),
methamphetamine (METH), cocaine (COC), and methylphenidate (Ritalin®). DAT
is the major clearance mechanism for synaptic dopamine (DA)
(1) and thereby regulates the
strength and duration of dopaminergic signaling. AMPH and METH are substrates
for DAT and competitively inhibit DA uptake
(2,
3) and release DA through
reverse transport
(4–9).
AMPH- and METH-induced elevations in extracellular DA result in complex
neurochemical changes and profound psychiatric effects
(2,
10–16).
Despite their structural and pharmacokinetic similarities, a recent National
Institute on Drug Abuse report describes METH as a more potent stimulant than
AMPH with longer lasting effects at comparable doses
(17). Although the route of
METH administration and its availability must contribute to the almost four
times higher lifetime nonmedical use of METH compared with AMPH
(18), there may also be
differences in the mechanisms that underlie the actions of these two drugs on
the dopamine transporter.Recent studies by Joyce et al.
(19) have shown that compared
with d-AMPH alone, the combination of d- and
l-AMPH in Adderall® significantly prolonged the time course of
extracellular DA in vivo. These experiments demonstrate that subtle
structural features of AMPH, such as chirality, can affect its action on
dopamine transporters. Here we investigate whether METH, a more lipophilic
analog of AMPH, affects DAT differently than AMPH, particularly in regard to
stimulated DA efflux.METH and AMPH have been reported as equally effective in increasing
extracellular DA levels in rodent dorsal striatum (dSTR), nucleus accumbens
(NAc) (10,
14,
20), striatal synaptosomes,
and DAT-expressing cells in vitro
(3,
6). John and Jones
(21), however, have recently
shown in mouse striatal and substantia nigra slices, that AMPH is a more
potent inhibitor of DA uptake than METH. On the other hand, in synaptosomes
METH inhibits DA uptake three times more effectively than AMPH
(14), and in DAT-expressing
COS-7 cells, METH releases DA more potently than AMPH (EC50 = 0.2
μm for METH versus EC50 = 1.7
μm for AMPH) (5).
However, these differences do not hold up under all conditions. For example,
in a study utilizing C6 cells, the disparity between AMPH and METH was not
found (12).The variations in AMPH and METH data extend to animal models. AMPH- and
METH-mediated behavior has been reported as similar
(22), lower
(20), or higher
(23) for AMPH compared with
METH. Furthermore, although the maximal locomotor activation response was less
for METH than for AMPH at a lower dose (2 mg/kg, intraperitoneal), both drugs
decreased locomotor activity at a higher dose (4 mg/kg)
(20). In contrast, in the
presence of a salient stimuli, METH is more potent in increasing the overall
magnitude of locomotor activity in rats yet is equipotent with AMPH in the
absence of these stimuli
(23).The simultaneous regulation of DA uptake and efflux by DAT substrates such
as AMPH and METH, as well as the voltage dependence of DAT
(24), may confound the
interpretation of existing data describing the action of these drugs. Our
biophysical approaches allowed us to significantly decrease the contribution
of DA uptake and more accurately determine DAT-mediated DA efflux with
millisecond time resolution. We have thus exploited time-resolved, whole-cell
voltage clamp in combination with in vitro and in vivo
microamperometry and Ca2+ imaging to compare the impact of METH and
AMPH on DAT function and determine the consequence of these interactions on
cell physiology.We find that near the resting potential, METH is more effective than AMPH
in stimulating DAT to release DA. In addition, at efficacious concentrations
METH generates more current, greater DA efflux, and higher Ca2+
release from internal stores than AMPH. Both METH-induced or the lesser
AMPH-induced increase in intracellular Ca2+ are independent of
membrane potential. The additional Ca2+ response induced by METH
requires intact phosphorylation sites in the N-terminal domain of DAT.
Finally, our in vivo voltammetry data indicate that METH inhibits
clearance of locally applied DA more effectively than AMPH in the rat nucleus
accumbens, which plays an important role in reward and addiction, but not in
the dorsal striatum, which is involved in a variety of cognitive functions.
Taken together these data imply that AMPH and METH have distinguishable
effects on DAT that can be shown both at the molecular level and in
vivo, and are likely to be implicated in the relative euphoric and
addictive properties of these two psychostimulants. 相似文献
4.
Quang-Kim Tran Jared Leonard D. J. Black Owen W. Nadeau Igor G. Boulatnikov Anthony Persechini 《The Journal of biological chemistry》2009,284(18):11892-11899
We have investigated the possible biochemical basis for enhancements in NO
production in endothelial cells that have been correlated with agonist- or
shear stress-evoked phosphorylation at Ser-1179. We have found that a
phosphomimetic substitution at Ser-1179 doubles maximal synthase activity,
partially disinhibits cytochrome c reductase activity, and lowers the
EC50(Ca2+) values for calmodulin binding and enzyme
activation from the control values of 182 ± 2 and 422 ± 22
nm to 116 ± 2 and 300 ± 10 nm. These are
similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q.
K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry
47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179
has no additional effect on maximal synthase activity, cooperativity between
the two substitutions completely disinhibits reductase activity and further
reduces the EC50(Ca2+) values for calmodulin binding and
enzyme activation to 77 ± 2 and 130 ± 5 nm. We have
confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179
and phosphomimetic substitutions at these positions have similar functional
effects. Changes in the biochemical properties of eNOS produced by combined
phosphorylation at Ser-617 and Ser-1179 are predicted to substantially
increase synthase activity in cells at a typical basal free Ca2+
concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and
l-citrulline from l-arginine and O2, with
NADPH as the electron donor
(1). The role of NO generated
by endothelial nitricoxide synthase
(eNOS)2 in the
regulation of smooth muscle tone is well established and was the first of
several physiological roles for this small molecule that have so far been
identified (2). The
nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each
subunit contains a reductase and oxygenase domain
(1). A significant difference
between the reductase domains in eNOS and nNOS and the homologous P450
reductases is the presence of inserts in these synthase isoforms that appear
to maintain them in their inactive states
(3,
4). A calmodulin (CaM)-binding
domain is located in the linker that connects the reductase and oxygenase
domains, and the endothelial and neuronal synthases both require
Ca2+ and exogenous CaM for activity
(5,
6). When CaM is bound, it
somehow counteracts the effects of the autoinhibitory insert(s) in the
reductase. The high resolution structure for the complex between
(Ca2+)4-CaM and the isolated CaM-binding domain from
eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a
pair of Ca2+-binding sites, enfold the domain, as has been observed
in several other such CaM-peptide complexes
(7). Consistent with this
structure, investigations of CaM-dependent activation of the neuronal synthase
suggest that both CaM lobes must participate
(8,
9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497,
Ser-617, Ser-635, and Ser-1179
(10–12).
There are equivalent phosphorylation sites in the human enzyme
(10–12).
Phosphorylation of the bovine enzyme at Thr-497, which is located in the
CaM-binding domain, blocks CaM binding and enzyme activation
(7,
11,
13,
14). Ser-116 can be basally
phosphorylated in cells (10,
11,
13,
15), and dephosphorylation of
this site has been correlated with increased NO production
(13,
15). However, it has also been
reported that a phosphomimetic substitution at this position has no effect on
enzyme activity measured in vitro
(13). Ser-1179 is
phosphorylated in response to a variety of stimuli, and this has been reliably
correlated with enhanced NO production in cells
(10,
11). Indeed, NO production is
elevated in transgenic endothelium expressing an eNOS mutant containing an
S1179D substitution, but not in tissue expressing an S1179A mutant
(16). Shear stress or insulin
treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in
endothelial cells, and this is correlated with increased NO production in the
absence of extracellular Ca2+
(17–19).
Akt-catalyzed phosphorylation or an S1179D substitution has also been
correlated with increased synthase activity in cell extracts at low
intracellular free [Ca2+]
(17). Increased NO production
has also been observed in cells expressing an eNOS mutant containing an S617D
substitution, and physiological stimuli such as shear-stress, bradykinin,
VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and
Ser-1179 (12,
13,
20). Although S617D eNOS has
been reported to have the same maximum activity in vitro as the wild
type enzyme (20), in our hands
an S617D substitution increases the maximal CaM-dependent synthase activity of
purified mutant enzyme ∼2-fold, partially disinhibits reductase activity,
and reduces the EC50(Ca2+) values for CaM binding and
enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp
substitution at Ser-1179 in eNOS on the Ca2+ dependence of CaM
binding and CaM-dependent activation of reductase and synthase activities. We
also describe the effects on these properties of combining this substitution
with one at Ser-617. Finally, we demonstrate that Akt-catalyzed
phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar
functional effects. Our results suggest that phosphorylation of eNOS at
Ser-617 and Ser-1179 can substantially increase synthase activity in cells at
a typical basal free Ca2+ concentration of 50–100
nm, while single phosphorylations at these sites produce smaller
activity increases, and can do so only at higher free Ca2+
concentrations. 相似文献
5.
6.
7.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
8.
Karla Frydenvang L. Leanne Lash Peter Naur Pekka A. Postila Darryl S. Pickering Caleb M. Smith Michael Gajhede Makoto Sasaki Ryuichi Sakai Olli T. Pentika?nen Geoffrey T. Swanson Jette S. Kastrup 《The Journal of biological chemistry》2009,284(21):14219-14229
The prevailing structural model for ligand activation of ionotropic
glutamate receptors posits that agonist efficacy arises from the stability and
magnitude of induced domain closure in the ligand-binding core structure. Here
we describe an exception to the correlation between ligand efficacy and domain
closure. A weakly efficacious partial agonist of very low potency for
homomeric iGluR5 kainate receptors, 8,9-dideoxyneodysiherbaine (MSVIII-19),
induced a fully closed iGluR5 ligand-binding core. The degree of relative
domain closure, ∼30°, was similar to that we resolved with the
structurally related high affinity agonist dysiherbaine and to that of
l-glutamate. The pharmacological activity of MSVIII-19 was
confirmed in patch clamp recordings from transfected HEK293 cells, where
MSVIII-19 predominantly inhibits iGluR5-2a, with little activation apparent at
a high concentration (1 mm) of MSVIII-19 (<1% of mean
glutamate-evoked currents). To determine the efficacy of the ligand
quantitatively, we constructed concentration-response relationships for
MSVIII-19 following potentiation of steady-state currents with concanavalin A
(EC50 = 3.6 μm) and on the nondesensitizing receptor
mutant iGluR5-2b(Y506C/L768C) (EC50 = 8.1 μm).
MSVIII-19 exhibited a maximum of 16% of full agonist efficacy, as measured in
parallel recordings with glutamate. Molecular dynamics simulations and
electrophysiological recordings confirm that the specificity of MSVIII-19 for
iGluR5 is partly attributable to interdomain hydrogen bond residues
Glu441 and Ser721 in the iGluR5-S1S2 structure. The
weaker interactions of MSVIII-19 with iGluR5 compared with dysiherbaine,
together with altered stability of the interdomain interaction, may be
responsible for the apparent uncoupling of domain closure and channel opening
in this kainate receptor subunit.Ionotropic glutamate receptors
(iGluRs)3 are central
to fast excitatory synaptic transmission in the central nervous system and are
involved in numerous physiological and pathophysiological processes. The
iGluRs consist of three different classes of receptors,
N-methyl-d-aspartic acid (NMDA),
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate
receptors (1), which are
assembled as tetramers in a dimer of dimers configuration
(2,
3). These receptors can be
considered as multidomain proteins, composed of an extracellular
N-terminal domain, a ligand-binding core made of discontinuous S1 and
S2 segments that form two lobes (domains D1 and D2), three
transmembrane-spanning regions (M1–M3) with a re-entrant loop between M1
and M2, and finally a cytoplasmic region
(1).Ligand-binding cores of iGluRs assume tertiary structures in solution that
reproduce the pharmacological profiles of full-length receptors.
Crystallographic studies of ligand-binding core complexes from representative
members of all three iGluR subtypes
(4–6)
as well as the ligand-binding core of the structurally related δ2
subunit in complex with d-serine
(7) have yielded unprecedented
insight into structural correlates of iGluR function. Binding of agonists to
iGluR ligand-binding cores can be described as a “Venus flytrap”
mechanism. In the resting state, the ligand-binding core is present in an open
form that is stabilized by antagonists
(4,
8,
9). When an agonist binds to
the ligand-binding core, a rotational change in conformation occurs, resulting
in domain closure of the D1 and D2 lobes around a central hinge region
(4,
6). In full-length receptors,
this domain closure is thought to result in the opening of the ion channel
(receptor activation). The extents of domain closure of ligand-binding cores
of AMPA and kainate receptor subunits are correlated with the activation and
the desensitization of the receptor
(9,
10).However, previous studies have questioned the association between the
degree of domain closure of the ligand-binding core and channel opening or
agonist efficacy. For example, AMPA was shown to induce a more closed
structure of the ligand-binding core of the mutated iGluR2(L650T) than was
expected from its partial agonist efficacy
(11,
12). Also, no correlation
between domain closure and agonist efficacy has been demonstrated for the NR1
subunit of NMDA receptors
(13).In this study, we present the first example of a nonmutated kainate
receptor that lacks the correlation between domain closure and efficacy. We
tested if two structurally related kainate receptor ligands, one an agonist
and one described previously as an antagonist
(14), conformed to the
prevailing structural model of ligand-induced activity. The high affinity
agonist dysiherbaine (DH) is a natural excitotoxin originally isolated from a
marine sponge (15,
16), whereas
8,9-dideoxyneodysiherbaine (MSVIII-19) is a synthetic analog that inhibits
activation of iGluR5 receptors
(14). To investigate receptor
interactions with the two closely related compounds as well as the degree of
domain closure introduced by the compounds, we determined the crystal
structures of DH and MSVIII-19 in complex with the ligand-binding core of the
kainate receptor subunit iGluR5 (iGluR5-S1S2). These two structures, along
with functional studies, provide novel insights into the mechanism of kainate
receptor activation, inhibition, and desensitization. 相似文献
9.
Maika Deffieu Ingrid Bhatia-Ki??ová Bénédicte Salin Anne Galinier Stéphen Manon Nadine Camougrand 《The Journal of biological chemistry》2009,284(22):14828-14837
The antioxidant N-acetyl-l-cysteine prevented the
autophagy-dependent delivery of mitochondria to the vacuoles, as examined by
fluorescence microscopy of mitochondria-targeted green fluorescent protein,
transmission electron microscopy, and Western blot analysis of mitochondrial
proteins. The effect of N-acetyl-l-cysteine was specific
to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation
of alkaline phosphatase and the presence of hallmarks of non-selective
microautophagy were not altered by N-acetyl-l-cysteine.
The effect of N-acetyl-l-cysteine was not related to its
scavenging properties, but rather to its fueling effect of the glutathione
pool. As a matter of fact, the decrease of the glutathione pool induced by
chemical or genetical manipulation did stimulate mitophagy but not general
autophagy. Conversely, the addition of a cell-permeable form of glutathione
inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the
strain Δuth1, which is deficient in selective mitochondrial
degradation. These data show that mitophagy can be regulated independently of
general autophagy, and that its implementation may depend on the cellular
redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of
long-lived proteins and organelles, where they are degraded and recycled.
Autophagy plays a crucial role in differentiation and cellular response to
stress and is conserved in eukaryotic cells from yeast to mammals
(1,
2). The main form of autophagy,
macroautophagy, involves the non-selective sequestration of large portions of
the cytoplasm into double-membrane structures termed autophagosomes, and their
delivery to the vacuole/lysosome for degradation. Another process,
microautophagy, involves the direct sequestration of parts of the cytoplasm by
vacuole/lysosomes. The two processes coexist in yeast cells but their extent
may depend on different factors including metabolic state: for example, we
have observed that nitrogen-starved lactate-grown yeast cells develop
microautophagy, whereas nitrogen-starved glucose-grown cells preferentially
develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in
the way that autophagosomes and vacuole invaginations do not appear to
discriminate the sequestered material. However, selective forms of autophagy
have been observed (4) that
target namely peroxisomes (5,
6), chromatin
(7,
8), endoplasmic reticulum
(9), ribosomes
(10), and mitochondria
(3,
11–13).
Although non-selective autophagy plays an essential role in survival by
nitrogen starvation, by providing amino acids to the cell, selective autophagy
is more likely to have a function in the maintenance of cellular structures,
both under normal conditions as a “housecleaning” process, and
under stress conditions by eliminating altered organelles and macromolecular
structures
(14–16).
Selective autophagy targeting mitochondria, termed mitophagy, may be
particularly relevant to stress conditions. The mitochondrial respiratory
chain is both the main site and target of
ROS4 production
(17). Consequently, the
maintenance of a pool of healthy mitochondria is a crucial challenge for the
cells. The progressive accumulation of altered mitochondria
(18) caused by the loss of
efficiency of the maintenance process (degradation/biogenesis de
novo) is often considered as a major cause of cellular aging
(19–23).
In mammalian cells, autophagic removal of mitochondria has been shown to be
triggered following induction/blockade of apoptosis
(23), suggesting that
autophagy of mitochondria was required for cell survival following
mitochondria injury (14).
Consistent with this idea, a direct alteration of mitochondrial permeability
properties has been shown to induce mitochondrial autophagy
(13,
24,
25). Furthermore, inactivation
of catalase induced the autophagic elimination of altered mitochondria
(26). In the yeast
Saccharomyces cerevisiae, the alteration of
F0F1-ATPase biogenesis in a conditional mutant has been
shown to trigger autophagy
(27). Alterations of
mitochondrial ion homeostasis caused by the inactivation of the
K+/H+ exchanger was shown to cause both autophagy and
mitophagy (28). We have
reported that treatment of cells with rapamycin induced early ROS production
and mitochondrial lipid oxidation that could be inhibited by the hydrophobic
antioxidant resveratrol (29).
Furthermore, resveratrol treatment impaired autophagic degradation of both
cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death,
suggesting that mitochondrial oxidation events may play a crucial role in the
regulation of autophagy. This existence of regulation of autophagy by ROS has
received molecular support in HeLa cells
(30): these authors showed
that starvation stimulated ROS production, namely H2O2,
which was essential for autophagy. Furthermore, they identified the cysteine
protease hsAtg4 as a direct target for oxidation by
H2O2. This provided a possible connection between the
mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown
yeast cells have established the existence of two distinct processes: the
first one occurring very early, is selective for mitochondria and is dependent
on the presence of the mitochondrial protein Uth1p; the second one occurring
later, is not selective for mitochondria, is not dependent on Uth1p, and is a
form of bulk microautophagy
(3). The absence of the
selective process in the Δuth1 mutant strongly delays and
decreases mitochondrial protein degradation
(3,
12). The putative protein
phosphatase Aup1p has been also shown to be essential in inducing mitophagy
(31). Additionally several Atg
proteins were shown to be involved in vacuolar sequestration of mitochondrial
GFP (3,
12,
32,
33). Recently, the protein
Atg11p, which had been already identified as an essential protein for
selective autophagy has also been reported as being essential for mitophagy
(33).The question remains as to identify of the signals that trigger selective
mitophagy. It is particularly intriguing that selective mitophagy is activated
very early after the shift to a nitrogen-deprived medium
(3). Furthermore, selective
mitophagy is very active on lactate-grown cells (with fully differentiated
mitochondria) but is nearly absent in glucose-grown cells
(3). In the present paper, we
investigated the relationships between the redox status of the cells and
selective mitophagy, namely by manipulating glutathione. Our results support
the view that redox imbalance is a trigger for the selective elimination of
mitochondria. 相似文献
10.
11.
Neeliyath A. Ramakrishnan Marian J. Drescher Dennis G. Drescher 《The Journal of biological chemistry》2009,284(3):1364-1372
The molecular mechanisms underlying synaptic exocytosis in the hair cell,
the auditory and vestibular receptor cell, are not well understood. Otoferlin,
a C2 domain-containing Ca2+-binding protein, has been implicated as
having a role in vesicular release. Mutations in the OTOF gene cause
nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In
the present study, we generated otoferlin fusion proteins containing two of
the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F
and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and
SNAP-25 in a Ca2+-dependent manner (with optimal 61
μm free Ca2+ required for binding). These
interactions were greatly diminished for C2F with the P1825A mutation,
possibly because of a reduction in tertiary structural change, induced by
Ca2+, for the mutated C2F compared with the native C2F. The
otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity
(Kd = 1.7 × 10–5 m) than
for the C2F interaction (Kd = 2.6 ×
10–9 m). In contrast, it was the otoferlin C2D
domain that bound the Cav1.3 II-III loop, in a
Ca2+-dependent manner. The L1011P mutation in C2D rendered this
binding insensitive to Ca2+ and considerably diminished. Overall,
we demonstrated that otoferlin interacts with two main target-SNARE proteins
of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the
calcium channel, with the otoferlin C2F and C2D domains of central importance
for binding. Because mutations in the otoferlin C2 domains that cause deafness
in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the
Cav1.3 calcium channel, it is these interactions that may mediate
regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear
hair cell function.Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref.
1). In mechanosensory hair
cells, calcium microdomains (2)
and possibly nanodomains (3)
are formed when voltage-gated calcium channels open upon depolarization.
Calcium at these sites is thought to activate protein interactions, leading to
vesicle fusion. Some of the key players in this process are the
target-SNARE2
proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin
(4). Vesicle-SNARE
synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal
synapse, modulating calcium channels and vesicle release by a
Ca2+-dependent interaction with other SNARE proteins in the
presence of lipid molecules
(4–6).
However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not
detected (7). Instead, fast
neurotransmitter release in auditory and vestibular hair cells, facilitated
largely by an L-type voltagegated calcium channel, Cav1.3
(8,
9), is thought to be modulated
by a newly discovered protein, otoferlin, acting as the Ca2+ sensor
and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic
deafness (10). Gene sequences
of different deaf families show that the OTOF gene can undergo
mutation at multiple locations
(11–13).
Recently, it has been demonstrated that otoferlin is necessary for synaptic
exocytosis from hair cells
(14). Further, an engineered
mutation in the C2B domain of otoferlin has been shown to cause deafness in
mice (15). However, the
precise function of otoferlin as a synaptic protein is not well
understood.Specific mutations in the otoferlin C2F (P1825A) or C2D (L1011P) domains in
humans have been documented to cause DFNB9 deafness
(11,
12). Previous studies
suggested that a region of otoferlin containing all three C2 domains, D, E,
and F, binds directly to the t-SNARE molecules syntaxin 1A and SNAP-25 in
response to an increase in Ca2+ concentration
(14). However, it is not
understood how a single amino acid substitution in one domain of otoferlin,
such as C2F (11) or C2D
(12), might independently lead
to deafness. Here, we examine the role of otoferlin as a Ca2+
sensor as well as a facilitator of vesicle fusion, as indicated by
protein-protein interactions and their [Ca2+] dependence. 相似文献
12.
Takafumi Tasaki Adriana Zakrzewska Drew D. Dudgeon Yonghua Jiang John S. Lazo Yong Tae Kwon 《The Journal of biological chemistry》2009,284(3):1884-1895
The N-end rule pathway is a ubiquitin-dependent system where E3 ligases
called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and
type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have
recently reported an E3 family (termed UBR1 through UBR7) characterized by the
70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured
during affinity-based proteomics with synthetic degrons. Here we characterized
substrate binding specificity and recognition domains of UBR proteins.
Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and
300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding
assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations
narrow down the degron-binding activity to a 72-residue UBR box-only fragment
that recognizes type-1 but not type-2 residues. A surface plasmon resonance
assay shows that the UBR box binds to the type-1 substrate Arg-peptide with
Kd of ∼3.4 μm. Downstream from the UBR
box, we identify a second substrate recognition domain, termed the N-domain,
required for type-2 substrate recognition. The ∼80-residue N-domain shows
structural and functional similarity to 106-residue Escherichia coli
ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box
functions as a common structural element essential for binding to all known
destabilizing N-terminal residues, whereas specific residues localized in the
UBR box (for type 1) or the N-domain (for type 2) provide substrate
selectivity through interaction with the side group of an N-terminal amino
acid. Our work provides new insights into substrate recognition in the N-end
rule pathway.The N-end rule pathway is a ubiquitin
(Ub)2-dependent
proteolytic system in which N-terminal residues of short-lived proteins
function as an essential component of degradation signals (degrons) called
N-degrons (Fig. 1A)
(1-15).
An N-degron can be created from a pre-N-degron through specific N-terminal
modifications (12).
Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing
residues that function through their deamidation by N-terminal amidohydrolases
into the secondary destabilizing N-terminal residues Asp and Glu, respectively
(6,
16)
(Fig. 1A). N-terminal
Asp and Glu are secondary destabilizing residues that function through their
arginylation by ATE1 R-transferase, which creates the primary
destabilizing residue Arg at the N terminus
(4,
8)
(Fig. 1A). N-terminal
Cys can also function as a tertiary destabilizing residue through its
oxidation in a manner depending on nitric oxide and oxygen (O2);
the oxidized Cys residue is subsequently arginylated by ATE1
(8,
13,
17).Open in a separate windowFIGURE 1.A, the mammalian N-end rule pathway. N-terminal residues are
indicated by single-letter abbreviations for amino acids. Yellow
ovals denote the rest of a protein substrate. C*
denotes oxidized N-terminal Cys, either Cys-sulfinic acid
[CysO2(H)] or Cys-sulfonic acid [CysO3(H)]. The Cys
oxidation requires nitric oxide and oxygen (O2) or its derivatives.
The oxidized Cys is arginylated by ATE1 Arg-tRNA-protein transferase
(R-transferase). N-recognins also recognize internal (non-N-terminal)
degrons in other substrates of the N-end rule pathway. B, the
X-peptide pull-down assay. Left, a 12-mer peptide bearing N-terminal
Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residue
was cross-linked through its C-terminal Cys residue to Ultralink Iodoacetyl
beads. Right, the otherwise identical 12-mer peptide, bearing
C-terminal biotinylated Lys instead of Cys, was conjugated, via biotin, to the
streptavidin-Sepharose beads. C, the X-peptide pull-down assay of
endogenous UBR proteins using testes extracts. Extracts from mouse testes were
mixed with bead-conjugated X-peptides bearing N-terminal Phe (F), Gly
(G), or Arg (R). After centrifugation, captured proteins
were separated and subjected to anti-UBR immunoblotting. Mo, a
pull-down reaction with mock beads. D, the X-peptide pull-down assays
using rat testis extracts were performed in the presence of varying
concentrations of NaCl. After incubation and washing, bound proteins were
eluted by 10 mm Tyr-Ala for Phe-peptide, 10 mm Arg-Ala
for Arg-peptide, and 5 mm Tyr-Ala and 5 mm Arg-Ala for
Val-peptide. Eluted proteins were subjected to immunoblotting for UBR1 and
UBR5. E, cytoplasmic fractions of wild-type (+/+),
Ubr1-/-, Ubr2-/-,
Ubr1-/-Ubr2-/-, and
Ubr1-/-Ubr2-/-Ubr4RNAi
MEFs were subjected to X-peptide pull-down assay. Precipitated proteins were
separated and analyzed by immunoblotting for UBR1 and UBR4.N-terminal Arg together with other primary destabilizing N-terminal
residues are directly bound by specific E3 Ub ligases called N-recognins
(3,
7,
9). Destabilizing N-terminal
residues can be created through the removal of N-terminal Met or the
endoproteolytic cleavage of a protein, which exposes a new amino acid at the N
terminus (12,
13). N-terminal degradation
signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2
(bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues
(2,
12). In addition to a
destabilizing N-terminal residue, a functional N-degron requires at least one
internal Lys residue (the site of a poly-Ub chain formation) and a
conformational feature required for optimal ubiquitylation
(1,
2,
18). UBR1 and UBR2 are
functionally overlapping N-recognins
(3,
7,
9). Our proteomic approach
using synthetic peptides bearing destabilizing N-terminal residues captured a
set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa
UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR
box
(10-12).
UBR5 is a HECT E3 ligase known as EDD (E3 identified by
differential display)
(19) and a homolog of
Drosophila hyperplastic discs
(20). The mammalian genome
encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7
(10). UBR box proteins are
generally heterogeneous in size and sequence but contain, with the exception
of UBR4, specific signatures unique to E3s or a substrate recognition subunit
of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in
UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7
(Fig. 2B). The
biochemical properties of more recently identified UBR box proteins, such as
UBR3 through UBR7, are largely unknown.Open in a separate windowFIGURE 2.The binding properties of the UBR box family members to type-1 and
type-2 destabilizing N-terminal residues. A, the X-peptide
pull-down assay with overexpressed, full-length UBR proteins: UBR2, UBR3 (in
S. cerevisiae cells), UBR4, UBR5 (in COS7 cells), and UBR6 and UBR7
(in the wheat germ lysates). Precipitates were analyzed by immunoblotting (for
UBR2, UBR3, UBR4, and UBR5) with tag-specific antibodies as indicated in
B or autoradiography (for UBR6 and UBR7). B, the structures
of UBR box proteins. Shown are locations of the UBR box, the N-domain, and
other E3-related domains. UBR, UBR box; RING, RING finger;
UAIN, UBR-specific autoinhibitory domain; CRD, cysteine-rich
domain; PHD, plant homeodomain; HECT, HECT domain.Studies using knock-out mice implicated the N-end rule pathway in cardiac
development and signaling, angiogenesis
(8,
15), meiosis
(9), DNA repair
(21), neurogenesis
(15), pancreatic functions
(22), learning and memory
(23,
24), female development
(9), muscle atrophy
(25), and olfaction
(11). Mutations in human
UBR1 is a cause of Johanson-Blizzard syndrome
(22), an autosomal recessive
disorder with multiple developmental abnormalities
(26). Other functions of the
pathway include: (i) a nitric oxide and oxygen (O2) sensor
controlling the proteolysis of RGS4, RGS5, and RGS16
(8,
13,
17), (ii) a heme sensor
through hemin-dependent inhibition of ATE1 function
(27), (iii) the regulation of
short peptide import through the peptide-modulated degradation of the
repressor of the import (28,
29), (iv) the control of
chromosome segregation through the degradation of a separate produced cohesin
fragment (30), (v) the
regulation of apoptosis through the degradation of a caspase-processed
inhibitor of apoptosis (31,
32), (vi) the control of the
human immunodeficiency virus replication cycle through the degradation of
human immunodeficiency virus integrase
(10,
33), and (vii) the regulation
of leaf senescence in plants
(34).In the present study we characterized substrate binding specificities and
recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4,
and UBR5 were captured by N-terminal degradation determinants, whereas UBR3,
UBR6, and UBR7 were not. We also report that in contrast to other E3 systems
that usually recognize substrates through protein-protein interface, UBR1 and
UBR2 have a general substrate recognition domain termed the UBR box.
Remarkably, a 72-residue UBR box-only fragment fully retains its structural
integrity and thereby the ability to recognize type-1 N-end rule substrates.
We also report that the N-domain, structurally and functionally related with
bacterial N-recognins, is required for recognizing type-2 N-end rule
substrates. We discuss the evolutionary relationship between eukaryotic and
prokaryotic N-recognins. 相似文献
13.
14.
15.
16.
Graham H. Diering John Church Masayuki Numata 《The Journal of biological chemistry》2009,284(20):13892-13903
NHE5 is a brain-enriched Na+/H+ exchanger that
dynamically shuttles between the plasma membrane and recycling endosomes,
serving as a mechanism that acutely controls the local pH environment. In the
current study we show that secretory carrier membrane proteins (SCAMPs), a
group of tetraspanning integral membrane proteins that reside in multiple
secretory and endocytic organelles, bind to NHE5 and co-localize predominantly
in the recycling endosomes. In vitro protein-protein interaction
assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic
extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5
increased cell-surface abundance as well as transporter activity of NHE5
across the plasma membrane. Expression of a deletion mutant lacking the
SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the
N-terminal extension, reduced the transporter activity. Although both Arf6 and
Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across
the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by
dominant-negative Arf6 but not by dominant-negative Rab11. Together, these
results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes
and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are
especially sensitive to perturbations of pH
(1). Many voltage- and
ligand-gated ion channels that control membrane excitability are sensitive to
changes in cellular pH
(1-3).
Neurotransmitter release and uptake are also influenced by cellular and
organellar pH (4,
5). Moreover, the intra- and
extracellular pH of both neurons and glia are modulated in a highly transient
and localized manner by neuronal activity
(6,
7). Thus, neurons and glia
require sophisticated mechanisms to finely tune ion and pH homeostasis to
maintain their normal functions.Na+/H+ exchangers
(NHEs)3 were
originally identified as a class of plasma membrane-bound ion transporters
that exchange extracellular Na+ for intracellular H+,
and thereby regulate cellular pH and volume. Since the discovery of NHE1 as
the first mammalian NHE (8),
eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have
been isolated in mammals (9,
10). NHE1-5 commonly exhibit
transporter activity across the plasma membrane, whereas NHE6-9 are mostly
found in organelle membranes and are believed to regulate organellar pH in
most cell types at steady state
(11). More recently, NHE10 was
identified in human and mouse osteoclasts
(12,
13). However, the cDNA
encoding NHE10 shares only a low degree of sequence similarity with other
known members of the NHE gene family, raising the possibility that
this sodium-proton exchanger may belong to a separate gene family distantly
related to NHE1-9 (see Ref.
9).NHE gene family members contain 12 putative transmembrane domains
at the N terminus followed by a C-terminal cytosolic extension that plays a
role in regulation of the transporter activity by protein-protein interactions
and phosphorylation. NHEs have been shown to regulate the pH environment of
synaptic nerve terminals and to regulate the release of neurotransmitters from
multiple neuronal populations
(14-16).
The importance of NHEs in brain function is further exemplified by the
findings that spontaneous or directed mutations of the ubiquitously expressed
NHE1 gene lead to the progression of epileptic seizures, ataxia, and
increased mortality in mice
(17,
18). The progression of the
disease phenotype is associated with loss of specific neuron populations and
increased neuronal excitability. However, NHE1-null mice appear to
develop normally until 2 weeks after birth when symptoms begin to appear.
Therefore, other mechanisms may compensate for the loss of NHE1
during early development and play a protective role in the surviving neurons
after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene
family whose mRNA is expressed almost exclusively in the brain
(19,
20), although more recent
studies have suggested that NHE5 might be functional in other cell
types such as sperm (21,
22) and osteosarcoma cells
(23). Curiously, mutations
found in several forms of congenital neurological disorders such as
spinocerebellar ataxia type 4
(24-26)
and autosomal dominant cerebellar ataxia
(27-29)
have been mapped to chromosome 16q22.1, a region containing NHE5.
However, much remains unknown as to the molecular regulation of NHE5 and its
role in brain function.Very few if any proteins work in isolation. Therefore identification and
characterization of binding proteins often reveal novel functions and
regulation mechanisms of the protein of interest. To begin to elucidate the
biological role of NHE5, we have started to explore NHE5-binding proteins.
Previously, β-arrestins, multifunctional scaffold proteins that play a
key role in desensitization of G-protein-coupled receptors, were shown to
directly bind to NHE5 and promote its endocytosis
(30). This study demonstrated
that NHE5 trafficking between endosomes and the plasma membrane is regulated
by protein-protein interactions with scaffold proteins. More recently, we
demonstrated that receptor for activated
C-kinase 1 (RACK1), a scaffold protein that links
signaling molecules such as activated protein kinase C, integrins, and Src
kinase (31), directly
interacts with and activates NHE5 via integrin-dependent and independent
pathways (32). These results
further indicate that NHE5 is partly associated with focal adhesions and that
its targeting to the specialized microdomain of the plasma membrane may be
regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily
conserved tetra-spanning integral membrane proteins. SCAMPs are found in
multiple organelles such as the Golgi apparatus, trans-Golgi network,
recycling endosomes, synaptic vesicles, and the plasma membrane
(33,
34) and have been shown to
play a role in exocytosis
(35-38)
and endocytosis (39).
Currently, five isoforms of SCAMP have been identified in mammals. The
extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats,
which may allow these isoforms to participate in clathrin coat assembly and
vesicle budding by binding to Eps15 homology (EH)-domain proteins
(40,
41). Further, SCAMP2 was shown
recently to bind to the small GTPase Arf6
(38), which is believed to
participate in traffic between the recycling endosomes and the cell surface
(42,
43). More recent studies have
suggested that SCAMPs bind to organellar membrane type NHE7
(44) and the serotonin
transporter SERT (45) and
facilitate targeting of these integral membrane proteins to specific
intracellular compartments. We show in the current study that SCAMP2 binds to
NHE5, facilitates the cell-surface targeting of NHE5, and elevates
Na+/H+ exchange activity at the plasma membrane, whereas
expression of a SCAMP2 deletion mutant lacking the N-terminal domain
containing the NPF repeats suppresses the effect. Further we show that this
activity of SCAMP2 requires an active form of a small GTPase Arf6, but not
Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal
compartment and control its cell-surface abundance via an Arf6-dependent
pathway. 相似文献
17.
Hongjie Yuan Katie M. Vance Candice E. Junge Matthew T. Geballe James P. Snyder John R. Hepler Manuel Yepes Chian-Ming Low Stephen F. Traynelis 《The Journal of biological chemistry》2009,284(19):12862-12873
Zinc is hypothesized to be co-released with glutamate at synapses of the
central nervous system. Zinc binds to NR1/NR2A
N-methyl-d-aspartate (NMDA) receptors with high affinity
and inhibits NMDAR function in a voltage-independent manner. The serine
protease plasmin can cleave a number of substrates, including
protease-activated receptors, and may play an important role in several
disorders of the central nervous system, including ischemia and spinal cord
injury. Here, we demonstrate that plasmin can cleave the native NR2A
amino-terminal domain (NR2AATD), removing the functional high
affinity Zn2+ binding site. Plasmin also cleaves recombinant
NR2AATD at lysine 317 (Lys317), thereby producing a
∼40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments
observed in rat brain membrane preparations. A homology model of the
NR2AATD predicts that Lys317 is near the surface of the
protein and is accessible to plasmin. Recombinant expression of NR2A with an
amino-terminal deletion at Lys317 is functional and Zn2+
insensitive. Whole cell voltage-clamp recordings show that Zn2+
inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected
HEK 293 cells and cultured cortical neurons is significantly reduced by
plasmin treatment. Mutating the plasmin cleavage site Lys317 on
NR2A to alanine blocks the effect of plasmin on Zn2+ inhibition.
The relief of Zn2+ inhibition by plasmin occurs in
PAR1-/- cortical neurons and thus is independent of interaction
with protease-activated receptors. These results suggest that plasmin can
directly interact with NMDA receptors, and plasmin may increase NMDA receptor
responses through disruption or removal of the amino-terminal domain and
relief of Zn2+ inhibition.N-Methyl-d-aspartate
(NMDA)2 receptors are
one of three types of ionotropic glutamate receptors that play critical roles
in excitatory neurotransmission, synaptic plasticity, and neuronal death
(1–3).
NMDA receptors are comprised of glycine-binding NR1 subunits in combination
with at least one type of glutamate-binding NR2 subunit
(1,
4). Each subunit contains three
transmembrane domains, one cytoplasmic re-entrant membrane loop, one bi-lobed
domain that forms the ligand binding site, and one bi-lobed amino-terminal
domain (ATD), thought to share structural homology to periplasmic amino
acid-binding proteins
(4–6).
Activation of NMDA receptors requires combined stimulation by glutamate and
the co-agonist glycine in addition to membrane depolarization to overcome
voltage-dependent Mg2+ block of the ion channel
(7). The activity of NMDA
receptors is negatively modulated by a variety of extracellular ions,
including Mg2+, polyamines, protons, and Zn2+ ions,
which can exert tonic inhibition under physiological conditions
(1,
4). Several extracellular
modulators such as Zn2+ and ifenprodil are thought to act at the
ATD of the NMDA receptor
(8–14).Zinc is a transition metal that plays key roles in both catalytic and
structural capacities in all mammalian cells
(15). Zinc is required for
normal growth and survival of cells. In addition, neuronal death in
hypoxia-ischemia and epilepsy has been associated with Zn2+
(16–18).
Abnormal metabolism of zinc may contribute to induction of cytotoxicity in
neurodegenerative diseases, such as Alzheimer''s disease, Parkinson''s disease,
and amyotrophic lateral sclerosis
(19). Zinc is co-released with
glutamate at excitatory presynaptic terminals and inhibits native NMDA
receptor activation (20,
21). Zn2+ inhibits
NMDA receptor function through a dual mechanism, which includes
voltage-dependent block and voltage-independent inhibition
(22–24).
Voltage-independent Zn2+ inhibition at low nanomolar concentrations
(IC50, 20 nm) is observed for NR2A-containing NMDA
receptors
(25–28).
Evidence has accumulated that the amino-terminal domain of the NR2A subunit
controls high-affinity Zn2+ inhibition of NMDA receptors, and
several histidine residues in this region may constitute part of an
NR2A-specific Zn2+ binding site
(8,
9,
11,
12). For the NR2A subunit,
several lines of evidence suggest that Zn2+ acts by enhancing
proton inhibition (8,
11,
29,
30).Serine proteases present in the circulation, mast cells, and elsewhere
signal directly to cells by cleaving protease-activated receptors (PARs),
members of a subfamily of G-protein-coupled receptors. Cleavage exposes a
tethered ligand domain that binds to and activates the cleaved receptors
(31,
32). Protease receptor
activation has been studied extensively in relation to coagulation and
thrombolysis (33). In addition
to their circulation in the bloodstream, some serine proteases and PARs are
expressed in the central nervous system, and have been suggested to play roles
in physiological conditions (e.g. long-term potentiation or memory)
and pathophysiological states such as glial scarring, edema, seizure, and
neuronal death (31,
34–36).Functional interactions between proteases and NMDA receptors have
previously been suggested. Earlier studies reported that the blood-derived
serine protease thrombin potentiates NMDA receptor response more than 2-fold
through activation of PAR1
(37). Plasmin, another serine
protease, similarly potentiates NMDA receptor response
(38). Tissue-plasminogen
activator (tPA), which catalyzes the conversion of the zymogen precursor
plasminogen to plasmin and results in PAR1 activation, also interacts with and
cleaves the ATD of the NR1 subunit of the NMDA receptor
(39,
40). This raises the
possibility that plasmin may also interact directly with the NMDA receptor
subunits to modulate receptor response. We therefore investigated the ability
of plasmin to cleave the NR2A NMDA receptor subunit. We found that nanomolar
concentrations of plasmin can cleave within the ATD, a region that mediates
tonic voltage-independent Zn2+ inhibition of NR2A-containing NMDA
receptors. We hypothesized that plasmin cleavage reduces the
Zn2+-mediated inhibition of NMDA receptors by removing the
Zn2+ binding domain. In the present study, we have demonstrated
that Zn2+ inhibition of agonist-evoked NMDA currents is decreased
significantly by plasmin treatment in recombinant NR1/NR2A-transfected HEK 293
cells and cultured cortical neurons. These concentrations of plasmin may be
pathophysiologically relevant in situations in which the blood-brain barrier
is compromised, which could allow blood-derived plasmin to enter brain
parenchyma at concentrations in excess of these that can cleave NR2A. Thus,
ability of plasmin to potentiate NMDA function through the relief of the
Zn2+ inhibition could exacerbate the harmful actions of NMDA
receptor overactivation in pathological situations. In addition, if newly
cleaved NR2AATD enters the bloodstream during ischemic injury, it
could serve as a biomarker of central nervous system injury. 相似文献
18.
Xiaojun Li C. T. Ranjith-Kumar Monica T. Brooks S. Dharmaiah Andrew B. Herr Cheng Kao Pingwei Li 《The Journal of biological chemistry》2009,284(20):13881-13891
The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded
RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate
innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that
lacks signaling capability, regulates the signaling of the RLRs. To establish
the structural basis of dsRNA recognition by the RLRs, we have determined the
2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound
to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of
dsRNA with minimal contacts between the protein molecules. Gel filtration
chromatography and analytical ultracentrifugation demonstrated that LGP2 binds
blunt-ended dsRNA of different lengths, forming complexes with 2:1
stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do
not stimulate the activation of RIG-I efficiently in cells. Surprisingly,
full-length LGP2 containing mutations that abolish dsRNA binding retained the
ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading
pathogens; it is the ubiquitous system of defense against microbial infections
(1). Toll-like receptors
(TLRs)3 and RIG-I
(retinoic acid-inducible gene
1)-like receptors (RLRs) play key roles in innate immune response
toward viral infection
(2-5).
Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the
endosome following phagocytosis of the pathogens
(6). RIG-I-like receptors RIG-I
and MDA5 detect viral RNA from replicating viruses in infected cells
(3,
7,
8). Stimulation of these
receptors leads to the induction of type I interferons (IFNs) and other
proinflammatory cytokines, conferring antiviral activity to the host cells and
activating the acquired immune responses
(4,
9).RIG-I discriminates between viral and host RNA through specific recognition
of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp
ssRNA) generated by viral RNA polymerases
(10,
11). In addition, RIG-I also
recognizes double-stranded RNA generated during RNA virus replication
(7,
12). Transfection of cells
with synthetic double-stranded RNA stimulates the activation of RIG-I
(13,
14). Synthetic dsRNA mimics,
such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5
when introduced into the cytoplasm of cells. Digestion of poly(I·C)
with RNase III transforms poly(I·C) from a ligand for MDA5 into a
ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I
recognizes short dsRNA (15).
Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of
these receptors in antiviral immune responses and demonstrated that they sense
different sets of RNA viruses
(12,
16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N
termini, a DEX(D/H) box RNA helicase domain, and a C-terminal
regulatory or repressor domain (CTD). The helicase domain and the CTD are
responsible for viral RNA binding, whereas the CARDs are required for
signaling (3,
8). The current model of RIG-I
activation suggests that under resting conditions RIG-I is in a suppressed
conformation, and viral RNA binding triggers a conformation change that leads
to the exposure of the CARDs for the recruitment of the downstream protein
IPS-1 (also known as MAVS, Cardif, or VISA)
(14,
17). Limited proteolysis of
the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD
are involved in dsRNA and 5′ ppp ssRNA binding
(14). The CTD of RIG-I
overlaps with the C terminus of the previously identified repressor domain
(18). The structures of RIG-I
and LGP2 (laboratory of genetics and
physiology 2) CTD in isolation have been determined by
x-ray crystallography and NMR spectroscopy
(14,
19,
20). A large, positively
charged surface on RIG-I recognizes the 5′ triphosphate group of viral
ssRNA (14,
19). RNA binding studies by
titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that
overlapping sets of residues on this charged surface are involved in RNA
binding (14). Mutagenesis of
several positively charged residues on this surface either reduces or disrupts
RNA binding by RIG-I, and these mutations also affect the induction of
IFN-β in vivo
(14,
19). However, the exact nature
of how the RLRs recognize viral RNA and how RNA binding activates these
receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no
signaling capability (21,
22). The expression of LGP2 is
inducible by dsRNA or IFN treatment as well as virus infection
(21). Overexpression of LGP2
inhibits Sendai virus and Newcastle disease virus signaling
(21). When coexpressed with
RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD
with the CARD and the helicase domain of RIG-I
(18). LGP2 could suppress
RIG-I signaling by three possible ways
(23): 1) binding RNA with high
affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly
with RIG-I to block the assembly of the signaling complex; and 3) competing
with IKKi (IκB kinase ε) in the NF-κB signaling pathway for a
common binding site on IPS-1. To elucidate the structural basis of dsRNA
recognition by the RLRs, we have crystallized human LGP2 CTD (residues
541-678) bound to an 8-bp double-stranded RNA and determined the structure of
the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD
binds to the termini of dsRNA. Mutagenesis and functional studies showed that
dsRNA binding is likely not required for the inhibition of RIG-I signaling by
LGP2. 相似文献
19.
Parmil K. Bansal Amanda Nourse Rashid Abdulle Katsumi Kitagawa 《The Journal of biological chemistry》2009,284(6):3586-3592
The kinetochore, which consists of DNA sequence elements and structural
proteins, is essential for high-fidelity chromosome transmission during cell
division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore
complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study,
we show that Sgt1 forms homodimers by performing in vitro and in
vivo immunoprecipitation and analytical ultracentrifugation analyses.
Analyses of the dimerization of Sgt1 deletion proteins showed that the
Skp1-binding domain (amino acids 1–211) contains the Sgt1
homodimerization domain. Also, the Sgt1 mutant proteins that were unable to
dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is
important for Sgt1-Skp1 binding. Restoring dimerization activity of a
dimerization-deficient sgt1 mutant (sgt1-L31P) by using the
CENP-B (centromere protein-B) dimerization
domain suppressed the temperature sensitivity, the benomyl sensitivity, and
the chromosome missegregation phenotype of sgt1-L31P. These results
strongly suggest that Sgt1 dimerization is required for kinetochore
assembly.Spindle microtubules are coupled to the centromeric region of the
chromosome by a structural protein complex called the kinetochore
(1,
2). The kinetochore is thought
to generate a signal that arrests cells during mitosis when it is not properly
attached to microtubules, thereby preventing aberrant chromosome transmission
to the daughter cells, which can lead to tumorigenesis
(3,
4). The kinetochore of the
budding yeast Saccharomyces cerevisiae has been characterized
thoroughly, genetically and biochemically; thus, its molecular structure is
the most well detailed to date. More than 70 different proteins comprise the
budding yeast kinetochore, and several of those are conserved in mammals
(2).The budding yeast centromere DNA is a 125-bp region that contains three
conserved regions, CDEI, CDEII, and CDEIII
(5,
6). CDEI is bound by Cbf1
(7–9).
CDEIII (25 bp) is essential for centromere function
(10) and is the site where
CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13
(11–18),
and Skp1 (17,
18), all of which are
essential for viability. Mutations in any of the four CBF3 proteins abolish
the ability of CDEIII to bind to CBF3
(19,
20). All of the described
kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores
dependent on the CBF3 complex
(2). Therefore, the CBF3
complex is the fundamental structure of the kinetochore, and the mechanism of
CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4
kinetochore-defective mutant dosage suppressor
(21). Sgt1 and Skp1 activate
Ctf13; thus, they are required for assembly of the CBF3 complex
(21). The molecular chaperone
Hsp90 is also required for the formation of the Skp1-Ctf13 complex
(22). Sgt1 has two highly
conserved motifs that are required for protein-protein interaction, the
tetratricopeptide repeat
(TPR)2
(21) and the CS
(CHORD protein- and Sgt1-specific) motif. We and others
(23–26)
have found that both domains are important for the interaction with Hsp90. The
Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore
complex; this interaction is an initial step in kinetochore assembly
(24,
26,
27) that is conserved between
yeast and humans (28,
29).In this study, we further characterized the molecular mechanism of this
assembly process. We found that Sgt1 forms dimers in vivo, and our
results strongly suggest that Sgt1 dimerization is required for kinetochore
assembly in budding yeast. 相似文献