共查询到20条相似文献,搜索用时 93 毫秒
1.
Wenli Li Jianhua Ju Scott R. Rajski Hiroyuki Osada Ben Shen 《The Journal of biological chemistry》2008,283(42):28607-28617
Tautomycin (TTM) is a highly potent and specific protein phosphatase
inhibitor isolated from Streptomyces spiroverticillatus. The
biological activity of TTM makes it an important lead for drug discovery,
whereas its spiroketal-containing polyketide chain and rare dialkylmaleic
anhydride moiety draw attention to novel biosynthetic chemistries responsible
for its production. To elucidate the biosynthetic machinery associated with
these novel molecular features, the ttm biosynthetic gene cluster
from S. spiroverticillatus was isolated and characterized, and its
involvement in TTM biosynthesis was confirmed by gene inactivation and
complementation experiments. The ttm cluster was localized to a 86-kb
DNA region, consisting of 20 open reading frames that encode three modular
type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five
proteins for methoxymalonyl-S-acyl carrier protein biosynthesis
(Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and
regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF
and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is
proposed based on functional assignments from sequence analysis, which agrees
well with previous feeding experiments, and has been further supported by
in vivo gene inactivation experiments. These findings set the stage
to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM
analogs.Tautomycin (TTM)2
is a polyketide natural product first isolated in 1987 from Streptomyces
spiroverticillatus (1).
The structure and stereochemistry of TTM were established on the basis of
chemical degradation and spectroscopic evidence
(2-4).
TTM contains several features not common to polyketide natural products,
including a spiroketal group, a methoxymalonate-derived unit, and an acyl
chain bearing a dialkylmaleic anhydride moiety. Structurally related to TTM is
tautomycetin (TTN), which was first isolated in 1989 from Streptomyces
griseochromogenes following the discovery of TTM
(5,
6). The structure of TTN was
deduced by chemical degradation and spectroscopic analysis
(6), and its stereochemistry
was established by comparison of spectral data with those of TTN degradation
products and synthetic fragments
(7). Both TTM and TTN exist as
tautomeric mixtures composed of two interconverting anhydride and diacid forms
in approximately a 5:4 ratio under neutral conditions
(Fig. 1A)
(1,
2).Open in a separate windowFIGURE 1.A, structures of TTM and TTN in anhydride or diacid forms, and
biosynthetic origin of the dialkylmaleic anhydride by feeding experiments
using 13C-labeled acetate and propionate. The
methoxymalonate-derived unit in TTM is highlighted by the dotted oval.
R, polyketide moiety of TTM or TTN. B, selected natural product
inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric
anhydride moiety. C, selected natural products containing a
dialkylmaleic anhydride moiety.Early studies of TTM revealed its ability to induce morphological changes
in leukemia cells (8). However,
it was later realized that TTM is a potent and specific inhibitor of protein
phosphatases (PPs) PP-1 and PP-2A
(9). PP-1 and PP-2A are two of
the four major serine/threonine protein phosphatases that regulate diverse
cellular events such as cell division, gene expression, muscle contraction,
glycogen metabolism, and neuronal signaling in eukaryotic cells
(10-12).
Many natural product PP-1 and PP-2A inhibitors are known, including okadaic
acid (13), calyculin-A
(14), phoslactomycin,
spirastrellolide, and cantharidin
(15)
(Fig. 1B), as well as
TTM (16,
17), and TTN
(18). They have served as
useful tools to study PP-involved intracellular events in vivo and as
novel leads for drug discovery
(10-12).
Among these PP inhibitors, TTM and TTN are unique because of their PP-1
selectivity. Despite their structural similarities, TTM exhibits potent
specific inhibition of PP-1 and PP-2A with IC50 values of 22-32
nm and only a slight preference for PP-1
(18). Conversely, TTN shows
nearly a 40-fold higher binding affinity to PP-1 (IC50 = 1.6
nm) than to PP-2A (IC50 = 62 nm)
(18). Because the major
structural differences between TTM and TTN reside in the region distal to the
dialkylmaleic anhydride moiety (Fig.
1A), it has been proposed that differences in these
moieties might be responsible for the PP-1 selectivity
(17-19).
Finally, TTN also has an impressive immuno-suppressive activity
(20,
21), which is apparently
devoid for TTM. Clearly, the structural differences between these two
polyketides translate into large, exploitable differences in bio-activities,
yet an understanding of the biosynthetic origins of these differences remains
elusive.The spiroketal and dialkylmaleic anhydride features of TTM are uncommon for
polyketide natural products, as is the methoxymalonate-derived unit
(Fig. 1A). Few studies
have been carried out for spiroketal biosynthesis, yet it is reasonably common
among the phosphatase inhibitors such as calyculin A, okadaic acid, and a few
others (Fig. 1B). Less
common, but still found in the phosphatase inhibitor cantharidin, as well as
TTM and TTN, is the dialkylmaleic anhydride moiety
(Fig. 1B); this unit
appears in a number of other natural products
(Fig. 1C), although
the biosynthetic steps leading to this reactive moiety (a protected version of
a dicarboxylate) have not been rigorously investigated. Feeding experiments
with 13C-labeled precursors indicated that the anhydride of TTM and
TTN is assembled from a propionate and an as yet undefined C-5 unit
(Fig. 1A), which would
require novel chemistry for polyketide biosynthesis
(22). TTM differentiates
itself from all known PP-1 and PP-2A inhibitors by virtue of its unique
combination of both the dialkymaleic anhydride and spiroketal
functionalities.Multiple total syntheses of TTM and a small number of analogs have been
reported, confirming the predicted structure and absolute stereochemistry and
facilitating structure-activity relationship studies on PP inhibition and
apoptosis induction (19,
23-25).
These studies revealed that: (i) the C22-C26 carbon chain and the
dialkylmaleic anhydride are the minimum requirements for TTM bioactivity; (ii)
the C18-C21 carbon chain and 22-hydroxy group are important for PP inhibition;
(iii) the spiroketal moiety determines the affinity to specific protein
phosphatases; (iv) the active form is most likely the dicarboxylate; and (v)
3′-epi-TTM exhibits 1,000-fold less activity than TTM. However, taken as
a whole, none of the analogs had an improved potency or selectivity for PP-1
inhibition than the natural TTM
(19,
22-25).
As a result, a more specific inhibitor of PP-1 is urgently awaited to
differentiate the physiological roles of PP-1 and PP-2A in vivo and
to explore PPs as therapeutic targets for drug discovery.We have undertaken the cloning and characterization of the TTM biosynthetic
gene cluster from S. spiroverticillatus as the first step toward
engineering TTM biosynthesis for novel analogs
(26). We report here: (i)
cloning and sequencing of the complete ttm gene cluster, (ii)
determination of the ttm gene cluster boundaries, (iii)
bioinformatics analysis of the ttm cluster and a proposal for TTM
biosynthesis, and (iv) genetic characterization of the TTM pathway to support
the proposed pathway. Of particular interest has been the identification of
genes possibly related to dialkylmaleic anhydride biosynthesis, the unveiling
of the ttm polyketide synthase (PKS) genes predicted to select and
incorporate four different starter and extender units for TTM production, and
the apparent lack of candidate genes associated with spiroketal formation.
These findings now set the stage to engineer TTM analogs for novel PP-1- and
PP-2A-specific inhibitors by applying combinatorial biosynthetic methods to
the TTM biosynthetic machinery. 相似文献
2.
Tsuneo Ferguson Jitesh A. Soares Tanja Lienard Gerhard Gottschalk Joseph A. Krzycki 《The Journal of biological chemistry》2009,284(4):2285-2295
Archaeal methane formation from methylamines is initiated by distinct
methyltransferases with specificity for monomethylamine, dimethylamine, or
trimethylamine. Each methylamine methyltransferase methylates a cognate
corrinoid protein, which is subsequently demethylated by a second
methyltransferase to form methyl-coenzyme M, the direct methane precursor.
Methylation of the corrinoid protein requires reduction of the central cobalt
to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric
iron-sulfur protein, was isolated from Methanosarcina barkeri and is
required for in vitro ATP-dependent reductive activation of
methylamine:CoM methyl transfer from all three methylamines. In the absence of
the methyltransferases, highly purified RamA was shown to mediate the
ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for
the monomethylamine corrinoid protein, MtmC. The ramA gene is located
near a cluster of genes required for monomethylamine methyltransferase
activity, including MtbA, the methylamine-specific CoM methylase and the
pyl operon required for co-translational insertion of pyrrolysine
into the active site of methylamine methyltransferases. RamA possesses a
C-terminal ferredoxin-like domain capable of binding two tetranuclear
iron-sulfur proteins. Mutliple ramA homologs were identified in
genomes of methanogenic Archaea, often encoded near methyltrophic
methyltransferase genes. RamA homologs are also encoded in a diverse selection
of bacterial genomes, often located near genes for corrinoid-dependent
methyltransferases. These results suggest that RamA mediates reductive
activation of corrinoid proteins and that it is the first functional archetype
of COG3894, a family of redox proteins of unknown function.Most methanogenic Archaea are capable of producing methane only from carbon
dioxide. The Methanosarcinaceae are a notable exception as representatives are
capable of methylotrophic methanogenesis from methylated amines, methylated
thiols, or methanol. Methanogenesis from these substrates requires methylation
of 2-mercaptoethanesulfonic acid (coenzyme M or CoM) that is subsequently used
by methylreductase to generate methane and a mixed disulfide whose reduction
leads to energy conservation
(1–4).Methylation of CoM with trimethylamine
(TMA),4 dimethylamine
(DMA), or monomethylamine (MMA) is initiated by three distinct
methyltransferases that methylate cognate corrinoid-binding proteins
(3). MtmB, the MMA
methyltransferase, specifically methylates cognate corrinoid protein, MtmC,
with MMA (see Fig. 1)
(5,
6). The DMA methyltransferase,
MtbB, and its cognate corrinoid protein, MtbC, interact specifically to
demethylate DMA (7,
8). TMA is demethylated by the
TMA methyltransferase (MttB) in conjunction with the TMA corrinoid protein
(MttC) (8,
9). Each of the methylated
corrinoid proteins is a substrate for a methylcobamide:CoM methyltransferase,
MtbA, which produces methyl-CoM
(10–12).Open in a separate windowFIGURE 1.MMA:CoM methyl transfer. A schematic of the reactions catalyzed by
MtmB, MtmC, and MtbA is shown that emphasizes the key role of MtmC in the
catalytic cycle of both methyltransferases. Oxidation to Co(II)-MtmC of the
supernucleophilic Co(I)-MtmC catalytic intermediate inactivates methyl
transfer from MMA to the thiolate of coenzyme M (HSCoM). In
vitro reduction of the Co(II)-MtmC with either methyl viologen reduced to
the neutral species or with RamA in an ATP-dependent reaction can regenerate
the Co(I) species. In either case in vitro Ti(III)-citrate is the
ultimate source of reducing power.CoM methylation with methanol requires the methyltransferase MtaB and the
corrinoid protein MtaC, which is then demethylated by another
methylcobamide:CoM methyltransferase, MtaA
(13–15).
The methylation of CoM with methylated thiols such as dimethyl sulfide in
Methanosarcina barkeri is catalyzed by a corrinoid protein that is
methylated by dimethyl sulfide and demethylated by CoM, but in this case an
associated CoM methylase carries out both methylation reactions
(16).In bacteria, analogous methyltransferase systems relying on small corrinoid
proteins are used to achieve methylation of tetrahydrofolate. In
Methylobacterium spp., CmuA, a single methyltransferase with a
corrinoid binding domain, along with a separate pterin methylase, effect the
methylation of tetrahydrofolate with chloromethane
(17,
18). In Acetobacterium
dehalogenans and Moorella thermoacetica various three-component
systems exist for specific demethylation of different phenylmethyl ethers,
such as vanillate (19) and
veratrol (20), again for the
methylation of tetrahydrofolate. Sequencing of the genes encoding the
corrinoid proteins central to the archaeal and bacterial methylotrophic
pathways revealed they are close homologs. Furthermore, genes predicted to
encode such corrinoid proteins and pterin methyltransferases are widespread in
bacterial genomes, often without demonstrated metabolic function. All of these
corrinoid proteins are similar to the well characterized cobalamin binding
domain of methionine synthase
(21,
22).In contrast, the TMA, DMA, MMA, and methanol methyltransferases are not
homologous proteins. The methylamine methyltransferases do share the common
distinction of having in-frame amber codons
(6,
8) within their encoding genes
that corresponds to the genetically encoded amino acid pyrrolysine
(23–25).
Pyrrolysine has been proposed to act in presenting a methylammonium adduct to
the central cobalt ion of the corrinoid protein for methyl transfer
(3,
23,
26). However, nucleophilic
attack on a methyl donor requires the central cobalt ion of a corrinoid
cofactor is in the nucleophilic Co(I) state rather than the inactive Co(II)
state (27). Subsequent
demethylation of the methyl-Co(III) corrinoid cofactor regenerates the
nucleophilic Co(I) cofactor. The Co(I)/Co(II) in the cobalamin binding domain
of methionine synthase has an Em value of -525 mV at pH 7.5
(28). It is likely to be
similarly low in the homologous methyltrophic corrinoid proteins. These low
redox potentials make the corrinoid cofactor subject to adventitious oxidation
to the inactive Co(II) state (Fig.
1).During isolation, these corrinoid proteins are usually recovered in a
mixture of Co(II) or hydroxy-Co(III) states. For in vitro studies,
chemical reduction can maintain the corrinoid protein in the active Co(I)
form. The methanol:CoM or the phenylmethyl ether:tetrahydrofolate
methyltransferase systems can be activated in vitro by the addition
of Ti(III) alone as an artificial reductant
(14,
19). In contrast, activation
of the methylamine corrinoid proteins further requires the addition of methyl
viologen as a redox mediator. Ti(III) reduces methyl viologen to the extremely
low potential neutral species. In vitro activation with these agents
does not require ATP (5,
7,
9).Cellular mechanisms also exist to achieve the reductive activation of
corrinoid cofactors in methyltransferase systems. Activation of human
methionine synthase involves reduction of the co(II)balamin by methionine
synthase reductase (29),
whereas the Escherichia coli enzyme requires flavodoxin
(30). The endergonic reduction
is coupled with the exergonic methylation of the corrinoid with
S-adenosylmethionine
(27). An activation system
exists in cellular extracts of A. dehalogenans that can activate the
veratrol:tetrahydrofolate three-component system and catalyze the direct
reduction of the veratrol-specific corrinoid protein to the Co(I) state;
however, the activating protein has not been purified
(31).For the methanogen methylamine and methanol methyltransferase systems, an
activation process is readily detectable in cell extracts that is ATP- and
hydrogen-dependent (32,
33). Daas et al.
(34,
35) examined the activation of
the methanol methyltransferase system in M. barkeri and purified in
low yield a methyltransferase activation protein (MAP) which in the presence
of a preparation of hydrogenase and uncharacterized proteins was required for
ATP-dependent reductive activation of methanol:CoM methyl transfer. MAP was
found to be a heterodimeric protein without a UV-visible detectable prosthetic
group. Unfortunately, no protein sequence has been reported for MAP, leaving
the identity of the gene in question. The same MAP protein was also suggested
to activate methylamine:CoM methyl transfer, but this suggestion was based on
results with crude protein fractions containing many cellular proteins other
than MAP (36).Here we report of the identification and purification to near-homogeneity
of RamA (reductive activation of
methyltransfer, amines), a protein mediating activation
of methylamine:CoM methyl transfer in a highly purified system
(Fig. 1). Quite unlike MAP,
which was reported to lack prosthetic groups, RamA is an iron-sulfur protein
that can catalyze reduction of a corrinoid protein such as MtmC to the Co(I)
state in an ATP-dependent reaction (Fig.
1). Peptide mapping of RamA allowed identification of the gene
encoding RamA and its homologs in the genomes of Methanosarcina spp.
RamA belongs to COG3894, a group of uncharacterized metal-binding proteins
found in a number of genomes. RamA, thus, provides a functional example for a
family of proteins widespread among bacteria and Archaea whose physiological
role had been largely unknown. 相似文献
3.
Hans Bakker Takuji Oka Angel Ashikov Ajit Yadav Monika Berger Nadia A. Rana Xiaomei Bai Yoshifumi Jigami Robert S. Haltiwanger Jeffrey D. Esko Rita Gerardy-Schahn 《The Journal of biological chemistry》2009,284(4):2576-2583
In mammals, xylose is found as the first sugar residue of the
tetrasaccharide
GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the
formation of the glycosaminoglycans heparin/heparan sulfate and
chondroitin/dermatan sulfate. It is also found in the trisaccharide
Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor
repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes
the formation of the UDP-xylose substrate for the different
xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in
the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place
in these organelles, no obvious requirement exists for membrane transport of
UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has
been documented, and we recently succeeded with the cloning of a human
UDP-xylose transporter (SLC25B4). Here we provide new evidence for a
functional role of UDP-xylose transport by characterization of a new Chinese
hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The
mutant fails to initiate glycosaminoglycan synthesis and is not capable of
xylosylating Notch. Complementation was achieved by expression of a
cytoplasmic variant of UXS, which proves the existence of a functional Golgi
UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid
occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated
control of the cytoplasmically localized UDP-glucose dehydrogenase in the
mutant. The data presented in this study suggest the bidirectional transport
of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in
controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.Xylose is only known to occur in two different mammalian glycans. First,
xylose is the starting sugar residue of the common tetrasaccharide,
GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, attached to
proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans
(GAGs)2
(1). Second, xylose is found in
the trisaccharide Xylα1,3Xylα1,3Glcβ1-O-Ser in
epidermal growth factor (EGF)-like repeats of proteins, such as blood
coagulation factors VII and IX
(2) and Notch
(3)
(Fig. 1). Two variants of
O-xylosyltransferases (XylT1 and XylT2) are responsible for the
initiation of glycosaminoglycan biosynthesis, which differ in terms of
acceptor specificity and tissue distribution
(4-7),
and two different enzymatic activities have been identified that catalyze
xylosylation of O-glucose residues added to EGF repeats
(8-10).
On Notch, O-glucose occurs on EGF repeats in a similar fashion as
O-fucose, which modifications have been shown to influence
ligand-mediated Notch signaling
(11-16).
Recently, rumi, the gene encoding the Notch
O-glucosyltransferase in Drosophila, has been identified,
and inactivation of the gene was found to cause a temperature-sensitive
Notch phenotype (17).
Although this finding clearly demonstrated that O-glucosylation is
essential for Notch signaling, the importance of xylosylation for Notch
functions remains ambiguous.Open in a separate windowFIGURE 1.UDP-xylose metabolism in mammalian cells. A, UDP-Xyl is
synthesized in two steps from UDP-Glc by the enzymes UGDH, forming UDP-GlcA,
and UXS, also referred to as UDP-glucuronic acid decarboxylase. UGDH is
inhibited by the product of the second enzyme, UDP-Xyl
(42). B, in mammals,
UDP-Xyl is synthesized within the lumen of the ER/Golgi, where it is substrate
for different xylosyltransferases incorporating xylose in the
glycosaminoglycan core (XylT1 and XylT2) or in O-glucose-linked
glycans. The nucleotide sugar transporter SLC35D1
(52) has been shown to
transport UDP-GlcA over the ER membrane and SLC35B4
(29) to transport UDP-Xyl over
the Golgi membrane. The function of this latter transporter is unclear.Several different Chinese hamster ovary (CHO) cell lines with defects in
GAG biosynthesis have been isolated by screening for reduced incorporation of
sulfate (18) and reduced
binding of fibroblast growth factor 2 (FGF-2)
(19,
20) and by direct selection
with FGF-2 conjugated to the plant cytotoxin saporin
(21). Isolated cells (called
pgs, for proteoglycan synthesis mutants)
(21) exhibited defects in
various stages of GAG biosynthesis, ranging from the initiating
xylosyltransferase to specific sulfation reactions
(18,
19,
21-25).
Mutants that affect overall GAG biosynthesis were shown to have a defect in
the assembly of the common core tetrasaccharide. Interestingly, these latter
mutants could be separated into clones in which GAG biosynthesis can be
restored by the external addition of xylosides as artificial primers and those
that cannot (18). The two
mutants belonging to the first group are pgsA-745 and pgsB-761. Although
pgs-745 is defective in XylT2
(4-6,
18), pgsB-761 exhibits a
defect in galactosyltransferase I (B4GalT7), the enzyme that catalyzes the
first step in the elongation of the xylosylated protein (25 (see
Fig. 1B). Restoration
of GAG biosynthesis in the latter mutant presumably occurs through a second
β1-4-galactosyltransferase, able to act on xylosides when provided at
high concentration but not on the endogenous protein-linked xylose.Here we describe the isolation of a third CHO cell line (pgsI-208) with the
xyloside-correctable phenotype. The mutant is deficient in UDP-xylose synthase
(UXS), also known as UDP-glucuronic acid decarboxylase. This enzyme catalyzes
the synthesis of UDP-Xyl, the common donor substrate for the different
xylosyltransferases, by decarboxylation of UDP-glucuronic acid. Importantly,
UXS in the animal cell is localized in the lumen of the ER and/or Golgi
(26-28),
superseding at first sight the need for the Golgi UDP-xylose transporter,
which has been recently cloned and characterized
(29). Using this cell variant,
experiments were designed that establish the functional significance of
UDP-Xyl transport with respect to UDP-glucuronic acid production and
xylosylation. 相似文献
4.
5.
6.
Daniel Lingwood Sebastian Schuck Charles Ferguson Mathias J. Gerl Kai Simons 《The Journal of biological chemistry》2009,284(18):12041-12048
Cell membranes predominantly consist of lamellar lipid bilayers. When
studied in vitro, however, many membrane lipids can exhibit
non-lamellar morphologies, often with cubic symmetries. An open issue is how
lipid polymorphisms influence organelle and cell shape. Here, we used
controlled dimerization of artificial membrane proteins in mammalian tissue
culture cells to induce an expansion of the endoplasmic reticulum (ER) with
cubic symmetry. Although this observation emphasizes ER architectural
plasticity, we found that the changed ER membrane became sequestered into
large autophagic vacuoles, positive for the autophagy protein LC3. Autophagy
may be targeting irregular membrane shapes and/or aggregated protein. We
suggest that membrane morphology can be controlled in cells.The observation that simple mixtures of amphiphilic (polar) lipids and
water yield a rich flora of phase structures has opened a long-standing debate
as to whether such membrane polymorphisms are relevant for living organisms
(1–7).
Lipid bilayers with planar geometry, termed lamellar symmetry, dominate the
membrane structure of cells. However, this architecture comprises only a
fraction of the structures seen with in vitro lipid-water systems
(7–11).
The propensity to form lamellar bilayers (a property exclusive to
cylindrically shaped lipids) is flanked by a continuum of lipid structures
that occur in a number of exotic and probably non-physiological
non-bilayer configurations
(3,
12). However, certain lipids,
particularly those with smaller head groups and more bulky hydrocarbon chains,
can adopt bilayered non-lamellar phases called cubic phases. Here the
bilayer is curved everywhere in the form of saddle shapes corresponding to an
energetically favorable minimal surface of zero mean curvature
(1,
7). Because a substantial
number of the lipids present in biological membranes, when studied as
individual pure lipids, form cubic phases
(13), cubic membranes have
received particular interest in cell biology.Since the application of electron microscopy
(EM)3 to the study of
cell ultrastructure, unusual membrane morphologies have been reported for
virtually every organelle (14,
15). However, interpretation
of three-dimensional structures from two-dimensional electron micrographs is
not easy (16). In seminal
work, Landh (17) developed the
method of direct template correlative matching, a technique that unequivocally
assesses the presence of cubic membranes in biological specimens
(16). Cubic phases adopt
mathematically well defined three-dimensional configurations whose
two-dimensional analogs have been derived
(4,
17). In direct template
correlative matching, electron micrographs are matched to these analogs. Cubic
cell membrane geometries and in vitro cubic phases of purified lipid
mixtures do differ in their lattice parameters; however, such deviations are
thought to relate to differences in water activity and lipid to protein ratios
(10,
14,
18). Direct template
correlative matching has revealed thousands of examples of cellular cubic
membranes in a broad survey of electron micrographs ranging from protozoa to
human cells (14,
17) and, more recently, in the
mitochondria of amoeba (19)
and in subcellular membrane compartments associated with severe acute
respiratory syndrome virus
(20). Analysis of cellular
cubic membranes has also been furthered by the development of EM tomography
that confirmed the presence of cubic bilayers in the mitochondrial membranes
of amoeba (21,
22).Although it is now clear that cubic membranes can exist in living cells,
the generation of such architecture would appear tightly regulated, as
evidenced by the dominance of lamellar bilayers in biology. In this light, we
examined the capability and implications of generating cubic membranes in the
endoplasmic reticulum (ER) of mammalian tissue culture cells. The ER is a
spatially interconnected complex consisting of two domains, the nuclear
envelope and the peripheral ER
(23–26).
The nuclear envelope surrounds the nucleus and is composed of two continuous
sheets of membranes, an inner and outer nuclear membrane connected to each
other at nuclear pores. The peripheral ER constitutes a network of branching
trijunctional tubules that are continuous with membrane sheet regions that
occur in closer proximity to the nucleus. Recently it has been suggested that
the classical morphological definition of rough ER (ribosome-studded) and
smooth ER (ribosome-free) may correspond to sheet-like and tubular ER domains,
respectively (27). The ER has
a strong potential for cubic architectures, as demonstrated by the fact that
the majority of cubic cell membranes in the EM record come from ER-derived
structures (14,
17). Furthermore, ER cubic
symmetries are an inducible class of organized smooth ER (OSER), a definition
collectively referring to ordered smooth ER membranes (=stacked cisternae on
the outer nuclear membrane, also called Karmelle
(28–30),
packed sinusoidal ER (31),
concentric membrane whorls
(30,
32–34),
and arrays of crystalloid ER
(35–37)).
Specifically, weak homotypic interactions between membrane proteins produce
both a whorled and a sinusoidal OSER phenotype
(38), the latter exhibiting a
cubic symmetry (16,
39).We were able to produce OSER with cubic membrane morphology via induction
of homo-dimerization of artificial membrane proteins. Interestingly, the
resultant cubic membrane architecture was removed from the ER system by
incorporation into large autophagic vacuoles. To assess whether these cubic
symmetries were favored in the absence of cellular energy, we depleted ATP. To
our surprise, the cells responded by forming large domains of tubulated
membrane, suggesting that a cubic symmetry was not the preferred conformation
of the system. Our results suggest that whereas the endoplasmic reticulum is
capable of adopting cubic symmetries, both the inherent properties of the ER
system and active cellular mechanisms, such as autophagy, can tightly control
their appearance. 相似文献
7.
John W. Hardin Francis E. Reyes Robert T. Batey 《The Journal of biological chemistry》2009,284(22):15317-15324
In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are
responsible for 2′-O-methylation of tRNAs and rRNAs. The
archaeal box C/D small RNP complex requires a small RNA component (sRNA)
possessing Watson-Crick complementarity to the target RNA along with three
proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from
S-adenosylmethionine to the target RNA is performed by fibrillarin,
which by itself has no affinity for the sRNA-target duplex. Instead, it is
targeted to the site of methylation through association with Nop5p, which in
turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a
bridge between the targeting and catalytic functions of the box C/D small RNP
complex, we have employed alanine scanning to evaluate the interaction between
the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA
complex. From these data, we were able to construct an isolated RNA-binding
domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography
and binds to the L7Ae box C/D RNA complex with near wild type affinity. These
data demonstrate that the Nop-RBD is an autonomously folding and functional
module important for protein assembly in a number of complexes centered on the
L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full
functionality in the cell (1).
Currently there are over 100 known RNA modification types ranging from small
functional group substitutions to the addition of large multi-cyclic ring
structures (2). Transfer RNA,
one of many functional RNAs targeted for modification
(3-6),
possesses the greatest modification type diversity, many of which are
important for proper biological function
(7). Ribosomal RNA, on the
other hand, contains predominantly two types of modified nucleotides:
pseudouridine and 2′-O-methylribose
(8). The crystal structures of
the ribosome suggest that these modifications are important for proper folding
(9,
10) and structural
stabilization (11) in
vivo as evidenced by their strong tendency to localize to regions
associated with function (8,
12,
13). These roles have been
verified biochemically in a number of cases
(14), whereas newly emerging
functional modifications are continually being investigated.Box C/D ribonucleoprotein
(RNP)3 complexes serve
as RNA-guided site-specific 2′-O-methyltransferases in both
archaea and eukaryotes (15,
16) where they are referred to
as small RNP complexes and small nucleolar RNPs, respectively. Target RNA
pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl
group of the nucleotide five bases upstream of either the D or D′ box
motif of the sRNA (Fig. 1,
star) (17,
18). In archaea, the internal
C′ and D′ motifs generally conform to a box C/D consensus sequence
(19), and each sRNA contains
two guide regions ∼12 nucleotides in length
(20). The bipartite
architecture of the RNP potentially enables the complex to methylate two
distinct RNA targets (21) and
has been shown to be essential for site-specific methylation
(22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components
of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D
sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D,
C′, and D′ boxes are labeled. The target RNA binds the sRNA
through Watson-Crick pairing and is methylated by fibrillarin at the fifth
nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three
proteins for activity (23):
the ribosomal protein L7Ae
(24,
25), fibrillarin, and the
Nop56/Nop58 homolog Nop5p (Fig.
1). L7Ae binds to both box C/D and the C′/D′ motifs
(26), which respectively
comprise kink-turn (27) or
k-loop structures (28), to
initiate the assembly of the RNP
(29,
30). Fibrillarin performs the
methyl group transfer from the cofactor S-adenosylmethionine to the
target RNA
(31-33).
For this to occur, the active site of fibrillarin must be positioned precisely
over the specific 2′-hydroxyl group to be methylated. Although
fibrillarin methylates this functional group in the context of a Watson-Crick
base-paired helix (guide/target), it has little to no binding affinity for
double-stranded RNA or for the L7Ae-sRNA complex
(22,
26,
33,
34). Nop5p serves as an
intermediary protein bringing fibrillarin to the complex through its
association with both the L7Ae-sRNA complex and fibrillarin
(22). Along with its role as
an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses
other functions not yet fully understood. For example, Nop5p self-dimerizes
through a coiled-coil domain
(35) that in most archaea and
eukaryotic homologs includes a small insertion sequence of unknown function
(36,
37). However, dimerization and
fibrillarin binding have been shown to be mutually exclusive in
Methanocaldococcus jannaschii Nop5p, potentially because of the
presence of this insertion sequence
(36). Thus, whether Nop5p is a
monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate
its interaction with a L7Ae box C/D RNA complex because both the
fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal
structures (29,
35,
38). Individual residues on
the surface of a monomeric form of Nop5p (referred to as mNop5p)
(22) were mutated to alanine,
and the effect on binding affinity for a L7Ae box C/D motif RNA complex was
assessed through the use of electrophoretic mobility shift assays. These data
reveal that residues important for binding cluster within the highly conserved
NOP domain (39,
40). To demonstrate that this
domain is solely responsible for the affinity of Nop5p for the preassembled
L7Ae box C/D RNA complex, we expressed and purified it in isolation from the
full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA
complex with nearly wild type affinity, demonstrating that the Nop-RBD is
truly an autonomously folding and functional module. Comparison of our data
with the crystal structure of the homologous spliceosomal hPrp31-15.5K
protein-U4 snRNA complex (41)
suggests the adoption of a similar mode of binding, further supporting a
crucial role for the NOP domain in RNP complex assembly. 相似文献
8.
Denise A. Berti Cain Morano Lilian C. Russo Leandro M. Castro Fernanda M. Cunha Xin Zhang Juan Sironi Cl��cio F. Klitzke Emer S. Ferro Lloyd D. Fricker 《The Journal of biological chemistry》2009,284(21):14105-14116
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme
that has been proposed to metabolize peptides within cells, thereby affecting
antigen presentation and G protein-coupled receptor signal transduction.
However, only a small number of intracellular substrates of EP24.15 have been
reported previously. Here we have identified over 100 peptides in human
embryonic kidney 293 (HEK293) cells that are derived from intracellular
proteins; many but not all of these peptides are substrates or products of
EP24.15. First, cellular peptides were extracted from HEK293 cells and
incubated in vitro with purified EP24.15. Then the peptides were
labeled with isotopic tags and analyzed by mass spectrometry to obtain
quantitative data on the extent of cleavage. A related series of experiments
tested the effect of overexpression of EP24.15 on the cellular levels of
peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10
of the cellular peptides were incubated with purified EP24.15 in
vitro, and the cleavage was monitored by high pressure liquid
chromatography and mass spectrometry. Many of the EP24.15 substrates
identified by these approaches are 9–11 amino acids in length,
supporting the proposal that EP24.15 can function in the degradation of
peptides that could be used for antigen presentation. However, EP24.15 also
converts some peptides into products that are 8–10 amino acids, thus
contributing to the formation of peptides for antigen presentation. In
addition, the intracellular peptides described here are potential candidates
to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and
if this process is impaired, the elevated levels of aged proteins usually lead
to the formation of intracellular insoluble aggregates that can cause severe
pathologies (1). In mammalian
cells, most proteins destined for degradation are initially tagged with a
polyubiquitin chain in an energy-dependent process and then digested to small
peptides by the 26 S proteasome, a large proteolytic complex involved in the
regulation of cell division, gene expression, and other key processes
(2,
3). In eukaryotes, 30–90%
of newly synthesized proteins may be degraded by proteasomes within minutes of
synthesis (3,
4). In addition to proteasomes,
other extralysosomal proteolytic systems have been reported
(5,
6). The proteasome cleaves
proteins into peptides that are typically 2–20 amino acids in length
(7). In most cases, these
peptides are thought to be rapidly hydrolyzed into amino acids by
aminopeptidases
(8–10).
However, some intracellular peptides escape complete degradation and are
imported into the endoplasmic reticulum where they associate with major
histocompatibility complex class I
(MHC-I)3 molecules and
traffic to the cell surface for presentation to the immune system
(10–12).
Additionally, based on the fact that free peptides added to the intracellular
milieu can regulate cellular functions mediated by protein interactions such
as gene regulation, metabolism, cell signaling, and protein targeting
(13,
14), intracellular peptides
generated by proteasomes that escape degradation have been suggested to play a
role in regulating protein interactions
(15). Indeed, oligopeptides
isolated from rat brain tissue using the catalytically inactive EP24.15 (EC
3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and
were found capable of altering G protein-coupled receptor signal transduction
(16). Moreover, EP24.15
overexpression itself changed both angiotensin II and isoproterenol signal
transduction, suggesting a physiological function for its intracellular
substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family
that contains the HEXXH motif
(17). This enzyme was first
described as a neuropeptide-degrading enzyme present in the soluble fraction
of brain homogenates (18).
Whereas EP24.15 can be secreted
(19,
20), its predominant location
in the cytosol and nucleus suggests that the primary function of this enzyme
is not the extracellular degradation of neuropeptides and hormones
(21,
22). EP24.15 was shown in
vivo to participate in antigen presentation through MHC-I
(23–25)
and in vitro to bind
(26) or degrade
(27) some MHC-I associated
peptides. EP24.15 has also been shown in vitro to degrade peptides
containing 5–17 amino acids produced after proteasome digestion of
β-casein (28). EP24.15
shows substrate size restriction to peptides containing from 5 to 17 amino
acids because of its catalytic center that is located in a deep channel
(29). Despite the size
restriction, EP24.15 has a broad substrate specificity
(30), probably because a
significant portion of the enzyme-binding site is lined with potentially
flexible loops that allow reorganization of the active site following
substrate binding (29).
Recently, it has also been suggested that certain substrates may be cleaved by
an open form of EP24.15 (31).
This characteristic is supported by the ability of EP24.15 to accommodate
different amino acid residues at subsites S4 to S3′, which even includes
the uncommon post-proline cleavage
(30). Such biochemical and
structural features make EP24.15 a versatile enzyme to degrade structurally
unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15
were isolated and identified using mass spectrometry
(22). The majority of peptides
captured by the inactive enzyme were intracellular protein fragments that
efficiently interacted with EP24.15; the smallest peptide isolated in these
assays contained 5 and the largest 17 amino acids
(15,
16,
22,
32), which is within the size
range previously reported for natural and synthetic substrates of EP24.15
(18,
30,
33,
34). Interestingly, the
peptides released by the proteasome are in the same size range of EP24.15
competitive inhibitors/substrates
(7,
35,
36). Taken altogether, these
data suggest that in the intracellular environment EP24.15 could further
cleave proteasome-generated peptides unrelated to MHC-I antigen presentation
(15).Although the mutated inactive enzyme “capture” assay was
successful in identifying several cellular protein fragments that were
substrates for EP24.15, it also found some interacting peptides that were not
substrates. In this study, we used several approaches to directly screen for
cellular peptides that were cleaved by EP24.15. The first approach involved
the extraction of cellular peptides from the HEK293 cell line, incubation
in vitro with purified EP24.15, labeling with isotopic tags, and
analysis by mass spectrometry to obtain quantitative data on the extent of
cleavage. The second approach examined the effect of EP24.15 overexpression on
the cellular levels of peptides in the HEK293 cell line. The third set of
experiments tested synthetic peptides with purified EP24.15 in vitro,
and examined cleavage by high pressure liquid chromatography and mass
spectrometry. Collectively, these studies have identified a large number of
intracellular peptides, including those that likely represent the endogenous
substrates and products of EP24.15, and this original information contributes
to a better understanding of the function of this enzyme in vivo. 相似文献
9.
Michael A. Gitcho Jeffrey Strider Deborah Carter Lisa Taylor-Reinwald Mark S. Forman Alison M. Goate Nigel J. Cairns 《The Journal of biological chemistry》2009,284(18):12384-12398
Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and
Paget disease of bone is a rare, autosomal dominant disorder caused by
mutations in the VCP (valosin-containing protein) gene. The disease
is characterized neuropathologically by frontal and temporal lobar atrophy,
neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are
distinct from those seen in other sporadic and familial FTLD-U entities. The
major component of the ubiquitinated inclusions of FTLD with VCP
mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy
links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most
familial forms of FTLD-U. Understanding the relationship between individual
gene defects and pathologic TDP-43 will facilitate the characterization of the
mechanisms leading to neurodegeneration. Using cell culture models, we have
investigated the role of mutant VCP in intracellular trafficking,
proteasomal function, and cell death and demonstrate that mutations in the
VCP gene 1) alter localization of TDP-43 between the nucleus and
cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum
stress, 4) increase markers of apoptosis, and 5) impair cell viability. These
results suggest that VCP mutation-induced neurodegeneration is
mediated by several mechanisms.Frontotemporal lobar degeneration
(FTLD)2
accounts for 10% of all late onset dementias and is the third most frequent
neurodegenerative disease after Alzheimer disease and dementia with Lewy
bodies (1). FTLD with
ubiquitin-immunoreactive inclusions is genetically, clinically, and
neuropathologically heterogeneous
(2,
3). FTLD-U comprises several
distinct entities, including sporadic forms and familial cases caused by
mutations in the genes encoding VCP (valosin-containing protein), GRN
(progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR
DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9
(2,
3). Frontotemporal dementia
with inclusion body myopathy and Paget disease of bone is a rare, autosomal
dominant disorder caused by mutations in the VCP gene located on
chromosome 9p13-p12
(4-10)
(Fig. 1). This multisystem
disease is characterized by progressive muscle weakness and atrophy, increased
osteoclastic bone resorption, and early onset frontotemporal dementia, also
called FTLD (9,
11). Mutations in VCP
are also associated with dilatative cardiomyopathy with ubiquitin-positive
inclusions (12).
Neuropathologic features of FTLD with VCP mutation include frontal
and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive
inclusions (FTLD-U). The majority of aggregates are ubiquitin- and
TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion
is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal
cytoplasmic inclusions (NCIs). A small number of inclusions are
VCP-immunoreactive (5,
13). Pathologic TDP-43 in
inclusions links a spectrum of diseases in which TDP-43 pathology is a primary
feature, including FTLD-U, motor neuron disease, including amyotrophic lateral
sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and
Paget disease of bone, as well as an expanding spectrum of other disorders in
which TDP-43 pathology is secondary
(14,
15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing
protein. CDC48 (magenta), located within the N terminus (residues
22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1
(23-25,
28). There are two AAA-ATPase
domains (AAA; blue) at residues 240-283 and 516-569, which
are joined by two linker regions (L1 and L2;
red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical
signature similar to that seen in other sporadic and familial cases of FTLD-U,
including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease,
FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome
9p (3,
16). TDP-43 proteinopathy in
these disorders is characterized by hyperphosphorylation of TDP-43,
ubiquitination, and cleavage to form C-terminal fragments detected only in
insoluble brain extracts from affected brain regions
(16). Identification of TDP-43
as the major component of the ubiquitin-immunoreactive inclusions of FTLD with
VCP mutation supports the hypothesis that VCP gene mutations
cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase
associated with diverse cellular activities) superfamily. The N-terminal
domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell
division cycle protein 48)) and two AAA-ATPase domains that form a hexameric
complex (Fig. 1)
(17). Recently, it has been
shown that the N-terminal domain of VCP binds phosphoinositides
(18,
19). AKT (activated
serine-threonine protein kinase) phosphorylates VCP and is required for
constitutive VCP function (20,
21). AKT is activated through
phospholipid binding and phosphorylation via the phosphoinositide 3-kinase
signaling pathway, which is involved in cell survival
(22). The lipid binding domain
may recruit VCP to the cell membrane where it is phosphorylated by AKT
(19).The diversity of VCP functions is modulated, in part, by a variety of
intracellular cofactors, including p47, gp78, and Npl4-Ufd1
(23). Cofactor p47 has been
shown to play a role in the maintenance and biogenesis of both the endoplasmic
reticulum (ER) and Golgi apparatus
(24). The structure of p47
contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and
together they act as a chaperone to deliver membrane fusion machinery to the
site of adjacent membranes
(25). The function of the
p47-VCP complex is dependent upon cell division cycle 2 (CDC2)
serine-threonine kinase phosphorylation of p47
(26,
27). Also, VCP has been found
to interact with the cytosolic tail of gp78, an ER membrane-spanning E3
ubiquitin ligase that exclusively binds VCP and enhances ER-associated
degradation (ERAD) (28). The
Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting
of proteins through the ubiquitin-proteasome system
(29,
30). The cell survival
response of this complex has been found to be important in DNA damage repair
though activation by phosphorylation and its recruitment to double-stranded
breaks (20,
31). The Npl4-Ufd1-VCP
cytosolic complex is also recruited to the ER membrane, interacting with
Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At
the ER membrane, these misfolded proteins are targeted to the proteasome via
ERAD
(32-34).
VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome
system, implicating VCP in the cell survival pathway and neuroprotection
(21,
35-37).To investigate the mechanism of neurodegeneration caused by VCP
mutations, we first tested the hypothesis that VCP mutations decrease
cell viability in vitro using a neuroblastoma SHSY-5Y cell line and
then investigated cellular pathways that are known to lead to
neurodegeneration, including decrease in proteasome activity, caspase-mediated
degeneration, and a change in cellular localization of TDP-43. 相似文献
10.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
11.
Tatsuhiro Sato Akio Nakashima Lea Guo Fuyuhiko Tamanoi 《The Journal of biological chemistry》2009,284(19):12783-12791
Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway
by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully
reproduced in vitro by using mTORC1 immunoprecipitated by the use of
anti-raptor antibody from mammalian cells starved for nutrients. The low
in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is
dramatically increased by the addition of recombinant Rheb. On the other hand,
the addition of Rheb does not activate mTORC2 immunoprecipitated from
mammalian cells by the use of anti-rictor antibody. The activation of mTORC1
is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42
did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition,
the activation is dependent on the presence of bound GTP. We also find that
the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a
recently proposed mediator of Rheb action, appears not to be involved in the
Rheb-dependent activation of mTORC1 in vitro, because the preparation
of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of
Rheb results in a significant increase of binding of the substrate protein
4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that
competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation
of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated
by Rheb. Rheb does not induce autophosphorylation of mTOR. These results
suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to
regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins
(1). We have shown that Rheb
proteins are conserved and are found from yeast to human
(2). Although yeast and fruit
fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or
simply Rheb) and Rheb2 (RhebL1)
(2). Structurally, these
proteins contain G1-G5 boxes, short stretches of amino acids that define the
function of the Ras superfamily G-proteins including guanine nucleotide
binding (1,
3,
4). Rheb proteins have a
conserved arginine at residue 15 that corresponds to residue 12 of Ras
(1). The effector domain
required for the binding with downstream effectors encompasses the G2 box and
its adjacent sequences (1,
5). Structural analysis by
x-ray crystallography further shows that the effector domain is exposed to
solvent, is located close to the phosphates of GTP especially at residues
35–38, and undergoes conformational change during GTP/GDP exchange
(6). In addition, all Rheb
proteins end with the CAAX (C is cysteine, A is an aliphatic amino
acid, and X is the C-terminal amino acid) motif that signals
farnesylation. In fact, we as well as others have shown that these proteins
are farnesylated
(7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling
pathway that plays central roles in regulating protein synthesis and growth in
response to nutrient, energy, and growth conditions
(10–14).
Rheb is down-regulated by a TSC1·TSC2 complex that acts as a
GTPase-activating protein for Rheb
(15–19).
Recent studies established that the GAP domain of TSC2 defines the functional
domain for the down-regulation of Rheb
(20). Mutations in the
Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms
include the appearance of benign tumors called hamartomas at different parts
of the body as well as neurological symptoms
(21,
22). Overexpression of Rheb
results in constitutive activation of mTOR even in the absence of nutrients
(15,
16). Two mTOR complexes,
mTORC1 and mTORC2, have been identified
(23,
24). Whereas mTORC1 is
involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is
involved in the phosphorylation of Akt in response to insulin. It has been
suggested that Rheb is involved in the activation of mTORC1 but not mTORC2
(25).Although Rheb is clearly involved in the activation of mTOR, the mechanism
of activation has not been established. We as well as others have suggested a
model that involves the interaction of Rheb with the TOR complex
(26–28).
Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was
reported (29). Rheb has been
shown to interact with mTOR
(27,
30), and this may involve
direct interaction of Rheb with the kinase domain of mTOR
(27). However, this Rheb/mTOR
interaction is a weak interaction and is not dependent on the presence of GTP
bound to Rheb (27,
28). Recently, a different
model proposing that FKBP38 (FK506-binding protein
38) mediates the activation of
mTORC1 by Rheb was proposed
(31,
32). In this model, FKBP38
binds mTOR and negatively regulates mTOR activity, and this negative
regulation is blocked by the binding of Rheb to FKBP38. However, recent
reports dispute this idea
(33).To further characterize Rheb activation of mTOR, we have utilized an in
vitro system that reproduces activation of mTORC1 by the addition of
recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved
cells using anti-raptor antibody and have shown that its kinase activity
against 4E-BP1 is dramatically increased by the addition of recombinant Rheb.
Importantly, the activation of mTORC1 is specific to Rheb and is dependent on
the presence of bound GTP as well as an intact effector domain. FKBP38 is not
detected in our preparation and further investigation suggests that FKBP38 is
not an essential component for the activation of mTORC1 by Rheb. Our study
revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1
rather than increasing the kinase activity of mTOR. 相似文献
12.
13.
14.
Leonard Kaysser Liane Lutsch Stefanie Siebenberg Emmanuel Wemakor Bernd Kammerer Bertolt Gust 《The Journal of biological chemistry》2009,284(22):14987-14996
Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics
isolated from Streptomyces sp. MK730-62F2 and belong to the
translocase I inhibitor family. Their complex structure is derived from
5′-(β-O-aminoribosyl)-glycyluridine and comprises a unique
N-methyldiazepanone ring. The biosynthetic gene cluster has been
identified, cloned, and sequenced, representing the first gene cluster of a
translocase I inhibitor. Sequence analysis revealed the presence of 23 open
reading frames putatively involved in export, resistance, regulation, and
biosynthesis of the caprazamycins. Heterologous expression of the gene cluster
in Streptomyces coelicolor M512 led to the production of
non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions
validated the boundaries of the cluster and inactivation of cpz21
resulted in the accumulation of novel simplified liponucleoside antibiotics
that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as
an acyltransferase in caprazamycin biosynthesis. In vivo and in
silico analysis of the caprazamycin biosynthetic gene cluster allows a
first proposal of the biosynthetic pathway and provides insights into the
biosynthesis of related uridyl-antibiotics.Caprazamycins
(CPZs)2
(Fig. 1, 1) are
liponucleoside antibiotics isolated from a fermentation broth of
Streptomyces sp. MK730-62F2
(1,
2). They show excellent
activity in vitro against Gram-positive bacteria, in particular
against the genus Mycobacterium including Mycobacterium
intracellulare, Mycobacterium avium, and Mycobacterium
tuberculosis (3). In a
pulmonary mouse model with M. tuberculosis H37Rv, administration of
caprazamycin B exhibited a therapeutic effect but no significant toxicity
(4). Structural elucidation
(2) revealed a complex and
unique composition of elements the CPZs share only with the closely related
liposidomycins (LPMs, 2)
(5). The core skeleton is the
(+)-caprazol (5)
composed of an N-alkylated
5′-(β-O-aminoribosyl)-glycyluridine, also known from
FR-900493 (6)
(6) and the muraymycins
(7)
(7), which is cyclized to form
a rare diazepanone ring. Attached to the 3′″-OH are β-hydroxy
fatty acids of different chain length resulting in CPZs A–G
(1). They differ from
the LPMs in the absence of a sulfate group at the 2″-position of the
aminoribose and the presence of a permethylated l-rhamnose
β-glycosidically linked to the 3-methylglutaryl (3-MG) moiety.Open in a separate windowFIGURE 1.Nucleoside antibiotics of the translocase I inhibitor family.The LPMs have been shown to inhibit biosynthesis of the bacterial cell wall
by targeting the formation of lipid I
(8). The CPZs are expected to
act in the same way and are assigned to the growing number of translocase I
inhibitors that include other nucleoside antibiotics, like the tunicamycins
and mureidomycins (9). During
peptidoglycan formation, translocase I catalyzes the transfer of
UDP-MurNAc-pentapeptide to the undecaprenyl phosphate carrier to
generate lipid I (10). This
reaction is considered an unexploited and promising target for new
anti-infective drugs (11).Recent investigations indicate that the 3″-OH group
(12), the amino group of the
aminoribosyl-glycyluridine, and an intact uracil moiety
(13) are essential for the
inhibition of the Escherichia coli translocase I MraY. The chemical
synthesis of the (+)-caprazol
(5) was recently
accomplished (14), however,
this compound only shows weak antibacterial activity. In contrast, the
acylated compounds 3 and 4 exhibit strong growth inhibition of
mycobacteria, suggesting a potential role of the fatty acid side chain in
penetration of the bacterial cell
(15,
16). Apparently, the
acyl-caprazols (4)
represent the most simplified antibiotically active liponucleosides and a good
starting point for further optimization of this class of potential
therapeutics.Although chemical synthesis and biological activity of CPZs and LPMs has
been studied in some detail, their biosynthesis remains speculative and only
few data exists about the formation of other translocase I inhibitors
(17,
18). Nevertheless, we assume
that the CPZ biosynthetic pathway is partially similar to that of LPMs,
FR-90043 (6), and
muraymycins (7) and
presents a model for the comprehension and manipulation of liponucleoside
formation. Considering the unique structural features of the CPZs we also
expect some unusual biotransformations to be involved in the formation of,
e.g. the (+)-caprazol.Here we report the identification and analysis of the CPZ gene cluster, the
first cluster of a translocase I inhibitor. A set of gene disruption
experiments provide insights into the biosynthetic origin of the CPZs and
moreover, heterologous expression of the gene cluster allows the generation of
novel bioactive derivatives by pathway engineering. 相似文献
15.
Jeffrey M. Boyd Jamie L. Sondelski Diana M. Downs 《The Journal of biological chemistry》2009,284(1):110-118
The ApbC protein has been shown previously to bind and rapidly transfer
iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J.,
Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47,
8195–8202. This study utilized both in vivo and in
vitro assays to examine the function of variant ApbC proteins. The in
vivo assays assessed the ability of ApbC proteins to function in pathways
with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins
were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S]
cluster, and transfer [Fe-S] cluster. This study details the first kinetic
analysis of ATP hydrolysis for a member of the ParA subfamily of
“deviant” Walker A proteins. Moreover, this study details the
first functional analysis of mutant variants of the ever expanding family of
ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that
ApbC protein needs ATPase activity and the ability to bind and rapidly
transfer [Fe-S] clusters for in vivo function.Proteins containing iron-sulfur ([Fe-S]) clusters are employed in a wide
array of metabolic functions (reviewed in Ref.
1). Research addressing the
biosynthesis of the iron-molybdenum cofactor of nitrogenase in Azotobacter
vinelandii led to the discovery of an operon
(iscAnifnifUSVcysE1) involved in the
biosynthesis of [Fe-S] clusters (reviewed in Ref.
2). Subsequent experiments led
to the finding of two more systems involved in the de novo
biosynthesis of [Fe-S] clusters, the isc and the suf systems
(3,
4). Like Escherichia
coli, the genome of Salmonella enterica serovar Typhimurium
encodes for the isc and suf [Fe-S] cluster biosynthesis
machinery.Recent studies have identified a number of additional or
non-isc/-suf-encoded proteins that are involved in bacterial
[Fe-S] cluster biosynthesis and repair. Examples include the following: CyaY,
an iron-binding protein believed to be involved in iron trafficking and iron
delivery
(5–7);
YggX, an Fe2+-binding protein that protects the cell from oxidative
stress (8,
9); ErpA, an alternate A-type
[Fe-S] cluster scaffolding protein
(10); NfuA, a proposed
intermediate [Fe-S] delivery protein
(11–13);
YtfE, a protein proposed to be involved in [Fe-S] cluster repair
(14,
15); and CsdA-CsdE, an
alternative cysteine desulferase
(16).Analysis of the metabolic network anchored to thiamine biosynthesis in
S. enterica identified lesions in three non-isc or
-suf loci that compromise Fe-S metabolism as follows: apbC,
apbE, and rseC
(17–21).
This metabolic system was subsequently used to dissect a role for
cyaY and gshA in [Fe-S] cluster metabolism
(6,
22,
23). Of these, the
apbC (mrp in E. coli) locus was identified as the
predominant site of lesions that altered thiamine synthesis by disrupting
[Fe-S] cluster metabolism (17,
18).ApbC is a member of the ParA subfamily of proteins that have a wide array
of functions, including electron transfer
(24), initiation of cell
division (25), and DNA
segregation (26,
27). Importantly, ATP
hydrolysis is required for function of all well characterized members of this
subfamily, and all members contain a “deviant” Walker A motif,
which contains two lysine residues instead of one (GKXXXGK(S/T))
(28). ApbC has been shown to
hydrolyze ATP (17).Recently, five proteins with a high degree of identity to ApbC have been
shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence
alignments of the central portion of these proteins and bacterial ApbC are
shown in Fig. 1. HCF101 was
demonstrated to be involved in chloroplast [Fe-S] cluster metabolism
(29,
30). The CFD1, Npb35, and
huNbp35 (formally Nubp1) proteins were demonstrated to be involved in
cytoplasmic [Fe-S] cluster metabolism
(31,
32). Ind1 was demonstrated to
be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme
NADH:ubiquinone oxidoreductase
(33). There is currently no
report of any of these proteins hydrolyzing ATP.Open in a separate windowFIGURE 1.Protein sequence alignments of members of the ApbC/Nbp35 subfamily of
ParA family of proteins. Protein alignments were assembled using the
Clustal_W method in the Lasergene® software and show only the central
portion of the proteins, which have the highest sequence conservation. The
three boxed areas highlight the Walker A box, conserved Ser residue,
and CXXC motif. Proteins listed are as follows: ApbC (S.
enterica serovar Typhimurium LT2), CFD1 (S. cerevisiae), Nbp35
(S. cerevisiae), HCF101 (Arabidopsis thaliana), huNpb35
(formally Nubp1) (Homo sapiens), and Ind1 (Candida
albicans).Biochemical analysis of ApbC indicated that it could bind and transfer
[Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate
isomerase (34). Additional
genetic studies indicated that ApbC has a degree of functional redundancy with
IscU, a known [Fe-S] cluster scaffolding protein
(35,
36).In this study we investigate the correlation between the biochemical
properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and
[Fe-S] cluster transfer rates) and the in vivo function of this
protein. This is the first detailed kinetic analysis of ATP hydrolysis for a
member of the ParA subfamily of deviant Walker A proteins and the first
functional analysis of a member of the ever expanding family of ApbC/Nbp35
proteins. Data presented indicate that noncomplementing variants have distinct
biochemical properties that place them in three distinct classes. 相似文献
16.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
17.
Eva Brombacher Simon Urwyler Curdin Ragaz Stefan S. Weber Keiichiro Kami Michael Overduin Hubert Hilbi 《The Journal of biological chemistry》2009,284(8):4846-4856
The causative agent of Legionnaires disease, Legionella
pneumophila, forms a replicative vacuole in phagocytes by means of the
intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV
secretion system and translocated effector proteins, some of which subvert
host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC
anchors to the membrane of Legionella-containing vacuoles (LCVs) by
specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a
nonbiased screen for novel L. pneumophila PI-binding proteins, we
identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the
predominant PtdIns(4)P-binding protein. Purified SidM specifically and
directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate
LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L.
pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding
domain of SidM was mapped to the 12-kDa C-terminal sequence, termed
“P4M” (PtdIns4P binding of
SidM/DrrA). The isolated P4M domain is largely helical and
displayed higher PtdIns(4)P binding activity in the context of the
α-helical, monomeric full-length protein. SidM constructs containing P4M
were translocated by Icm/Dot-proficient L. pneumophila and localized
to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via
its P4M domain. An L. pneumophila ΔsidM mutant strain
displayed significantly higher amounts of SidC on LCVs, suggesting that SidM
and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally,
RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by
host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits
PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC
and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative
agent of Legionnaires disease, but it evolved as a parasite of various species
of environmental predatory protozoa, including the social amoeba
Dictyostelium discoideum
(1,
2). The human disease is linked
to the inhalation of contaminated aerosols, followed by replication in
alveolar macrophages. To accommodate the transfer between host cells, L.
pneumophila alternates between replicative and transmissive phases, the
regulation of which includes an apparent quorum-sensing system
(3–5).In macrophages and amoebae, L. pneumophila forms a replicative
compartment, the Legionella-containing vacuole
(LCV).3 LCVs avoid
fusion with lysosomes (6),
intercept vesicular traffic at endoplasmic reticulum (ER) exit sites
(7), and fuse with the ER
(8–10).
The uptake of L. pneumophila and formation of LCVs in macrophages and
amoebae depends on the Icm/Dot type IV secretion system (T4SS)
(11–14).
Although more than 100 Icm/Dot substrates (“effector” proteins)
have been identified to date, only few are functionally characterized,
including effectors that interfere with host cell signal transduction, vesicle
trafficking, or apoptotic pathways
(15–18).Two Icm/Dot-translocated substrates, SidM/DrrA
(19,
20) and RalF
(21), have been characterized
as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small
GTPases. These bacterial GEFs are recruited to and activate their targets on
LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic
signal transduction pathways and in actin cytoskeleton regulation
(22). Inactive Rho GTPases
bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases
are activated by removal of the GDI and the exchange of GDP with GTP by GEFs,
which promotes the interaction with downstream effector proteins, such as
protein or lipid kinases and various adaptor proteins. The cycle is closed by
hydrolysis of the bound GTP, which is mediated by GTPase-activating
proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle
transport, and additionally, SidM acts as a GDI displacement factor (GDF) to
activate Rab1 (23,
24). The function of SidM is
assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA
preferentially binds to activated Rab1, thus supporting the recruitment of
early secretory vesicles by SidM
(19,
20,
23,
25,
26). Another Icm/Dot
substrate, LepB (27),
contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its
GTPase-activating protein function, thus acting as an antagonist of SidM
(24).The Icm/Dot substrate RalF recruits and activates the small GTPase
ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle
transport from Golgi to ER
(21). Dominant negative Arf1
(7,
28) or knockdown of Arf1 by
RNA interference (29) impairs
the formation of LCVs, as well as the recruitment of the Icm/Dot substrate
SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane
(31), where the proteins
specifically bind to the host cell lipid phosphatidylinositol 4-phosphate
(PtdIns(4)P) (32,
33). Phosphoinositides (PIs)
regulate eukaryotic receptor-mediated signal transduction, actin remodeling,
and membrane dynamics (34,
35). PtdIns(4)P is present on
the cytoplasmic membrane, but localizes preferentially to the
trans-Golgi network (TGN), where this PI is produced by an
Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ)
(36) to promote trafficking
along the secretory pathway. Recently, PtdIns(4)P was found to also mediate
the export of early secretory vesicles from ER exit sites
(37). At present, the L.
pneumophila effector proteins that mediate exploitation of host PI
signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using
different PIs coupled to agarose beads, we identified SidM as a major
PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a
novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs,
including the P4M domain, were found to be translocated and bind the LCV
membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ. 相似文献
18.
19.
20.
ATP-binding cassette (ABC) transporters transduce the free energy of ATP
hydrolysis to power the mechanical work of substrate translocation across cell
membranes. MsbA is an ABC transporter implicated in trafficking lipid A across
the inner membrane of Escherichia coli. It has sequence similarity
and overlapping substrate specificity with multidrug ABC transporters that
export cytotoxic molecules in humans and prokaryotes. Despite rapid advances
in structure determination of ABC efflux transporters, little is known
regarding the location of substrate-binding sites in the transmembrane segment
and the translocation pathway across the membrane. In this study, we have
mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using
site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin
labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster
at the cytoplasmic end of helices 3 and 6 at a site accessible from the
membrane/water interface and extending into an aqueous chamber formed at the
interface between the two transmembrane domains. Binding of a nonhydrolyzable
ATP analog inverts the transporter to an outward-facing conformation and
relieves DNR quenching by spin labels suggesting DNR exclusion from proximity
to the spin labels. The simplest model consistent with our data has DNR
entering near an elbow helix parallel to the water/membrane interface,
partitioning into the open chamber, and then translocating toward the
periplasm upon ATP binding.ATP-binding cassette
(ABC)2 transporters
transduce the energy of ATP hydrolysis to power the movement of a wide range
of substrates across the cell membranes
(1,
2). They constitute the largest
family of prokaryotic transporters, import essential cell nutrients, flip
lipids, and export toxic molecules
(3). Forty eight human ABC
transporters have been identified, including ABCB1, or P-glycoprotein, which
is implicated in cross-resistance to drugs and cytotoxic molecules
(4,
5). Inherited mutations in
these proteins are linked to diseases such as cystic fibrosis, persistent
hypoglycemia of infancy, and immune deficiency
(6).The functional unit of an ABC transporter consists of four modules. Two
highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze
ATP to supply the active energy for transport
(7). ABCs drive the mechanical
work of proteins with diverse functions ranging from membrane transport to DNA
repair (3,
5). Substrate specificity is
determined by two transmembrane domains (TMDs) that also provide the
translocation pathway across the bilayer
(7). Bacterial ABC exporters
are expressed as monomers, each consisting of one NBD and one TMD, that
dimerize to form the active transporter
(3). The number of
transmembrane helices and their organization differ significantly between ABC
importers and exporters reflecting the divergent structural and chemical
nature of their substrates (1,
8,
9). Furthermore, ABC exporters
bind substrates directly from the cytoplasm or bilayer inner leaflet and
release them to the periplasm or bilayer outer leaflet
(10,
11). In contrast, bacterial
importers have their substrates delivered to the TMD by a dedicated high
affinity substrate-binding protein
(12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on
the inner membrane to its final destination in the outer membrane requires the
ABC transporter MsbA (13).
Although MsbA has not been directly shown to transport lipid A, suppression of
MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits
bacterial growth strongly suggesting a role in translocation
(14-16).
In addition to this role in lipid A transport, MsbA shares sequence similarity
with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus
lactis, and Sav1866 of Staphylococcus aureus
(16-19).
ABCB1, a prototype of the ABC family, is a plasma membrane protein whose
overexpression provides resistance to chemotherapeutic agents in cancer cells
(1). LmrA and MsbA have
overlapping substrate specificity with ABCB1 suggesting that both proteins can
function as drug exporters
(18,
20). Indeed, cells expressing
MsbA confer resistance to erythromycin and ethidium bromide
(21). MsbA can be photolabeled
with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342
() across membrane vesicles in an energy-dependent manner
( H3334221).The structural mechanics of ABC exporters was revealed from comparison of
the MsbA crystal structures in the apo- and nucleotide-bound states as well as
from analysis by spin labeling EPR spectroscopy in liposomes
(17,
19,
22,
23). The energy harnessed from
ATP binding and hydrolysis drives a cycle of NBD association and dissociation
that is transmitted to induce reorientation of the TMD from an inward- to
outward-facing conformation
(17,
19,
22). Large amplitude motion
closes the cytoplasmic end of a chamber found at the interface between the two
TMDs and opens it to the periplasm
(23). These rearrangements
lead to significant changes in chamber hydration, which may drive substrate
translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile,
wasting the energy of ATP hydrolysis without substrate extrusion
(7). Consistent with this
model, ATP binding reduces ABCB1 substrate affinity, potentially through
binding site occlusion
(24-26).
Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP
hydrolysis increasing transport efficiency
(1,
27,
28). However, there is a
paucity of information regarding the location of substrate binding, the
transport pathway, and the structural basis of substrate recognition by ABC
exporters. In vitro studies of MsbA substrate specificity identify a
broad range of substrates that stimulate ATPase activity
(29). In addition to the
putative physiological substrates lipid A and lipopolysaccharide (LPS), the
ABCB1 substrates Ilmofosine, , and verapamil differentially enhance ATP
hydrolysis of MsbA ( H3334229,
30). Intrinsic MsbA tryptophan
(Trp) fluorescence quenching by these putative substrate molecules provides
further support of interaction
(29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model
of substrate binding to ABC efflux exporters. This so-called
“hydrophobic cleaner model” describes substrates binding from the
inner leaflet of the bilayer and then translocating through the TMD
(10,
31,
32). These studies also
identified a large number of residues involved in substrate binding and
selectivity (33). When these
crucial residues are mapped onto the crystal structures of MsbA, a subset of
homologous residues clusters to helices 3 and 6 lining the putative substrate
pathway (34). Consistent with
a role in substrate binding and specificity, simultaneous replacement of two
serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport
of ethidium and taxol, although and erythromycin interactions remain
unaffected ( H3334234).The tendency of lipophilic substrates to partition into membranes confounds
direct analysis of substrate interactions with ABC exporters
(35,
36). Such partitioning may
promote dynamic collisions with exposed Trp residues and nonspecific
cross-linking in photo-affinity labeling experiments. In this study, we
utilize a site-specific quenching approach to identify residues in the
vicinity of the daunorubicin (DNR)-binding site
(37). Although the data on DNR
stimulation of ATP hydrolysis is inconclusive
(20,
29,
30), the quenching of MsbA Trp
fluorescence suggests a specific interaction. Spin labels were introduced
along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent
quenching of DNR fluorescence. Residues that quench DNR cluster along the
cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR.
Furthermore, many of these residues are not lipid-exposed but face the
putative substrate chamber formed between the two TMDs. These residues are
proximal to two Trps, which likely explains the previously reported quenching
(29). Our results suggest DNR
partitions to the membrane and then binds MsbA in a manner consistent with the
hydrophobic cleaner model. Interpretation in the context of the crystal
structures of MsbA identifies a putative translocation pathway through the
transmembrane segment. 相似文献