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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
4.
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
5.
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
6.
Maika Deffieu Ingrid Bhatia-Ki??ová Bénédicte Salin Anne Galinier Stéphen Manon Nadine Camougrand 《The Journal of biological chemistry》2009,284(22):14828-14837
The antioxidant N-acetyl-l-cysteine prevented the
autophagy-dependent delivery of mitochondria to the vacuoles, as examined by
fluorescence microscopy of mitochondria-targeted green fluorescent protein,
transmission electron microscopy, and Western blot analysis of mitochondrial
proteins. The effect of N-acetyl-l-cysteine was specific
to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation
of alkaline phosphatase and the presence of hallmarks of non-selective
microautophagy were not altered by N-acetyl-l-cysteine.
The effect of N-acetyl-l-cysteine was not related to its
scavenging properties, but rather to its fueling effect of the glutathione
pool. As a matter of fact, the decrease of the glutathione pool induced by
chemical or genetical manipulation did stimulate mitophagy but not general
autophagy. Conversely, the addition of a cell-permeable form of glutathione
inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the
strain Δuth1, which is deficient in selective mitochondrial
degradation. These data show that mitophagy can be regulated independently of
general autophagy, and that its implementation may depend on the cellular
redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of
long-lived proteins and organelles, where they are degraded and recycled.
Autophagy plays a crucial role in differentiation and cellular response to
stress and is conserved in eukaryotic cells from yeast to mammals
(1,
2). The main form of autophagy,
macroautophagy, involves the non-selective sequestration of large portions of
the cytoplasm into double-membrane structures termed autophagosomes, and their
delivery to the vacuole/lysosome for degradation. Another process,
microautophagy, involves the direct sequestration of parts of the cytoplasm by
vacuole/lysosomes. The two processes coexist in yeast cells but their extent
may depend on different factors including metabolic state: for example, we
have observed that nitrogen-starved lactate-grown yeast cells develop
microautophagy, whereas nitrogen-starved glucose-grown cells preferentially
develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in
the way that autophagosomes and vacuole invaginations do not appear to
discriminate the sequestered material. However, selective forms of autophagy
have been observed (4) that
target namely peroxisomes (5,
6), chromatin
(7,
8), endoplasmic reticulum
(9), ribosomes
(10), and mitochondria
(3,
11–13).
Although non-selective autophagy plays an essential role in survival by
nitrogen starvation, by providing amino acids to the cell, selective autophagy
is more likely to have a function in the maintenance of cellular structures,
both under normal conditions as a “housecleaning” process, and
under stress conditions by eliminating altered organelles and macromolecular
structures
(14–16).
Selective autophagy targeting mitochondria, termed mitophagy, may be
particularly relevant to stress conditions. The mitochondrial respiratory
chain is both the main site and target of
ROS4 production
(17). Consequently, the
maintenance of a pool of healthy mitochondria is a crucial challenge for the
cells. The progressive accumulation of altered mitochondria
(18) caused by the loss of
efficiency of the maintenance process (degradation/biogenesis de
novo) is often considered as a major cause of cellular aging
(19–23).
In mammalian cells, autophagic removal of mitochondria has been shown to be
triggered following induction/blockade of apoptosis
(23), suggesting that
autophagy of mitochondria was required for cell survival following
mitochondria injury (14).
Consistent with this idea, a direct alteration of mitochondrial permeability
properties has been shown to induce mitochondrial autophagy
(13,
24,
25). Furthermore, inactivation
of catalase induced the autophagic elimination of altered mitochondria
(26). In the yeast
Saccharomyces cerevisiae, the alteration of
F0F1-ATPase biogenesis in a conditional mutant has been
shown to trigger autophagy
(27). Alterations of
mitochondrial ion homeostasis caused by the inactivation of the
K+/H+ exchanger was shown to cause both autophagy and
mitophagy (28). We have
reported that treatment of cells with rapamycin induced early ROS production
and mitochondrial lipid oxidation that could be inhibited by the hydrophobic
antioxidant resveratrol (29).
Furthermore, resveratrol treatment impaired autophagic degradation of both
cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death,
suggesting that mitochondrial oxidation events may play a crucial role in the
regulation of autophagy. This existence of regulation of autophagy by ROS has
received molecular support in HeLa cells
(30): these authors showed
that starvation stimulated ROS production, namely H2O2,
which was essential for autophagy. Furthermore, they identified the cysteine
protease hsAtg4 as a direct target for oxidation by
H2O2. This provided a possible connection between the
mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown
yeast cells have established the existence of two distinct processes: the
first one occurring very early, is selective for mitochondria and is dependent
on the presence of the mitochondrial protein Uth1p; the second one occurring
later, is not selective for mitochondria, is not dependent on Uth1p, and is a
form of bulk microautophagy
(3). The absence of the
selective process in the Δuth1 mutant strongly delays and
decreases mitochondrial protein degradation
(3,
12). The putative protein
phosphatase Aup1p has been also shown to be essential in inducing mitophagy
(31). Additionally several Atg
proteins were shown to be involved in vacuolar sequestration of mitochondrial
GFP (3,
12,
32,
33). Recently, the protein
Atg11p, which had been already identified as an essential protein for
selective autophagy has also been reported as being essential for mitophagy
(33).The question remains as to identify of the signals that trigger selective
mitophagy. It is particularly intriguing that selective mitophagy is activated
very early after the shift to a nitrogen-deprived medium
(3). Furthermore, selective
mitophagy is very active on lactate-grown cells (with fully differentiated
mitochondria) but is nearly absent in glucose-grown cells
(3). In the present paper, we
investigated the relationships between the redox status of the cells and
selective mitophagy, namely by manipulating glutathione. Our results support
the view that redox imbalance is a trigger for the selective elimination of
mitochondria. 相似文献
7.
8.
Motoki Takaku Shinichi Machida Noriko Hosoya Shugo Nakayama Yoshimasa Takizawa Isao Sakane Takehiko Shibata Kiyoshi Miyagawa Hitoshi Kurumizaka 《The Journal of biological chemistry》2009,284(21):14326-14336
The RAD51 protein is a central player in homologous recombinational repair.
The RAD51B protein is one of five RAD51 paralogs that function in the
homologous recombinational repair pathway in higher eukaryotes. In the present
study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested
to be involved in actin-remodeling processes, unexpectedly binds to the RAD51
and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and
strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged
DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone
promotes single-stranded DNA annealing, and the recombination activities of
the EVL protein are further enhanced by the RAD51B protein. The expression of
the EVL protein is not ubiquitous, but it is significantly expressed in breast
cancer-derived MCF7 cells. These results suggest that the EVL protein is a
novel recombination factor that may be required for repairing specific DNA
lesions, and that may cause tumor malignancy by its inappropriate
expression.Chromosomal DNA double strand breaks
(DSBs)2 are potential
inducers of chromosomal aberrations and tumorigenesis, and they are accurately
repaired by the homologous recombinational repair (HRR) pathway, without base
substitutions, deletions, and insertions
(1–3).
In the HRR pathway (4,
5), single-stranded DNA (ssDNA)
tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue
of the bacterial RecA protein, binds to the ssDNA tail and forms a helical
nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact
double-stranded DNA (dsDNA) to form a three-component complex, containing
ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the
RAD51 protein promotes recombination reactions, such as homologous pairing and
strand exchange
(6–9).The RAD51 protein requires auxiliary proteins to promote the homologous
pairing and strand exchange reactions efficiently in cells
(10–12).
In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the
RAD51 protein
(13–17)
and stimulate the RAD51-mediated homologous pairing and/or strand exchange
reactions in vitro
(18–21).
The human RAD51AP1 protein, which directly binds to the RAD51 protein
(22), was also found to
stimulate RAD51-mediated homologous pairing in vitro
(23,
24). The BRCA2 protein
contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs
(25–33),
and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated
RAD51-mediated strand exchange
(34,
35). Most of these
RAD51-interacting factors are known to be required for efficient RAD51
assembly onto DSB sites in cells treated with ionizing radiation
(10–12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which
share about 20–30% amino acid sequence similarity with the RAD51 protein
(36–38).
RAD51B-deficient cells are hypersensitive to DSB-inducing agents,
such as cisplatin, mitomycin C (MMC), and γ-rays, indicating that the
RAD51B protein is involved in the HRR pathway
(39–44).
Genetic experiments revealed that RAD51B-deficient cells exhibited
impaired RAD51 assembly onto DSB sites
(39,
44), suggesting that the
RAD51B protein functions in the early stage of the HRR pathway. Biochemical
experiments also suggested that the RAD51B protein participates in the early
to late stages of the HRR pathway
(45–47).In the present study, we found that the human EVL (Ena/Vasp-like) protein
binds to the RAD51 and RAD51B proteins in a HeLa cell extract. The EVL protein
is known to be involved in cytoplasmic actin remodeling
(48) and is also overexpressed
in breast cancer (49). Like
the RAD51B knockdown cells, the EVL knockdown cells partially impaired RAD51
foci formation after DSB induction, suggesting that the EVL protein enhances
RAD51 assembly onto DSB sites. The purified EVL protein preferentially bound
to ssDNA and stimulated RAD51-mediated homologous pairing and strand exchange.
The EVL protein also promoted the annealing of complementary strands. These
recombination reactions that were stimulated or promoted by the EVL protein
were further enhanced by the RAD51B protein. These results strongly suggested
that the EVL protein is a novel factor that activates RAD51-mediated
recombination reactions, probably with the RAD51B protein. We anticipate that,
in addition to its involvement in cytoplasmic actin dynamics, the EVL protein
may be required in homologous recombination for repairing specific DNA
lesions, and it may cause tumor malignancy by inappropriate recombination
enhanced by EVL overexpression in certain types of tumor cells. 相似文献
9.
10.
Greg Brown Alexander Singer Vladimir V. Lunin Michael Proudfoot Tatiana Skarina Robert Flick Samvel Kochinyan Ruslan Sanishvili Andrzej Joachimiak Aled M. Edwards Alexei Savchenko Alexander F. Yakunin 《The Journal of biological chemistry》2009,284(6):3784-3792
Gluconeogenesis is an important metabolic pathway, which produces glucose
from noncarbohydrate precursors such as organic acids, fatty acids, amino
acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of
gluconeogenesis, is found in all organisms, and five different classes of
these enzymes have been identified. Here we demonstrate that Escherichia
coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which
show different catalytic properties. We present the first crystal structure of
a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and
in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor
(phosphate). The crystal structure of the ligand-free GlpX revealed a compact,
globular shape with two α/β-sandwich domains. The core fold of GlpX
is structurally similar to that of Li+-sensitive phosphatases
implying that they have a common evolutionary origin and catalytic mechanism.
The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that
the active site is located between two domains and accommodates several
conserved residues coordinating two metal ions and the substrate. The third
metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate
strongly inhibited activity of both GlpX and YggF, and the crystal structure
of the GlpX complex with phosphate demonstrated that the inhibitor molecule
binds to the active site. Alanine replacement mutagenesis of GlpX identified
12 conserved residues important for activity and suggested that
Thr90 is the primary catalytic residue. Our data provide insight
into the molecular mechanisms of the substrate specificity and catalysis of
GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase
(FBPase,2 EC
3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of
fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It
is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis,
and the product, fructose 6-phosphate, is an important precursor in various
biosynthetic pathways (1). In
all organisms, gluconeogenesis is an important metabolic pathway that allows
the cells to synthesize glucose from noncarbohydrate precursors, such as
organic acids, amino acids, and glycerol. FBPases are members of the large
superfamily of lithium-sensitive phosphatases, which includes three families
of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167
sequences, Pfam data base). These enzymes show metal-dependent and
lithium-sensitive phosphomonoesterase activity and include inositol
polyphosphate 1-phosphatases, inositol monophosphatases (IMPases),
3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting
on both inositol 1,4-bisphosphate and PAP (PIPases)
(2). They possess a common
structural core with the active site lying between α+β and
α/β domains (3).
Li+-sensitive phosphatases are putative targets for lithium therapy
in the treatment of manic depressive patients
(4), whereas FBPases are
targets for the development of drugs for the treatment of noninsulin-dependent
diabetes (5,
6). In addition, FBPase is
required for virulence in Mycobacterium tuberculosis and
Leishmania major and plays an important role in the production of
lysine and glutamate by Corynebacterium glutamicum
(7,
8).Presently, five different classes of FBPases have been proposed based on
their amino acid sequences (FBPases I to V)
(9–11).
Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in
various prokaryotes. Types I, II, and III are primarily in bacteria, type IV
in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in
thermophilic prokaryotes from both domains
(11). Many organisms have more
than one FBPase, mostly the combination of types I + II or II + III, but no
bacterial genome has a combination of types I and III FBPases
(9). The type I FBPase is the
most widely distributed among living organisms and is the primary FBPase in
Escherichia coli, most bacteria, a few archaea, and all
eukaryotes (9,
11–15).
The type II FBPases are represented by the E. coli GlpX and FBPase
F-I from Synechocystis PCC6803
(9,
16); type III is represented
by the Bacillus subtilis FBPase
(17); type IV is represented
by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus
furiosus (18), MJ0109
from Methanococcus jannaschii
(19), and AF2372 from
Archaeoglobus fulgidus
(20); and type V is
represented by the FBPases TK2164 from Pyrococcus
(Thermococcus) kodakaraensis and ST0318 from Sulfolobus
tokodai (10,
21).Three-dimensional structures of the type I (from pig kidney, spinach
chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V
(ST0318) FBPases have been solved
(10,
11,
19,
20,
22,
23). FBPases I and IV and
inositol monophosphatases share a common sugar phosphatase fold organized in
five layered interleaved α-helices and β-sheets
(α-β-α-β-α)
(2,
19,
24). ST0318 (an FBPase V
enzyme) is composed of one domain with a completely different four-layer
α-β-β-α fold
(10). The FBPases from these
three classes (I, IV, and V) require divalent cations for activity
(Mg2+, Mn2+, or Zn2+), and their structures
have revealed the presence of three or four metal ions in the active site.E. coli has five Li+-sensitive phosphatases as follows:
CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase
II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a
3′-phosphoadenosine 5′-phosphatase involved in the cysteine
biosynthesis pathway (25,
26), whereas SuhB is an
inositol monophosphatase (IMPase) that is also known as a suppressor of
temperature-sensitive growth phenotypes in E. coli
(27,
28). Fbp is required for
growth on gluconeogenic substrates and probably represents the main
gluconeogenic FBPase (12).
This enzyme has been characterized both biochemically and structurally and
shown to be inhibited by low concentrations of AMP (IC50 15
μm) (11,
29,
30). The E. coli
GlpX, a class II enzyme FBPase, has been shown to possess a
Mn2+-dependent FBPase activity
(9). The increased expression
of glpX from a multicopy plasmid complemented the Fbp-
phenotype; however, the glpX knock-out strain grew normally on
gluconeogenic substrates (succinate or glycerol)
(9).In this study, we present the first structure of a class II FBPase, the
E. coli GlpX, in a free state and in the complex with FBP + metals or
phosphate. We have demonstrated that the fold of GlpX is similar to that of
the lithium-sensitive phosphatases. We have identified the GlpX residues
important for activity and proposed a catalytic mechanism. We have also showed
that YggF is a third FBPase in E. coli, which has distinct catalytic
properties and is more sensitive than GlpX to the inhibition by lithium or
phosphate. 相似文献
11.
Gaetan Pascreau Frank Eckerdt Andrea L. Lewellyn Claude Prigent James L. Maller 《The Journal of biological chemistry》2009,284(9):5497-5505
p53 is an important tumor suppressor regulating the cell cycle at multiple
stages in higher vertebrates. The p53 gene is frequently deleted or mutated in
human cancers, resulting in loss of p53 activity. This leads to centrosome
amplification, aneuploidy, and tumorigenesis, three phenotypes also observed
after overexpression of the oncogenic kinase Aurora A. Accordingly, recent
studies have focused on the relationship between these two proteins. p53 and
Aurora A have been reported to interact in mammalian cells, but the function
of this interaction remains unclear. We recently reported that
Xenopus p53 can inhibit Aurora A activity in vitro but only
in the absence of TPX2. Here we investigate the interplay between
Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2
is required for nearly all Aurora A activation and for full p53 synthesis and
phosphorylation in vivo during oocyte maturation. In vitro,
phosphorylation mediated by Aurora A targets serines 129 and 190 within the
DNA binding domain of p53. Glutathione S-transferase pull-down
studies indicate that the interaction occurs via the p53 transactivation
domain and the Aurora A catalytic domain around the T-loop. Our studies
suggest that targeting of TPX2 might be an effective strategy for specifically
inhibiting the phosphorylation of Aurora A substrates, including p53.Aurora A is an oncogenic protein kinase that is active in mitosis and plays
important roles in spindle assembly and centrosome function
(1). Overexpression of either
human or Xenopus Aurora A transforms mammalian cells, but only when
the p53 pathway is altered
(2–4).
Aurora A is localized on centrosomes during mitosis, and overexpression of the
protein leads to centrosome amplification and aneuploidy
(2,
3,
5,
6), two likely contributors to
genomic instability (7,
8). Because of its oncogenic
potential and amplification in human tumors, considerable attention has been
focused on the mechanism of Aurora A activation in mitosis. Evidence from
several laboratories indicates that activation occurs as a result of
phosphorylation of a threonine residue in the T-loop of the kinase
(4,
9,
10). Purification of Aurora
A-activating activity from M phase Xenopus egg extracts led to an
apparent activation mechanism in which autophosphorylation at the T-loop is
stimulated by binding of the targeting protein for Xklp2 (TPX2)
(11–14).
On the other hand, it has been shown that Aurora A activity can be inhibited
by interaction with several proteins, including PP1 (protein phosphatase 1),
AIP (Aurora A kinase-interacting protein), and, more recently, p53
(9,
15–17).p53 is a well known tumor suppressor able to drive cell cycle arrest,
apoptosis, or senescence when DNA is damaged or cell integrity is threatened
(18,
19). In human cancers, the p53
gene is frequently deleted or mutated, leading to inactivation of p53
functions (20). p53 protein is
almost undetectable in “normal cells,” mainly due to its
instability. Indeed, during a normal cell cycle, p53 associates with Mdm2 in
the nucleus and thereafter undergoes nuclear exclusion, allowing its
ubiquitination and subsequent degradation
(21). In cells under stress,
p53 is stabilized through the disruption of its interaction with Mdm2
(21), leading to p53
accumulation in the nucleus and triggering different responses, as described
above.Although p53 has mostly been characterized as a nuclear protein, it has
also been shown to localize on centrosomes
(22–24)
and regulate centrosome duplication
(23,
24). Centrosomes are believed
to act as scaffolds that concentrate many regulatory molecules involved in
signal transduction, including multiple protein kinases
(25). Thus, centrosomal
localization of p53 might be important for its own regulation by
phosphorylation/dephosphorylation, and one of its regulators could be the
mitotic kinase Aurora A. Indeed, phenotypes associated with the misexpression
of these two proteins are very similar. For example, overexpression of Aurora
A kinase leads to centrosome amplification, aneuploidy, and tumorigenesis, and
the same effects are often observed after down-regulation of p53
transactivation activity or deletion/mutation of its gene
(26,
27).Several recent studies performed in mammalian models show interplay between
p53 and Aurora A, with each protein having the ability to inhibit the other,
depending on the stage of the cell cycle and the stress level of the cell
(17,
28,
29). These studies reported
that p53 is a substrate of Aurora A, and serines 215 and 315 were demonstrated
to be the two major Aurora A phosphorylation sites in human p53 in
vitro and in vivo. Phosphorylation of Ser-215 within the DNA
binding domain of human p53 inhibited both p53 DNA binding and transactivation
activities (29). Recently, our
group showed that Xenopus p53 is able to inhibit Aurora A kinase
activity in vitro, but this inhibitory effect can be suppressed by
prior binding of Aurora A to TPX2
(9). Contrary to somatic cells,
where p53 is nuclear, unstable, and expressed at a very low level, p53 is
highly expressed in the cytoplasm of Xenopus oocytes and stable until
later stages of development
(30,
31). The high concentration of
both p53 and Aurora A in the oocyte provided a suitable basis for
investigating p53-Aurora A interaction and also evaluating Xenopus
p53 as a substrate of Aurora A. 相似文献
12.
13.
Hongjie Yuan Katie M. Vance Candice E. Junge Matthew T. Geballe James P. Snyder John R. Hepler Manuel Yepes Chian-Ming Low Stephen F. Traynelis 《The Journal of biological chemistry》2009,284(19):12862-12873
Zinc is hypothesized to be co-released with glutamate at synapses of the
central nervous system. Zinc binds to NR1/NR2A
N-methyl-d-aspartate (NMDA) receptors with high affinity
and inhibits NMDAR function in a voltage-independent manner. The serine
protease plasmin can cleave a number of substrates, including
protease-activated receptors, and may play an important role in several
disorders of the central nervous system, including ischemia and spinal cord
injury. Here, we demonstrate that plasmin can cleave the native NR2A
amino-terminal domain (NR2AATD), removing the functional high
affinity Zn2+ binding site. Plasmin also cleaves recombinant
NR2AATD at lysine 317 (Lys317), thereby producing a
∼40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments
observed in rat brain membrane preparations. A homology model of the
NR2AATD predicts that Lys317 is near the surface of the
protein and is accessible to plasmin. Recombinant expression of NR2A with an
amino-terminal deletion at Lys317 is functional and Zn2+
insensitive. Whole cell voltage-clamp recordings show that Zn2+
inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected
HEK 293 cells and cultured cortical neurons is significantly reduced by
plasmin treatment. Mutating the plasmin cleavage site Lys317 on
NR2A to alanine blocks the effect of plasmin on Zn2+ inhibition.
The relief of Zn2+ inhibition by plasmin occurs in
PAR1-/- cortical neurons and thus is independent of interaction
with protease-activated receptors. These results suggest that plasmin can
directly interact with NMDA receptors, and plasmin may increase NMDA receptor
responses through disruption or removal of the amino-terminal domain and
relief of Zn2+ inhibition.N-Methyl-d-aspartate
(NMDA)2 receptors are
one of three types of ionotropic glutamate receptors that play critical roles
in excitatory neurotransmission, synaptic plasticity, and neuronal death
(1–3).
NMDA receptors are comprised of glycine-binding NR1 subunits in combination
with at least one type of glutamate-binding NR2 subunit
(1,
4). Each subunit contains three
transmembrane domains, one cytoplasmic re-entrant membrane loop, one bi-lobed
domain that forms the ligand binding site, and one bi-lobed amino-terminal
domain (ATD), thought to share structural homology to periplasmic amino
acid-binding proteins
(4–6).
Activation of NMDA receptors requires combined stimulation by glutamate and
the co-agonist glycine in addition to membrane depolarization to overcome
voltage-dependent Mg2+ block of the ion channel
(7). The activity of NMDA
receptors is negatively modulated by a variety of extracellular ions,
including Mg2+, polyamines, protons, and Zn2+ ions,
which can exert tonic inhibition under physiological conditions
(1,
4). Several extracellular
modulators such as Zn2+ and ifenprodil are thought to act at the
ATD of the NMDA receptor
(8–14).Zinc is a transition metal that plays key roles in both catalytic and
structural capacities in all mammalian cells
(15). Zinc is required for
normal growth and survival of cells. In addition, neuronal death in
hypoxia-ischemia and epilepsy has been associated with Zn2+
(16–18).
Abnormal metabolism of zinc may contribute to induction of cytotoxicity in
neurodegenerative diseases, such as Alzheimer''s disease, Parkinson''s disease,
and amyotrophic lateral sclerosis
(19). Zinc is co-released with
glutamate at excitatory presynaptic terminals and inhibits native NMDA
receptor activation (20,
21). Zn2+ inhibits
NMDA receptor function through a dual mechanism, which includes
voltage-dependent block and voltage-independent inhibition
(22–24).
Voltage-independent Zn2+ inhibition at low nanomolar concentrations
(IC50, 20 nm) is observed for NR2A-containing NMDA
receptors
(25–28).
Evidence has accumulated that the amino-terminal domain of the NR2A subunit
controls high-affinity Zn2+ inhibition of NMDA receptors, and
several histidine residues in this region may constitute part of an
NR2A-specific Zn2+ binding site
(8,
9,
11,
12). For the NR2A subunit,
several lines of evidence suggest that Zn2+ acts by enhancing
proton inhibition (8,
11,
29,
30).Serine proteases present in the circulation, mast cells, and elsewhere
signal directly to cells by cleaving protease-activated receptors (PARs),
members of a subfamily of G-protein-coupled receptors. Cleavage exposes a
tethered ligand domain that binds to and activates the cleaved receptors
(31,
32). Protease receptor
activation has been studied extensively in relation to coagulation and
thrombolysis (33). In addition
to their circulation in the bloodstream, some serine proteases and PARs are
expressed in the central nervous system, and have been suggested to play roles
in physiological conditions (e.g. long-term potentiation or memory)
and pathophysiological states such as glial scarring, edema, seizure, and
neuronal death (31,
34–36).Functional interactions between proteases and NMDA receptors have
previously been suggested. Earlier studies reported that the blood-derived
serine protease thrombin potentiates NMDA receptor response more than 2-fold
through activation of PAR1
(37). Plasmin, another serine
protease, similarly potentiates NMDA receptor response
(38). Tissue-plasminogen
activator (tPA), which catalyzes the conversion of the zymogen precursor
plasminogen to plasmin and results in PAR1 activation, also interacts with and
cleaves the ATD of the NR1 subunit of the NMDA receptor
(39,
40). This raises the
possibility that plasmin may also interact directly with the NMDA receptor
subunits to modulate receptor response. We therefore investigated the ability
of plasmin to cleave the NR2A NMDA receptor subunit. We found that nanomolar
concentrations of plasmin can cleave within the ATD, a region that mediates
tonic voltage-independent Zn2+ inhibition of NR2A-containing NMDA
receptors. We hypothesized that plasmin cleavage reduces the
Zn2+-mediated inhibition of NMDA receptors by removing the
Zn2+ binding domain. In the present study, we have demonstrated
that Zn2+ inhibition of agonist-evoked NMDA currents is decreased
significantly by plasmin treatment in recombinant NR1/NR2A-transfected HEK 293
cells and cultured cortical neurons. These concentrations of plasmin may be
pathophysiologically relevant in situations in which the blood-brain barrier
is compromised, which could allow blood-derived plasmin to enter brain
parenchyma at concentrations in excess of these that can cleave NR2A. Thus,
ability of plasmin to potentiate NMDA function through the relief of the
Zn2+ inhibition could exacerbate the harmful actions of NMDA
receptor overactivation in pathological situations. In addition, if newly
cleaved NR2AATD enters the bloodstream during ischemic injury, it
could serve as a biomarker of central nervous system injury. 相似文献
14.
Eun-Yeong Bergsdorf Anselm A. Zdebik Thomas J. Jentsch 《The Journal of biological chemistry》2009,284(17):11184-11193
Members of the CLC gene family either function as chloride channels or as
anion/proton exchangers. The plant AtClC-a uses the pH gradient across the
vacuolar membrane to accumulate the nutrient
in this organelle. When AtClC-a was
expressed in Xenopus oocytes, it mediated
exchange
and less efficiently mediated Cl–/H+ exchange.
Mutating the “gating glutamate” Glu-203 to alanine resulted in an
uncoupled anion conductance that was larger for Cl– than
. Replacing the “proton
glutamate” Glu-270 by alanine abolished currents. These could be
restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4
and ClC-5 mediate stoichiometrically coupled
2Cl–/H+ exchange, their
transport is largely uncoupled from
protons. By contrast, the AtClC-a-mediated
accumulation in plant vacuoles
requires tight
coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in
AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this
proline was mutated to serine (P160S), Cl–/H+
exchange of AtClC-a proceeded as efficiently as
exchange, suggesting a role of this residue in
exchange. Indeed, when the corresponding serine of ClC-5 was replaced by
proline, this Cl–/H+ exchanger gained efficient
coupling. When inserted into the model Torpedo chloride channel
ClC-0, the equivalent mutation increased nitrate relative to chloride
conductance. Hence, proline in the CLC pore signature sequence is important
for
exchange and conductance both in
plants and mammals. Gating and proton glutamates play similar roles in
bacterial, plant, and mammalian CLC anion/proton exchangers.CLC proteins are found in all phyla from bacteria to humans and either
mediate electrogenic anion/proton exchange or function as chloride channels
(1). In mammals, the roles of
plasma membrane CLC Cl– channels include transepithelial
transport
(2–5)
and control of muscle excitability
(6), whereas vesicular CLC
exchangers may facilitate endocytosis
(7) and lysosomal function
(8–10)
by electrically shunting vesicular proton pump currents
(11). In the plant
Arabidopsis thaliana, there are seven CLC isoforms
(AtClC-a–AtClC-g)2
(12–15),
which may mostly reside in intracellular membranes. AtClC-a uses the pH
gradient across the vacuolar membrane to transport the nutrient nitrate into
that organelle (16). This
secondary active transport requires a tightly coupled
exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1
(one of the two CLC isoforms in Escherichia coli) display tightly
coupled Cl–/H+ exchange, but anion flux is largely
uncoupled from H+ when
is transported
(17–21).
The lack of appropriate expression systems for plant CLC transporters
(12) has so far impeded
structure-function analysis that may shed light on the ability of AtClC-a to
perform efficient
exchange. This dearth of data contrasts with the extensive mutagenesis work
performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues
(22,
23) and the investigation of
mutants (17,
19–21,
24–29)
have yielded important insights into their structure and function. CLC
proteins form dimers with two largely independent permeation pathways
(22,
25,
30,
31). Each of the monomers
displays two anion binding sites
(22). A third binding site is
observed when a certain key glutamate residue, which is located halfway in the
permeation pathway of almost all CLC proteins, is mutated to alanine
(23). Mutating this gating
glutamate in CLC Cl– channels strongly affects or even
completely suppresses single pore gating
(23), whereas CLC exchangers
are transformed by such mutations into pure anion conductances that are not
coupled to proton transport
(17,
19,
20). Another key glutamate,
located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark
of CLC anion/proton exchangers. Mutating this proton glutamate to
nontitratable amino acids uncouples anion transport from protons in the
bacterial EcClC-1 protein (27)
but seems to abolish transport altogether in mammalian ClC-4 and -5
(21). In those latter
proteins, anion transport could be restored by additionally introducing an
uncoupling mutation at the gating glutamate
(21).The functional complementation by AtClC-c and -d
(12,
32) of growth phenotypes of a
yeast strain deleted for the single yeast CLC Gef1
(33) suggested that these
plant CLC proteins function in anion transport but could not reveal details of
their biophysical properties. We report here the first functional expression
of a plant CLC in animal cells. Expression of wild-type (WT) and mutant
AtClC-a in Xenopus oocytes indicate a general role of gating and
proton glutamate residues in anion/proton coupling across different isoforms
and species. We identified a proline in the CLC signature sequence of AtClC-a
that plays a crucial role in
exchange. Mutating it to serine, the residue present in mammalian CLC proteins
at this position, rendered AtClC-a Cl–/H+ exchange
as efficient as
exchange. Conversely, changing the corresponding serine of ClC-5 to proline
converted it into an efficient
exchanger. When proline replaced the critical serine in Torpedo
ClC-0, the relative conductance of
this model Cl– channel was drastically increased, and
“fast” protopore gating was slowed. 相似文献
15.
Jianzhong Liu Shunqing Wang Ping Zhang Nasser Said-Al-Naief Suzanne M. Michalek Xu Feng 《The Journal of biological chemistry》2009,284(18):12512-12523
Lipopolysaccharide (LPS), a common bacteria-derived product, has long been
recognized as a key factor implicated in periodontal bone loss. However, the
precise cellular and molecular mechanisms by which LPS induces bone loss still
remains controversial. Here, we show that LPS inhibited osteoclastogenesis
from freshly isolated osteoclast precursors but stimulated osteoclast
formation from those pretreated with RANKL in vitro in tissue culture
dishes, bone slices, and a co-culture system containing osteoblasts,
indicating that RANKL-mediated lineage commitment is a prerequisite for
LPS-induced osteoclastogenesis. Moreover, the RANKL-mediated lineage
commitment is long term, irreversible, and TLR4-dependent. LPS exerts the dual
function primarily by modulating the expression of NFATc1, a master regulator
of osteoclastogenesis, in that it abolished RANKL-induced NFATc1 expression in
freshly isolated osteoclast precursors but stimulated its expression in
RANKL-pretreated cells. In addition, LPS prolonged osteoclast survival by
activating the Akt, NF-κB, and ERK pathways. Our current work has not
only unambiguously defined the role of LPS in osteoclastogenesis but also has
elucidated the molecular mechanism underlying its complex functions in
osteoclast formation and survival, thus laying a foundation for future
delineation of the precise mechanism of periodontal bone loss.LPS,2 a
common bacteria-derived product, has long been recognized as a key factor
implicated in the development of chronic periodontitis. LPS plays an important
role in periodontitis by initiating a local host response in gingival tissues
that involves recruitment of inflammatory cells, production of prostanoids and
cytokines, elaboration of lytic enzymes and activation of osteoclast formation
and function to induce bone loss
(1-3).Osteoclasts, the body''s sole bone-resorbing cells, are multinucleated giant
cells that differentiate from cells of hematopoietic lineage upon stimulation
by two critical factors: the macrophage/monocyte colony-forming factor (M-CSF)
and the receptor activator of NF-κB ligand (RANKL)
(4-6).
RANKL exerts its effects on osteoclast formation and function by binding to
its receptor, RANK (receptor activator of NF-κB) expressed on osteoclast
precursors and mature osteoclasts
(7-9).
RANKL also has a decoy receptor, osteoprotegerin, which inhibits RANKL action
by competing with RANK for binding RANKL
(10,
11).RANK is a member of the tumor necrosis factor receptor (TNFR) family
(12). Members of the TNFR
family lack intrinsic enzymatic activity, and hence they transduce
intracellular signals by recruiting various adaptor proteins including TNF
receptor-associated factors (TRAFs) through specific motifs in the cytoplasmic
domain (13,
14). It has been established
that RANK contains three functional TRAF-binding sites
(369PFQEP373, 559PVQEET564, and
604PVQEQG609) that, redundantly, play a role in
osteoclast formation and function
(15,
16). Collectively, through
these functional TRAF-binding motifs, RANK activates six major signaling
pathways, NF-κB, JNK, ERK, p38, NFATc1, and Akt, which play important
roles in osteoclast formation, function, and/or survival
(15,
17-19).
In particular, NFATc1 has been established as a master regulator of osteoclast
differentiation
(20-22).The involvement of osteoclasts in the pathogenesis of periodontal bone loss
is supported by observations that osteoclasts are physically present and
functionally involved in bone resorption in periodontal tissues
(23-27).
RANKL and RANK knockout mice develop osteopetrosis and show failure in tooth
eruption due to a lack of osteoclasts
(24,
25,
28). Moreover,
op/op mice, in which a mutation in the coding region of the
M-CSF gene generates a stop codon that leads to premature termination of
translation of M-CSF mRNA, also show osteopetrosis and failure in tooth
eruption due to a defect in osteoclast development
(26,
27).Whereas the role of osteoclasts in periodontal disease associated alveolar
bone destruction has been well established, the precise role of LPS in
osteoclastogenesis still remains controversial. The vast majority of the
previous studies demonstrated that LPS stimulates osteoclastogenesis. This is
consistent with the role that LPS, a well recognized pathogenic factor in
periodontitis, presumably plays in periodontal bone loss
(29-33).
However, two previous studies demonstrated, surprisingly, that LPS plays
bifunctional roles in osteoclastogenesis in that although it inhibits
osteoclast formation from normal osteoclast precursors, it reverses to promote
osteoclastogenesis from osteoclast precursors pretreated with RANKL
(34,
35). Given that this finding
is inconsistent with the presumed role of LPS as a pathogenic factor in
periodontal bone loss and lacks careful and further validation, the prevalent
view is still that LPS stimulates osteoclastogenesis
(1-3).
Importantly, if LPS indeed has a dual function in osteoclastogenesis, the
molecular mechanism by which LPS exerts a dual effect on osteoclastogenesis
need to be further elucidated.In the present work, using various in vitro assays, we have
demonstrated independently that LPS inhibits osteoclastogenesis from normal
osteoclast precursors but promotes the development of osteoclasts from
RANKL-pretreated cells in tissue culture dishes and bone slices in single-cell
and co-culture settings, confirming the two previous observations that LPS
play a bifunctional role in osteoclastogenesis
(34,
35). Moreover, we have further
shown that the RANKL-mediated lineage commitment is long term and irreversible
in LPS-mediated osteoclastogenesis. More importantly, we have revealed that
LPS inhibits osteoclastogenesis by suppressing NFATc1 expression and JNK
activation while it prolongs osteoclast survival by activating the Akt,
NF-κB, and ERK pathways. These studies have not only unambiguously and
precisely defined the role of LPS in osteoclastogenesis but, more importantly,
may also lead to a paradigm shift in future investigation of the molecular
mechanism of periodontal bone loss. 相似文献
16.
Lifu Wang John C. Lawrence Jr. Thomas W. Sturgill Thurl E. Harris 《The Journal of biological chemistry》2009,284(22):14693-14697
mTORC1 contains multiple proteins and plays a central role in cell growth
and metabolism. Raptor (regulatory-associated protein of mammalian target of
rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential
for mTORC1 activity and critical for the regulation of mTORC1 activity in
response to insulin signaling and nutrient and energy sufficiency. Herein we
demonstrate that mTOR phosphorylates raptor in vitro and in
vivo. The phosphorylated residues were identified by using phosphopeptide
mapping and mutagenesis. The phosphorylation of raptor is stimulated by
insulin and inhibited by rapamycin. Importantly, the site-directed mutation of
raptor at one phosphorylation site, Ser863, reduced mTORC1 activity
both in vitro and in vivo. Moreover, the Ser863
mutant prevented small GTP-binding protein Rheb from enhancing the
phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate
that mTOR-mediated raptor phosphorylation plays an important role on
activation of mTORC1.Mammalian target of rapamycin
(mTOR)2 has been shown
to function as a critical controller in cellular growth, survival, metabolism,
and development (1). mTOR, a
highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase,
structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR
complex 2 (mTORC2), each of which catalyzes the phosphorylation of different
substrates (1). The best
characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also
known as PHAS) and p70 S6 kinase (S6K)
(1), whereas mTORC2
phosphorylates the hydrophobic and turn motifs of protein kinase B
(Akt/protein kinase B) (2) and
protein kinase C (3,
4). mTORC1 constitutively
consists of mTOR, raptor, and mLst8/GβL
(1), whereas the proline-rich
Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that
disassociates after growth factor stimulation
(5,
6). Raptor is essential for
mTORC1 activity by providing a substrate binding function
(7) but also plays a regulatory
role on mTORC1 with stimuli of growth factors and nutrients
(8). In response to insulin,
raptor binding to substrates is elevated through the release of the
competitive inhibitor PRAS40 from mTORC1
(9,
10) because PRAS40 and the
substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus
sequence, the TOR signaling (TOS) motif
(10–14).
In response to amino acid sufficiency, raptor directly interacts with a
heterodimer of Rag GTPases and promotes mTORC1 localization to the
Rheb-containing vesicular compartment
(15).mTORC1 integrates signaling pathways from growth factors, nutrients,
energy, and stress, all of which generally converge on the tuberous sclerosis
complex (TSC1-TSC2) through the phosphorylation of TSC2
(1). Growth factors inhibit the
GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via
the PI3K/Akt pathway (16,
17), whereas energy depletion
activates TSC2 GTPase-activating protein activity by stimulating AMP-activated
protein kinase (AMPK) (18).
Rheb binds directly to mTOR, albeit with very low affinity
(19), and upon charging with
GTP, Rheb functions as an mTORC1 activator
(6). mTORC1 complexes isolated
from growth factor-stimulated cells show increased kinase activity yet do not
contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding
to mTOR leads to an increase in mTORC1 activity toward substrates, and what
the role of raptor is in this activation is currently unknown. More recently,
the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly
phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of
raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative
regulation mechanism independent of TSC2 in response to energy supply
(20). RSK-mediated raptor
phosphorylation enhances mTORC1 activity and provides a mechanism whereby
stress may activate mTORC1 independent of the PI3K/Akt pathway
(21). Therefore, the
phosphorylation status of raptor can be critical for the regulation of mTORC1
activity.In this study, we investigated phosphorylation sites in raptor catalyzed by
mTOR. Using two-dimensional phosphopeptide mapping, we found that
Ser863 and Ser859 in raptor were phosphorylated by mTOR
both in vivo and in vitro. mTORC1 activity in vitro
and in vivo is associated with the phosphorylation of
Ser863 in raptor. 相似文献
17.
18.
Sophie Pattingre Chantal Bauvy St��phane Carpentier Thierry Levade Beth Levine Patrice Codogno 《The Journal of biological chemistry》2009,284(5):2719-2728
Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated
during periods of nutrient starvation to preserve cell integrity. Ceramide is
a bioactive sphingolipid associated with a large range of cell processes. Here
we show that short-chain ceramides (C2-ceramide and
C6-ceramide) and stimulation of the de novo ceramide
synthesis by tamoxifen induce the dissociation of the complex formed between
the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This
dissociation is required for macroautophagy to be induced either in response
to ceramide or to starvation. Three potential phosphorylation sites,
Thr69, Ser70, and Ser87, located in the
non-structural N-terminal loop of Bcl-2, play major roles in the dissociation
of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal
protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to
stimulate macroautophagy. These findings reveal a new aspect of sphingolipid
signaling in up-regulating a major cell process involved in cell adaptation to
stress.Macroautophagy (referred to below as “autophagy”) is a
vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is
conserved in eukaryotic cells
(1–3).
Autophagy is initiated by the formation of a multimembrane-bound autophagosome
that engulfs cytoplasmic proteins and organelles. The last stage in the
process results in fusion with the lysosomal compartments, where the
autophagic cargo undergoes degradation. Basal autophagy is important in
controlling the quality of the cytoplasm by removing damaged organelles and
protein aggregates. Inhibition of basal autophagy in the brain is deleterious,
and leads to neurodegeneration in mouse models
(4,
5). Stimulation of autophagy
during periods of nutrient starvation is a physiological response present at
birth and has been shown to provide energy in various tissues of newborn pups
(6). In cultured cells,
starvation-induced autophagy is an autonomous cell survival mechanism, which
provides nutrients to maintain a metabolic rate and level of ATP compatible
with cell survival (7). In
addition, starvation-induced autophagy blocks the induction of apoptosis
(8). In other contexts, such as
drug treatment and a hypoxic environment, autophagy has also been shown to be
cytoprotective in cancer cells
(9,
10). However, autophagy is
also part of cell death pathways in certain situations
(11). Autophagy can be a
player in apoptosis-independent type-2 cell death (type-1 cell death is
apoptosis), also known as autophagic cell death. This situation has been shown
to occur when the apoptotic machinery is crippled in mammalian cells
(12,
13). Autophagy can also be
part of the apoptotic program, for instance in tumor necrosis
factor-α-induced cell death when NF-κB is inhibited
(14), or in human
immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T
cells (15). Moreover autophagy
has recently been shown to be required for the externalization of
phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of
apoptotic cells (16).The complex relationship between autophagy and apoptosis reflects the
intertwined regulation of these processes
(17,
18). Many signaling pathways
involved in the regulation of autophagy also regulate apoptosis. This
intertwining has recently been shown to occur at the level of the molecular
machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been
shown to inhibit starvation-induced autophagy by interacting with the
autophagy protein Beclin 1
(19). Beclin 1 is one of the
Atg proteins conserved from yeast to humans (it is the mammalian orthologue of
yeast Atg6) and is involved in autophagosome formation
(20). Beclin 1 is a platform
protein that interacts with several different partners, including hVps34
(class III phosphatidylinositol 3-kinase), which is responsible for the
synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is
important for events associated with the nucleation of the isolation membrane
before it elongates and closes to form autophagosomes in response to other Atg
proteins, including the Atg12 and
LC32
(microtubule-associated protein light chain 3 is the mammalian orthologue of
the yeast Atg8) ubiquitin-like conjugation systems
(3,
21). Various partners
associated with the Beclin 1 complex modulate the activity of hVps34. For
instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1,
and Bif-1 all up-regulate it
(22,
23).In view of the intertwining between autophagy and apoptosis, it is
noteworthy that Beclin 1 belongs to the BH3-only family of proteins
(24–26).
However, and unlike most of the proteins in this family, Beclin 1 is not able
to trigger apoptosis when its expression is forced in cells
(27). A BH3-mimetic drug,
ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger
autophagy by mirroring the effect of starvation
(25).The sphingolipids constitute a family of bioactive lipids
(28–32)
of which several members, such as ceramide and sphingosine 1-phosphate, are
signaling molecules. These molecules constitute a “sphingolipid
rheostat” that determines the fate of the cell, because in many settings
ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic
effect (31,
32). However, ceramide is also
engaged in a wide variety of other cell processes, such as the formation of
exosomes (33),
differentiation, cell proliferation, and senescence
(34). Recently we showed that
both ceramide and sphingosine 1-phosphate are able to stimulate autophagy
(35,
36). It has also been shown
that ceramide triggers autophagy in a large panel of mammalian cells
(37–39).
However, elucidation of the mechanism by which ceramide stimulates autophagy
is still in its infancy. We have previously demonstrated that ceramide induces
autophagy in breast and colon cancer cells by inhibiting the Class I
phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central
role in inhibiting autophagy
(36). Inhibition of mTOR is
another hallmark of starvation-induced autophagy
(17). This finding led us to
investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results
presented here show that ceramide is more potent than starvation in
dissociating the Beclin 1-Bcl-2 complex (see Ref.
40). This dissociation is
dependent on three phosphorylation sites (Thr69, Ser70,
and Ser87) located in a non-structural loop of Bcl-2. Ceramide
induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2.
Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation,
and thus the induction of autophagy by ceramide. These findings help to
explain how autophagy is regulated by a major lipid second messenger. 相似文献
19.
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
20.
Jayita Guhaniyogi Istvan Sohar Kalyan Das Ann M. Stock Peter Lobel 《The Journal of biological chemistry》2009,284(6):3985-3997
Late infantile neuronal ceroid lipofuscinosis is a fatal childhood
neurological disorder caused by a deficiency in the lysosomal protease
tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian
member of the S53 family of serine proteases, a group characterized by a
subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum.
TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is
proteolytically processed into the active enzyme after exposure to low pH
in vitro or targeting to the lysosome in vivo. In this
study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing
four Asn-linked N-acetylglucosamines that is indistinguishable from
fully glycosylated TPP1 in terms of autocatalytic processing of the proform
and enzymatic properties of the mature protease. The crystal structure of
deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large
151-residue C-shaped prodomain makes extensive contacts as it wraps around the
surface of the catalytic domain with the two domains connected by a 24-residue
flexible linker that passes through the substrate-binding groove. The
proenzyme structure reveals suboptimal catalytic triad geometry with its
propiece linker partially blocking the substrate-binding site, which together
serve to prevent premature activation of the protease. Finally, we have
identified numerous processing intermediates and propose a structural model
that explains the pathway for TPP1 activation in vitro. These data
provide new insights into TPP1 function and represent a valuable resource for
constructing improved TPP1 variants for treatment of late infantile neuronal
ceroid lipofuscinosis.Late infantile neuronal ceroid lipofuscinosis
(LINCL)3 (OMIM number
204500) is a neurodegenerative lysosomal storage disease of childhood that
presents typically between the ages of 2 and 4 years with the onset of
seizures. Disease progression is reflected by blindness, dementia, mental
retardation, and an increase in the severity of seizures. LINCL is always
fatal, and the life span of patients is typically 6-15 years. LINCL is caused
by mutations in TPP1 (previously named CLN2, for ceroid
lipofuscinosis neuronal type 2 gene)
(1), which normally encodes a
lysosomal protease, tripeptidyl-peptidase 1 (TPP1, EC 3.4.14.9)
(2,
3).There is currently no treatment of demonstrated efficacy for LINCL, but
promising progress is being made in some directions. Proof-of-principle for
virus-mediated gene therapy has been established in a mouse model of LINCL,
with a significant improvement in disease phenotype achieved with the use of
adeno-associated virus vectors expressing TPP1
(4-7).
Affected children have also been treated with adeno-associated virus vectors,
although it is too soon to determine whether significant clinical benefits
have been achieved in these early trials
(8). Enzyme replacement
therapy, an approach that has proven successful in a number of other lysosomal
storage diseases, has also been investigated in LINCL. Purified recombinant
human TPP1 that contains the mannose 6-phosphate lysosomal targeting
modification can be taken up by LINCL fibroblasts where it degrades storage
material (9), and the protein
has been introduced into the cerebrospinal fluid of the LINCL mouse model via
intraventricular injection, resulting in significant uptake into the brain and
some correction of neuropathology
(10).For therapeutic approaches that rely upon replacing a mutant gene product
with a functional protein via recombinant methods, e.g. gene and
enzyme replacement therapy, a thorough understanding of the biological and
biophysical properties of the protein in question are essential for success.
Thus, for LINCL, considerable effort has been directed toward the
investigation of TPP1, and as a result, this is a well characterized enzyme at
the functional and molecular levels (reviewed in Refs.
11,
12). TPP1 encodes a
563-residue preproprotein with a cleavable N-terminal 19-residue signal
sequence. The proenzyme (residues 20-563) is a soluble monomer that undergoes
proteolytic cleavage in the lysosome, converting the zymogen to an active,
mature protease (residues 196-563)
(1). Studies on purified
pro-TPP1 demonstrate that maturation is autocatalytic in vitro
(13,
14) but may involve other
proteases in vivo
(15). TPP1 is glycosylated,
and its N-linked oligosaccharides have been implicated in maturation,
activity, targeting, and stability of the processed enzyme
(16,
17).TPP1 is a serine protease
(14) that possesses two
catalytic functions as follows: a primary tripeptidyl exopeptidase activity
with a pH optimum of ∼5.0 that catalyzes the sequential release of
tripeptides from the unsubstituted N termini of substrates
(18), and a much weaker
endoproteolytic activity with a pH optimum of ∼3.0
(19). TPP1 exhibits broad
substrate specificity (20) and
is the only mammalian member of the S53 sedolisin family (reviewed in Ref.
21), which includes a number
of unusual bacterial serine peptidases
(22). High resolution crystal
structures of both free and inhibitor-bound complexes have been determined for
three bacterial members of this family (sedolisin
(23-26),
kumamolisin (27,
28), and kumamolisin-As
(29,
30)), and for one
(kumamolisin), the structure of a mutant, inactive precursor form has also
been obtained (28). These
proteins share a common subtilisin-like fold, an octahedrally coordinated
calcium-binding site, and an active site that contains an unusual Ser-Glu-Asp
(SED) catalytic triad, rather than the Ser-His-Asp (SHD) triad of subtilisin
(31,
32). Chemical modification
studies of TPP1 have revealed that Ser475 is the active site
nucleophile (14). Modeling
studies suggest that Glu272 and Asp276 complete the
catalytic triad and that Asp360 is homologous to the conserved Asn
in the subtilisin family in its role in stabilization of the oxyanion of the
tetrahedral intermediate during catalysis
(33). Site-directed
mutagenesis studies are consistent with these conclusions
(14,
34).A detailed understanding of the tertiary structure of TPP1 may have
implications for developing or improving therapeutic strategies. First, a high
resolution model would provide the basis for targeted protein engineering
efforts to design TPP1 derivatives with increased half-life prior to and/or
upon delivery to the lysosome. Successful creation of a longer lived TPP1
molecule could significantly enhance gene or enzyme replacement approaches to
LINCL. Second, a structural model for TPP1 could be valuable in designing
derivatives tagged with protein transduction domains to facilitate crossing of
the blood-brain barrier for delivery to the central nervous system from the
bloodstream. In this study, we present the crystal structure of the proform of
human TPP1 at 1.85 Å resolution. This model provides novel insights into
the structural basis for the pH-induced auto-activation of the proform of
TPP1. A structure of glycosylated pro-TPP1 has been independently determined,
displaying features similar to those of deglycosylated
TPP1.4 相似文献