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Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
4.
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Siddhartha Mitra Andrey S. Tsvetkov Steven Finkbeiner 《The Journal of biological chemistry》2009,284(7):4398-4403
The accumulation of mutant protein in intracellular aggregates is a common
feature of neurodegenerative disease. In Huntington disease, mutant huntingtin
leads to inclusion body (IB) formation and neuronal toxicity. Impairment of
the ubiquitin-proteasome system (UPS) has been implicated in IB formation and
Huntington disease pathogenesis. However, IBs form asynchronously in only a
subset of cells with mutant huntingtin, and the relationship between IB
formation and UPS function has been difficult to elucidate. Here, we applied
single-cell longitudinal acquisition and analysis to monitor mutant huntingtin
IB formation, UPS function, and neuronal toxicity. We found that proteasome
inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB
formation, the UPS is more impaired in neurons that go on to form IBs than in
those that do not. After forming IBs, impairment is lower in neurons with IBs
than in those without. These findings suggest IBs are a protective cellular
response to mutant protein mediated in part by improving intracellular protein
degradation.Huntington disease
(HD)4 is a progressive
incurable neurodegenerative disorder caused by the expansion of a
polyglutamine (polyQ) stretch in the N-terminal end of the huntingtin (htt)
protein above a threshold length of ∼36
(1). The deposition of
polyQ-expanded aggregated mutant htt in inclusion bodies (IBs) is a hallmark
of HD, and IBs are found in human post-mortem samples, transgenic mouse brain,
and cell-culture models (2).
The accumulation of ubiquitinated proteins in IBs has implicated the
ubiquitin-proteasome system (UPS) in the pathogenesis of HD, amyotrophic
lateral sclerosis, Parkinson disease, and polyQ-mediated disorders
(3).The UPS is a major pathway of intracellular protein degradation. After a
series of three reactions, each catalyzed by a different set of enzymes,
ubiquitin, a 76-amino acid polypeptide, forms an isopeptide bond with the
amino group of lysine residues on substrate proteins. Several lysine residues
within ubiquitin are sites for more ubiquitin additions. Once a protein
accumulates four or more ubiquitins, it is efficiently targeted to the
proteasome for degradation. The proteasome binds polyubiquitinated substrates
and hydrolyzes ubiquitin isopeptide bonds, releasing ubiquitin moieties before
degrading substrate proteins through chymotrypsin-like, trypsin-like, and
post-glutamyl peptidase activities
(3).Increased polyubiquitin levels and changes in ubiquitin linkages accompany
the accumulation of UPS substrates in the brains of HD patients and transgenic
mice and in cellular HD models
(4). UPS substrates accumulate
throughout the cell in polyQ models, even before IB formation
(5,
6). This has added to the
confusion over whether polyQ expansion leads to toxicity through direct
impairment of proteasomal degradation. Proteasomes have been reported to
cleave polyQ stretches efficiently
(7), inefficiently
(8), or essentially not at all
(9). In vivo,
polyQ-dependent degeneration occurs with no detectable proteasome inhibition
(10,
11) or is tightly linked to it
(12,
13). The inability of some
studies to detect UPS impairment in HD models may be due to the limited
sensitivity of conventional approaches to identify cell-to-cell variations in
UPS function.The relationship between IB formation and UPS function has been difficult
to determine. Protein turnover in cells with IBs is evidently reduced and
accompanied by the accumulation of cellular proteins
(14–16);
HEK293 cells containing mutant htt IBs have a greater degree of UPS impairment
than those without IBs (5).
Proteasome subunits and heat shock proteins colocalize with IBs, but it is
unclear if this colocalization facilitates protein delivery or unfolding at
the mouth of active proteasomes, or if it harms proteasome function by
sequestering essential cellular machinery
(18). Some IBs are relatively
static (8,
25), but the proteins in
others are dynamically exchanged with cytoplasmic and nuclear pools
(19,
20).UPS function is critical to cellular homeostasis. Deletion of one of the
two inducible polyubiquitin genes in mice leads to lower intracellular
ubiquitin levels in germ cells and hypothalamic neurons. These same
populations undergo cell-cycle arrest and hypothalamic neurodegeneration,
respectively (22,
23). Cell lines expressing
mutant huntingtin accumulate ubiquitinated proteins and undergo cell-cycle
arrest in G2/M (5). In neurons,
UPS impairment may lead to cell death through an accumulation of signals for
apoptosis, a decrease in NF-κB signaling, sensitization to other toxic
stimuli, remodeling of synapses, retraction of neurites, or other unidentified
mechanisms (24). The effect of
UPS impairment depends on cell type and cell cycle, and the relationship
between UPS impairment and striatal neuronal survival is largely unknown.Diffuse species of mutant htt induce IB formation and neuronal death in a
protein concentration-dependent manner
(2). IB formation delays
neuronal death, suggesting that IB formation helps neurons cope with toxic
diffuse mutant htt. Whether the effect of IB formation on survival is mediated
through UPS function has been difficult to determine. IB formation and
neuronal death occur asynchronously in overlapping but distinct subsets of
neurons that express mutant htt. The observation that IB formation is not
required for UPS impairment also complicates population analysis
(6,
26).To explore this problem, we applied single-cell analysis. We tracked single
neurons over their entire lifetimes, gaining spatial and temporal resolution
while simultaneously monitoring IB formation, UPS inhibition, and neuronal
toxicity. 相似文献
6.
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
7.
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
8.
As obligate intracellular parasites, viruses exploit diverse cellular
signaling machineries, including the mitogen-activated protein-kinase pathway,
during their infections. We have demonstrated previously that the open reading
frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90
ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities
(Kuang, E., Tang, Q., Maul, G. G., and Zhu, F.
(2008) J. Virol. 82
,1838
-1850). Here, we define the
mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45
to RSK increases the association of extracellular signal-regulated kinase
(ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass
protein complexes. We further demonstrated that the complexes shielded active
pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK
and ERK were activated and sustained at high levels. Finally, we provide
evidence that this mechanism contributes to the sustained activation of ERK
and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase
(ERK)2
mitogen-activated protein kinase (MAPK) signaling pathway has been implicated
in diverse cellular physiological processes including proliferation, survival,
growth, differentiation, and motility
(1-4)
and is also exploited by a variety of viruses such as Kaposi
sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human
immunodeficiency virus, respiratory syncytial virus, hepatitis B virus,
coxsackie, vaccinia, coronavirus, and influenza virus
(5-17).
The MAPK kinases relay the extracellular signaling through sequential
phosphorylation to an array of cytoplasmic and nuclear substrates to elicit
specific responses (1,
2,
18). Phosphorylation of MAPK
is reversible. The kinetics of deactivation or duration of signaling dictates
diverse biological outcomes
(19,
20). For example, sustained
but not transient activation of ERK signaling induces the differentiation of
PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells
(20-22).
During viral infection, a unique biphasic ERK activation has been observed for
some viruses (an early transient activation triggered by viral binding or
entry and a late sustained activation correlated with viral gene expression),
but the responsible viral factors and underlying mechanism for the sustained
ERK activation remain largely unknown
(5,
8,
13,
23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine
kinases that lie at the terminus of the ERK pathway
(1,
24-26).
In mammals, four isoforms are known, RSK1 to RSK4. Each one has two
catalytically functional kinase domains, the N-terminal kinase domain (NTKD)
and C-terminal kinase domain (CTKD) as well as a linker region between the
two. The NTKD is responsible for phosphorylation of exogenous substrates, and
the CTKD and linker region regulate RSK activation
(1,
24,
25). In quiescent cells ERK
binds to the docking site in the C terminus of RSK
(27-29).
Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase
(MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker
region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD
activation loop. The activated CTKD then phosphorylates Ser-380 in the linker
region, creating a docking site for 3-phosphoinositide-dependent protein
kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates
Ser-221 of RSK in the activation loop and activates the NTKD. The activated
NTKD autophosphorylates the serine residue near the ERK docking site, causing
a transient dissociation of active ERK from RSK
(25,
26,
28). The stimulation of
quiescent cells by a mitogen such as epidermal growth factor or a phorbol
ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually
results in a transient RSK activation that lasts less than 30 min. RSKs have
been implicated in regulating cell survival, growth, and proliferation.
Mutation or aberrant expression of RSK has been implicated in several human
diseases including Coffin-Lowry syndrome and prostate and breast cancers
(1,
24,
25,
30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma,
primary effusion lymphoma, and a subset of multicentric Castleman disease
(33,
34). Infection and
reactivation of KSHV activate multiple MAPK pathways
(6,
12,
35). Noticeably, the ERK/RSK
activation is sustained late during KSHV primary infection and reactivation
from latency (5,
6,
12,
23), but the mechanism of the
sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45,
an immediate early and also virion tegument protein of KSHV, interacts with
RSK1 and RSK2 and strongly stimulates their kinase activities
(23). We also demonstrated
that the activation of RSK plays an essential role in KSHV lytic replication
(23). In the present study we
determined the mechanism of ORF45-induced sustained ERK/RSK activation. We
found that ORF45 increases the association of RSK with ERK and protects them
from dephosphorylation, causing sustained activation of both ERK and RSK. 相似文献
9.
Il-Ha Lee Craig R. Campbell Sung-Hee Song Margot L. Day Sharad Kumar David I. Cook Anuwat Dinudom 《The Journal of biological chemistry》2009,284(19):12663-12669
It has recently been shown that the epithelial Na+ channel
(ENaC) is compartmentalized in caveolin-rich lipid rafts and that
pharmacological depletion of membrane cholesterol, which disrupts lipid raft
formation, decreases the activity of ENaC. Here we show, for the first time,
that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the
activity and membrane surface expression of ENaC. Physical interaction between
ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found
that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin
protein ligase of the Nedd4 family, which is known to induce ubiquitination
and internalization of ENaC. The effect of Cav-1 on ENaC requires the
proline-rich motifs at the C termini of the β- and γ-subunits of
ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken
together, our data suggest that Cav-1 inhibits the activity of ENaC by
decreasing expression of ENaC at the cell membrane via a mechanism that
involves the promotion of Nedd4-2-dependent internalization of the
channel.Amiloride-sensitive epithelial Na+ channels
(ENaC)3 are membrane
proteins that are expressed in salt-absorptive epithelia, including the distal
collecting tubules of the kidney, the mucosa of the distal colon, the
respiratory epithelium, and the excretory ducts of sweat and salivary glands
(1–4).
Na+ absorption via ENaC is critical to the normal regulation of
Na+ and fluid homeostasis and is important for maintaining blood
pressure (5) and the volume of
fluid in the respiratory passages
(6). Increased ENaC activity
has been implicated in the salt-sensitive inherited form of hypertension,
Liddle''s syndrome (7), and
dehydration of the surface of the airway epithelium in the pathology
associated with cystic fibrosis lung disease
(8).Expression of ENaC at the cell membrane surface is regulated by the E3
ubiquitin protein ligase, Nedd4-2 (neural precursor cell
expressed developmentally down-regulated
protein 4) (9). Interaction
between the WW domains of Nedd4-2 and the proline-rich PY motifs
(PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect
on the channel (10,
11). This interaction leads to
ubiquitination-dependent internalization of ENaC
(12,
13). Several regulators of
ENaC exert their effects on the channel by modulating the action of Nedd4-2.
For instance, serum and glucocorticoid-dependent protein kinase
(14), protein kinase B
(15), and G protein-coupled
receptor kinase (16)
up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of
cellular mechanisms that underlie internalization of ENaC remain to be
elucidated, the physiological significance of Nedd4-dependent internalization
of the channel has been well established. For instance, heritable mutations
that delete the cytosolic termini of the β-or γ-subunit of ENaC,
which contain the proline-rich motifs, are known to cause hyperactivity of
ENaC in the kidney (17) and
increase cell surface expression of the channel
(7,
18).The plasma membranes of most cell types contain lipid raft microdomains
that are enriched with glycosphingolipid and cholesterol
(19), that have distinctive
biophysical properties, and that selectively include or exclude signaling
molecules (20). These
microdomains promote clustering of an array of integral membrane proteins in
the membrane leaflets (21) and
may be important for organizing cascades of signaling molecules
(22,
23). Processes in which raft
microdomains are involved include the intracellular transport of proteins and
lipids to the cell membrane
(24), the endocytotic
retrieval of membrane proteins
(25,
26), and signal transduction
(27,
28). In addition, segregation
of signaling molecules within lipid rafts may facilitate cross-talk between
signal transduction pathways
(29), a phenomenon that may be
important in ensuring rapid and efficient integration of multiple cellular
signaling events (30,
31). Of particular interest is
the subpopulation of lipid rafts enriched with caveolin proteins. Caveolin-1
(Cav-1), a major caveolin isoform expressed in nonmuscle cells, has been
identified as being involved in diverse cellular functions, such as vesicular
transport, cholesterol homeostasis, and signal transduction
(32). Cav-1 also regulates the
activity and membrane expression of ion channels and transporters
(28).In epithelia, the majority of lipid rafts exist at the apical membrane
surface (22). Pools of ENaC
(33–36)
and several proteins that regulate activity of ENaC, such as Nedd4
(37), protein kinase B
(38), protein kinase C
(39), Go
(40), and the G
protein-coupled receptor kinase
(41), have been identified in
detergent-insoluble and cholesterol-rich membrane fractions from a variety of
cell types, consistent with localization of these proteins in lipid rafts.
Furthermore, detergent-free buoyant density separation of lipid rafts has
revealed the presence of Cav-1 with ENaC in the lipid raft-rich membrane
fraction (35). The
physiological role of lipid rafts in the regulation of ENaC has been the
subject of many recent investigations. Most of these studies used a
pharmacological agent, methyl-β-cyclodextrin (MβCD), to promote
redistribution of proteins away from the cholesterol-enriched membrane
domains. The results were, however, inconclusive. In some studies, MβCD
treatment was found to inhibit open probability
(42) or cell surface
expression of ENaC (35),
whereas others found no direct effect of MβCD on the channel
(33,
43).Despite a number of studies into the role of lipid rafts on the regulation
of ENaC, little is known about the physiological relevance of caveolins to the
function of this ion channel. In the present study, we use gene interference
and gene expression techniques to determine the role of Cav-1 in the
regulation of ENaC activity. We provide evidence of the association of Cav-1
with ENaC and evidence that Cav-1 negatively regulates both activity and
abundance of ENaC at the surface of epithelial cells. Importantly, we
demonstrate, for the first time, that the mechanism by which Cav-1 regulates
activity of ENaC involves the E3 ubiquitin protein ligase, Nedd4-2. 相似文献
10.
11.
Katrine M. Andersen Louise Madsen S?ren Prag Anders H. Johnsen Colin A. Semple Klavs B. Hendil Rasmus Hartmann-Petersen 《The Journal of biological chemistry》2009,284(22):15246-15254
The 26 S proteasome is a large proteolytic machine, which degrades most
intracellular proteins. We found that thioredoxin, Txnl1/TRP32, binds to
Rpn11, a subunit of the regulatory complex of the human 26 S proteasome. Txnl1
is abundant, metabolically stable, and widely expressed and is present in the
cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential
of about-250 mV. Mutant Txnl1 with one active site cysteine replaced by serine
formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26 S
proteasome. eEF1A1 is therefore a likely physiological substrate. In response
to knockdown of Txnl1, ubiquitin-protein conjugates were moderately
stabilized. Hence, Txnl1 is the first example of a direct connection between
protein reduction and proteolysis, two major intracellular protein quality
control mechanisms.Degradation of proteins in eukaryotic cells plays a pivotal role in the
regulation of several important processes, including cell division, antigen
presentation, and signal transduction
(1). Most intracellular
proteins are degraded by the 26 S proteasome, a 2.5-MDa protease complex
composed of more than 30 different subunits
(2).To become degraded, proteins are typically first conjugated to a chain of
ubiquitin moieties. This reaction is catalyzed by ubiquitin ligases. The
ubiquitin chains lend the proteins affinity for the 26 S proteasome
(3). For efficient degradation,
certain ubiquitylated proteins are shuttled to the 26 S proteasome by
substrate recruiting factors, such as Rad23, Dsk2, and eEF1A
(4,
5).The 26 S proteasome is composed of two stable subcomplexes, the
proteolytically active 20 S core and 19 S regulatory complexes, which bind to
one or both ends of the cylindrical 20 S core particle
(6). The regulatory complexes
first recognize the ubiquitylated substrates
(3), before the substrates are
deubiquitylated (7,
8), unfolded
(9,
10), and translocated into the
20 S particle for degradation.Although the 26 S proteasome has been known for more than 20 years
(11), novel subunits and
cofactors have been described recently
(12,
13). Here we report another
novel proteasome-associated protein, Txnl1 (thioredoxin-like protein 1), that
associates directly with the proteasome subunit Rpn11. Txnl1 exhibits
thioredoxin activity and targets eEF1A1 in vivo. Previous reports
have shown that eEF1A1 transfers misfolded nascent proteins from the ribosome
to the 26 S proteasome for degradation
(5,
14,
15). Accordingly,
ubiquitin-protein conjugates were stabilized upon knockdown of Txnl1
expression. Txnl1 therefore directly links protein reduction and proteolysis,
two major intracellular protein quality control mechanisms. 相似文献
12.
Marjelo A. Mines J. Shawn Goodwin Lee E. Limbird Fei-Fei Cui Guo-Huang Fan 《The Journal of biological chemistry》2009,284(9):5742-5752
The chemokine receptor CXCR4 plays important roles in the immune and
nervous systems. Abnormal expression of CXCR4 contributes to cancer and
inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4
ubiquitination is known to accelerate CXCR4 degradation, little is known about
counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist,
induces a time-dependent association of USP14 with CXCR4, or its C terminus,
that is not mimicked by USP2A, USP4, or USP7, other members of the
deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is
time-dependent following CXCL12 stimulation. The physical interaction of CXCR4
and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor;
knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4
deubiquitination, whereas overexpression of USP14 promotes CXCR4
deubiquitination. We also observed that ubiquitination of CXCR4 facilitated
receptor degradation, whereas overexpression of USP14 or RNAi-induced
knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most
interestingly, CXCR4-mediated chemotactic cell migration was blocked by either
overexpression or RNAi-mediated knockdown of USP14, implying that a
CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated
state of the receptor, is critical for the ligand gradient sensing and
directed motility required for chemokine-mediated chemotaxis. Our observation
that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does
not mediate a chemotactic response to CXCL12, indicates the importance of this
covalent modification not only in marking receptors for degradation but also
for permitting CXCR4-mediated signaling. Finally, the indistinguishable
activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in
response to CXCL12 may be independent of the ERK cascade.The CXCR4 (CXC chemokine receptor 4) is a member of the chemokine receptor
family, which belongs to the superfamily of G protein-coupled receptors
(GPCRs)2
(1). Its ligand, CXCL12, also
known as SDF-1α, also binds to RDC1, another chemokine receptor that is
being proposed to be renamed as CXCR7
(2). CXCR4 mediates
CXCL12-induced migration of peripheral blood lymphocytes
(3), CD34+
progenitor cells (4), and pre-
and pro-B cell lines (5). CXCR4
also plays an important role in the development of the immune system, because
mouse embryos lacking either expression of the CXCR4 receptor or of its CXCL12
ligand are embryonic lethal and also manifest abnormalities in B cell
lymphopoiesis and bone marrow myelopoiesis
(3,
6,
7). The altered cerebellar
neuron migration in mice null for the CXCR4 receptor also suggests a role for
this receptor in central nervous system development. Abnormal expression
and/or function of CXCR4 have been implicated in a number of diseases,
including human immunodeficiency virus infection
(8), cardiovascular disease
(9), allergic inflammatory
disease (10),
neuroinflammation (11),
neurodegenerative diseases
(12,
13), and cancers
(14-24).Stimulation of CXCR4 triggers various intracellular signaling cascades
(1,
14,
25-27),
such as extracellular signal-regulated kinase (ERK), which likely contribute
to CXCR4-induced cell proliferation, differentiation, and/or migration. Ligand
stimulation of CXCR4 also induces endocytosis of these receptors, which are
targeted to lysosomes for degradation through a pathway involving
ubiquitination of the C-terminal lysine residues
(28). CXCR4 ubiquitination can
be catalyzed by a member of the HECT family of E3 ligases, AIP4
(atrophin-interacting protein 4)
(29,
30). The ubiquitinated CXCR4
is delivered to the endosomal compartments via a regulated pathway involving
several adaptor proteins
(31).It has been noted that deubiquitination also regulates the fate and
function of ubiquitin-conjugated proteins. Deubiquitinating enzymes, which
catalyze the removal of ubiquitin from ubiquitin-conjugated proteins,
represent the largest family of enzymes in the ubiquitin system, implying the
possibility that substrate selectivity is even greater for these enzymes than
for those that catalyze ubiquitin ligation. Little is known about the
mechanisms of CXCR4 deubiquitination and their regulation by receptor ligands.
A proteomics study revealed that the steady state level of USP14 was increased
upon CXCL12 stimulation of target cells
(32), and preliminary studies
revealed that ligand stimulation led to enhanced association of USP14 with the
CXCR4. The present studies were undertaken to ascertain the functional
consequences of this interaction, the selectivity of CXCR4 for USP14, when
compared with three other deubiquitinating enzymes, USP2a, USP4, and USP7, and
the impact of modifying the ubiquitinated state of the receptor on CXCR4
turnover, CXCL12-evoked chemotaxis, and CXCL12-induced activation of ERK. 相似文献
13.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献
14.
Andrés Norambuena Claudia Metz Lucas Vicu?a Antonia Silva Evelyn Pardo Claudia Oyanadel Loreto Massardo Alfonso González Andrea Soza 《The Journal of biological chemistry》2009,284(19):12670-12679
Galectins have been implicated in T cell homeostasis playing complementary
pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent
pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase
D/phosphatidic acid signaling pathway that has not been reported for any
galectin before. Gal-8 increases phosphatidic signaling, which enhances the
activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a
subsequent decrease in basal protein kinase A activity. Strikingly, rolipram
inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4
activation releases a negative influence of cAMP/protein kinase A on ERK1/2.
The resulting strong ERK1/2 activation leads to expression of the death factor
Fas ligand and caspase-mediated apoptosis. Several conditions that decrease
ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking
antibodies. In addition, experiments with freshly isolated human peripheral
blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28,
show that Gal-8 is pro-apoptotic on activated T cells, most likely on a
subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic
lupus erythematosus block the apoptotic effect of Gal-8. These results
implicate Gal-8 as a novel T cell suppressive factor, which can be
counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly
studied as regulators of the immune response and potential therapeutic agents
for autoimmune disorders (1).
To date, 15 galectins have been identified and classified according with the
structural organization of their distinctive monomeric or dimeric carbohydrate
recognition domain for β-galactosides
(2,
3). Galectins are secreted by
unconventional mechanisms and once outside the cells bind to and cross-link
multiple glycoconjugates both at the cell surface and at the extracellular
matrix, modulating processes as diverse as cell adhesion, migration,
proliferation, differentiation, and apoptosis
(4–10).
Several galectins have been involved in T cell homeostasis because of their
capability to kill thymocytes, activated T cells, and T cell lines
(11–16).
Pro-apoptotic galectins might contribute to shape the T cell repertoire in the
thymus by negative selection, restrict the immune response by eliminating
activated T cells at the periphery
(1), and help cancer cells to
escape the immune system by eliminating cancer-infiltrating T cells
(17). They have also a
promising therapeutic potential to eliminate abnormally activated T cells and
inflammatory cells (1). Studies
on the mostly explored galectins, Gal-1, -3, and -9
(14,
15,
18–20),
as well as in Gal-2 (13),
suggest immunosuppressive complementary roles inducing different pathways to
apoptosis. Galectin-8
(Gal-8)4 is one of the
most widely expressed galectins in human tissues
(21,
22) and cancerous cells
(23,
24). Depending on the cell
context and mode of presentation, either as soluble stimulus or extracellular
matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis
(6,
7,
9,
10,
22,
25). Its role has been mostly
studied in relation to tumor malignancy
(23,
24). However, there is some
evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or
inflammatory disorders. For instance, the intrathymic expression and
pro-apoptotic effect of Gal-8 upon CD4highCD8high
thymocytes suggest a role for Gal-8 in shaping the T cell repertoire
(16). Gal-8 could also
modulate the inflammatory function of neutrophils
(26), Moreover Gal-8-blocking
agents have been detected in chronic autoimmune disorders
(10,
27,
28). In rheumatoid arthritis,
Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid
cells, but can be counteracted by a specific rheumatoid version of CD44
(CD44vRA) (27). In systemic
lupus erythematosus (SLE), a prototypic autoimmune disease, we recently
described function-blocking autoantibodies against Gal-8
(10,
28). Thus it is important to
define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in
immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with
specific integrins, such as α1β1, α3β1, and
α5β1 but not α4β1, and as a matrix protein promotes cell
adhesion and asymmetric spreading through activation of the extracellular
signal-regulated kinases 1 and 2 (ERK1/2)
(10). These early effects
occur within 5–30 min. However, ERK1/2 signaling supports long term
processes such as T cell survival or death, depending on the moment of the
immune response. During T cell activation, ERK1/2 contributes to enhance the
expression of interleukin-2 (IL-2) required for T cell clonal expansion
(29). It also supports T cell
survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by
other previously activated T cells
(30,
31). Later on, ERK1/2 is
required for activation-induced cell death, which controls the extension of
the immune response by eliminating recently activated and restimulated T cells
(32,
33). In activation-induced
cell death, ERK1/2 signaling contributes to enhance the expression of FasL and
its receptor Fas/CD95 (32,
33), which constitute a
preponderant pro-apoptotic system in T cells
(34). Here, we ask whether
Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to
participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling
(35) deserves special
attention. cAMP/PKA signaling plays an immunosuppressive role in T cells
(36) and is altered in SLE
(37). Phosphodiesterases
(PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA
during T cell activation (38,
39). PKA has been described to
control the activity of ERK1/2 either positively or negatively in different
cells and processes (35). A
little explored integration among ERK1/2 and PKA occurs via phosphatidic acid
(PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that
hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA
plays roles in signaling interacting with a variety of targeting proteins that
bear PA-binding domains (40).
In this way PA recruits Raf-1 to the plasma membrane
(41). It is also converted by
phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG),
which among other functions, recruits and activates the GTPase Ras
(42). Both Ras and Raf-1 are
upstream elements of the ERK1/2 activation pathway
(43). In addition, PA binds to
and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP
levels and PKA down-regulation
(44). The regulation and role
of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell
homeostasis, because it is also unknown whether galectins stimulate the PLD/PA
pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering
cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our
results for the first time show that a galectin increases the PA levels,
down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE
activity, and induces an ERK1/2-dependent expression of the pro-apoptotic
factor FasL. The enhanced PDE activity induced by Gal-8 is required for the
activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces
apoptosis in human peripheral blood mononuclear cells (PBMC), especially after
activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other
galectins the property of killing activated T cells contributing to the T cell
homeostasis. The pathway involves a particularly integrated signaling context,
engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for
galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its
susceptibility to inhibition by anti-Gal-8 autoantibodies. 相似文献
15.
16.
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
17.
Mario Perkovi? Stanislaw Schmidt Daniela Marino Rebecca A. Russell Benjamin Stauch Henning Hofmann Ferdinand Kopietz Bj?rn-Philipp Kloke J?rg Zielonka Heike Str?ver Johannes Hermle Dirk Lindemann Vinay K. Pathak Gisbert Schneider Martin L?chelt Klaus Cichutek Carsten Münk 《The Journal of biological chemistry》2009,284(9):5819-5826
18.
19.
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
20.
Tushar K. Beuria Srinivas Mullapudi Eugenia Mileykovskaya Mahalakshmi Sadasivam William Dowhan William Margolin 《The Journal of biological chemistry》2009,284(21):14079-14086
Cytokinesis in bacteria depends upon the contractile Z ring, which is
composed of dynamic polymers of the tubulin homolog FtsZ as well as other
membrane-associated proteins such as FtsA, a homolog of actin that is required
for membrane attachment of the Z ring and its subsequent constriction. Here we
show that a previously characterized hypermorphic mutant FtsA (FtsA*)
partially disassembled FtsZ polymers in vitro. This effect was
strictly dependent on ATP or ADP binding to FtsA* and occurred at
substoichiometric levels relative to FtsZ, similar to cellular levels.
Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical
concentration for FtsZ assembly but was able to disassemble preformed FtsZ
polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination
of the inhibited FtsZ polymers revealed a transition from long, straight
polymers and polymer bundles to mainly short, curved protofilaments. These
results indicate that a bacterial actin, when activated by adenine
nucleotides, can modify the length distribution of bacterial tubulin polymers,
analogous to the effects of actin-depolymerizing factor/cofilin on
F-actin.Bacterial cell division requires a large number of proteins that colocalize
to form a putative protein machine at the cell membrane
(1). This machine, sometimes
called the divisome, recruits enzymes to synthesize the septum cell wall and
to initiate and coordinate the invagination of the cytoplasmic membrane (and
in Gram-negative bacteria, the outer membrane). The most widely conserved and
key protein for this process is FtsZ, a homolog of tubulin that forms a ring
structure called the Z ring, which marks the site of septum formation
(2,
3). Like tubulin, FtsZ
assembles into filaments with GTP but does not form microtubules
(4). The precise assembly state
and conformation of these FtsZ filaments at the division ring is not clear,
although recent electron tomography work suggests that the FtsZ ring consists
of multiple short filaments tethered to the membrane at discrete junctures
(5), which may represent points
along the filaments bridged by membrane anchor proteins.In Escherichia coli, two of these anchor proteins are known. One
of these, ZipA, is not well conserved but is an essential protein in E.
coli. ZipA binds to the C-terminal tail of FtsZ
(6–8),
and purified ZipA promotes bundling of FtsZ filaments in vitro
(9,
10). The other, FtsA, is also
essential in E. coli and is more widely conserved among bacterial
species. FtsA is a member of the HSP70/actin superfamily
(11,
12), and like ZipA, it
interacts with the C-terminal tail of FtsZ
(7,
13–15).
FtsA can self-associate (16,
17) and bind ATP
(12,
18), but reports of ATPase
activity vary, with Bacillus subtilis FtsA having high activity
(19) and Streptococcus
pneumoniae FtsA exhibiting no detectable activity
(20). There are no reports of
any other in vitro activities of FtsA, including effects on FtsZ
assembly.Understanding how FtsA affects FtsZ assembly is important because FtsA has
a number of key activities in the cell. It is required for recruitment of a
number of divisome proteins
(21,
22) and helps to tether the Z
ring to the membrane via a C-terminal membrane-targeting sequence
(23). FtsA, like ZipA and
other divisome proteins, is necessary to activate the contraction of the Z
ring (24,
25). In E. coli, the
FtsA:FtsZ ratio is crucial for proper cell division, with either too high or
too low a ratio inhibiting septum formation
(26,
27). This ratio is roughly
1:5, with ∼700 molecules of FtsA and 3200 molecules of FtsZ per cell
(28), which works out to
concentrations of 1–2 and 5–10 μm, respectively.Another interesting property of FtsA is that single residue alterations in
the protein can result in significant enhancement of divisome activity. For
example, the R286W mutation of FtsA, also called FtsA*, can substitute for the
native FtsA and divide the cell. However, this mutant FtsA causes E.
coli cells to divide at less than 80% of their normal length
(29) and allows efficient
division of E. coli cells in the absence of ZipA
(30), indicating that it has
gain-of-function activity. FtsA* and other hypermorphic mutations such as
E124A and I143L can also increase division activity in cells lacking other
essential divisome components
(31–33).
The R286W and E124A mutants of FtsA also bypass the FtsA:FtsZ ratio rule,
allowing cell division to occur at higher ratios than with
WT2 FtsA. This may be
because the altered FtsA proteins self-associate more readily than WT FtsA,
which may cause different changes in FtsZ assembly state as compared with WT
FtsA (17,
34).In this study, we use an in vitro system with purified FtsZ and a
purified tagged version of FtsA* to elucidate the role of FtsA in activating
constriction of the Z ring in vivo. We show that FtsA*, at
physiological concentrations in the presence of ATP or ADP, has significant
effects on the assembly of FtsZ filaments. 相似文献