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1.
Bess Frost Julian Ollesch Holger Wille Marc I. Diamond 《The Journal of biological chemistry》2009,284(6):3546-3551
Tauopathies are sporadic and genetic neurodegenerative diseases
characterized by aggregation of the microtubule-associated protein Tau. Tau
pathology occurs in over 20 phenotypically distinct neurodegenerative
diseases, including Alzheimer disease and frontotemporal dementia. The
molecular basis of this diversity among sporadic tauopathies is unknown, but
distinct fibrillar wild-type (WT) Tau conformations could play a role. Using
Fourier transform infrared spectroscopy, circular dichroism, and electron
microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have
distinct secondary structures, fragilities, and morphologies. Furthermore,
P301L/V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar
conformation, termed WT*, that is maintained over multiple seeding reactions.
WT* has secondary structure, fragility, and morphology that are similar to
P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of
conformational diversity that arises via templated conformation change, as has
been described for amyloid β, β2-microglobulin, and prion
proteins.Tau filament deposition in Alzheimer disease, frontotemporal dementia, and
other tauopathies correlates closely with cognitive dysfunction and cell death
(1). About 10% of tauopathies
are due to dominant mutations in the Tau gene. These diseases are collectively
termed frontotemporal dementia with parkinsonism linked to chromosome 17,
FTDP-17
(2–4).
Most of the mutations occur in the microtubule-binding region of the Tau
protein, which is thought to be both its functional
(5) and pathogenic
(6) “core.”
Approximately 90% of tauopathies occur sporadically and involve only wild-type
(WT)2 Tau. Both
familial and sporadic tauopathies vary by regional involvement, disease
duration, age of onset, Tau isoform expression, and fibril morphology
(7). It is unknown how the
pathology of WT Tau might generate distinct disease phenotypes in sporadic
tauopathies, and whether conformational diversity of the protein could
potentially play a role in disease, as it does in prion disorders
(8,
9).Mutations in the Tau gene can generate conformationally distinct Tau
species. Structural differences between in vitro prepared WT, G272V,
N279K, P301L, V337M, and ΔK280 Tau fibrils have been observed using
Fourier transform infrared spectroscopy (FTIR)
(10), and differential
susceptibilities to protease cleavage in vitro have been described
for WT and P301L Tau fibrils
(11). Furthermore, Tau
filaments extracted from diseased brain are often morphologically distinct,
consisting of straight or paired helical filaments of various periodicities
and widths (12). It is unknown
whether WT Tau can assume self-propagating, structurally distinct fibrillar
conformations, as has been described for amyloid β peptide
(13),
β2-microglobulin
(14), and the prion protein
(15). In this study, we have
used biochemical and biophysical methods to test the hypothesis that WT Tau
fibrils exhibit conformational diversity that is maintained by templated
conformation change. 相似文献
2.
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
3.
4.
Dong Han Hamid Y. Qureshi Yifan Lu Hemant K. Paudel 《The Journal of biological chemistry》2009,284(20):13422-13433
In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked
to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms
paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is
phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa
bands on SDS-gel, and does not promote microtubule assembly. Upon
dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on
SDS-gels in a manner similar to tau that is isolated from normal brain and
promotes microtubule assembly. The site(s) that inhibits microtubule
assembly-promoting activity when phosphorylated in the diseased brain is not
known. In this study, when tau was phosphorylated by Cdk5 in vitro,
its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a
time-dependent manner. This mobility shift correlated with phosphorylation at
Ser202, and Ser202 phosphorylation inhibited tau
microtubule-assembly promoting activity. When several tau point mutants were
analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17,
but not nonspecific mutations S214A and S262A, promoted Ser202
phosphorylation and mobility shift to a ∼68-kDa band. Furthermore,
Ser202 phosphorylation inhibited the microtubule assembly-promoting
activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17
missense mutations, by promoting phosphorylation at Ser202, inhibit
the microtubule assembly-promoting activity of tau in vitro,
suggesting that Ser202 phosphorylation plays a major role in the
development of NFT pathology in AD and related tauopathies.Neurofibrillary tangles
(NFTs)4 and senile
plaques are the two characteristic neuropathological lesions found in the
brains of patients suffering from Alzheimer disease (AD). The major fibrous
component of NFTs are paired helical filaments (PHFs) (for reviews see Refs.
1–3).
Initially, PHFs were found to be composed of a protein component referred to
as “A68” (4).
Biochemical analysis reveled that A68 is identical to the
microtubule-associated protein, tau
(4,
5). Some characteristic
features of tau isolated from PHFs (PHF-tau) are that it is abnormally
hyperphosphorylated (phosphorylated on more sites than the normal brain tau),
does not bind to microtubules, and does not promote microtubule assembly
in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind
to and promote microtubule assembly
(6,
7). Tau hyperphosphorylation is
suggested to cause microtubule instability and PHF formation, leading to NFT
pathology in the brain
(1–3).PHF-tau is phosphorylated on at least 21 proline-directed and
non-proline-directed sites (8,
9). The individual contribution
of these sites in converting tau to PHFs is not entirely clear. However, some
sites are only partially phosphorylated in PHFs
(8), whereas phosphorylation on
specific sites inhibits the microtubule assembly-promoting activity of tau
(6,
10). These observations
suggest that phosphorylation on a few sites may be responsible and sufficient
for causing tau dysfunction in AD.Tau purified from the human brain migrates as ∼50–60-kDa bands on
SDS-gel due to the presence of six isoforms that are phosphorylated to
different extents (2). PHF-tau
isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68
kDa-bands on an SDS-gel (4,
5,
11). Studies have shown that
∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau
band as an ∼69-kDa band in these studies) are present only in brain areas
affected by NFT degeneration
(12,
13). Their amount is
correlated with the NFT densities at the affected brain regions. Moreover, the
increase in the amount of ∼64- and 68-kDa band tau in the brain correlated
with a decline in the intellectual status of the patient. The ∼64- and
68-kDa tau bands were suggested to be the pathological marker of AD
(12,
13). Biochemical analyses
determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which
upon dephosphorylation, migrated as normal tau on SDS-gel
(4,
5,
11). Tau sites involved in the
tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role
in AD pathology (12,
13). It is not known whether
phosphorylation at all 21 PHF-sites is required for the tau mobility shift in
AD. However, in vitro the tau mobility shift on SDS-gel is sensitive
to phosphorylation only on some sites
(6,
14). It is therefore possible
that in the AD brain, phosphorylation on some sites also causes a tau mobility
shift. Identification of such sites will significantly enhance our knowledge
of how NFT pathology develops in the brain.PHFs are also the major component of NFTs found in the brains of patients
suffering from a group of neurodegenerative disorders collectively called
tauopathies (2,
11). These disorders include
frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17),
corticobasal degeneration, progressive supranuclear palsy, and Pick disease.
Each PHF-tau isolated from autopsied brains of patients suffering from various
tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands
on SDS-gel, and is incapable of binding to microtubules. Upon
dephosphorylation, the above referenced PHF-tau migrates as a normal tau on
SDS-gel, binds to microtubules, and promotes microtubule assembly
(2,
11). These observations
suggest that the mechanisms of NFT pathology in various tauopathies may be
similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may
be an indicator of the disease. The tau gene is mutated in familial FTDP-17,
and these mutations accelerate NFT pathology in the brain
(15–18).
Understanding how FTDP-17 mutations promote tau phosphorylation can provide a
better understanding of how NFT pathology develops in AD and various
tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and
P301L tau mutations reduce tau phosphorylation
(19,
20). In COS cells, although
G272V, P301L, and V337M mutations do not show any significant affect, the
R406W mutation caused a reduction in tau phosphorylation
(21,
22). When expressed in SH-SY5Y
cells subsequently differentiated into neurons, the R406W, P301L, and V337M
mutations reduce tau phosphorylation
(23). In contrast, in
hippocampal neurons, R406W increases tau phosphorylation
(24). When phosphorylated by
recombinant GSK3β in vitro, the P301L and V337M mutations do not
have any effect, and the R406W mutation inhibits phosphorylation
(25). However, when incubated
with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations
stimulate tau phosphorylation
(26). The mechanism by which
FTDP-17 mutations promote tau phosphorylation leading to development of NFT
pathology has remained unclear.Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that
phosphorylates tau in the brain
(27,
28). In this study, to
determine how FTDP-17 missense mutations affect tau phosphorylation, we
phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by
Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility
shift to ∼64- and 68 kDa-bands. Although the mobility shift to a
∼64-kDa band is achieved by phosphorylation at Ser396/404 or
Ser202, the mobility shift to a 68-kDa band occurs only in response
to phosphorylation at Ser202. We show that in
vitro, FTDP-17 missense mutations, by promoting phosphorylation at
Ser202, enhance the mobility shift to ∼64- and 68-kDa bands and
inhibit the microtubule assembly-promoting activity of tau. Our data suggest
that Ser202 phosphorylation is the major event leading to NFT
pathology in AD and related tauopathies. 相似文献
5.
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
6.
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
7.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
8.
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
9.
10.
11.
12.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
13.
14.
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
15.
Jonathan M. Budzik So-Young Oh Olaf Schneewind 《The Journal of biological chemistry》2009,284(19):12989-12997
Bacillus cereus and other Gram-positive bacteria elaborate pili
via a sortase D-catalyzed transpeptidation mechanism from major and minor
pilin precursor substrates. After cleavage of the LPXTG sorting
signal of the major pilin, BcpA, sortase D forms an amide bond between the
C-terminal threonine and the amino group of lysine within the YPKN motif of
another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the
BcpA sorting signal, resulting in a covalent bond between BcpA and the cell
wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the
minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal
threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby
positioning BcpB at the tip of pili. Thus, unique attributes of the sorting
signals of minor pilins provide Gram-positive bacteria with a universal
mechanism ordering assembly of pili.Sortases catalyze transpeptidation reactions to assemble proteins in the
envelope of Gram-positive bacteria
(1). Secreted proteins require
a C-terminal sorting signal for sortase recognition such that sortase cleaves
the substrate at a short peptide motif and forms a thioester-linked
intermediate to its active site cysteine
(2–4).
Nucleophilic attack by an amino group within the bacterial envelope resolves
the thioester intermediate, generating an amide bond tethering surface
proteins at their C terminus onto Gram-positive bacteria
(5). Four classes of sortases
can be distinguished on the basis of sequence homology and substrate
recognition (6,
7). Sortase A cleaves secreted
protein at LPXTG sorting signals and recognizes the amino group of
lipid II peptidoglycan precursors as a nucleophile
(8,
9). Sortase B cleaves protein
substrates at NPQTN sorting signals
(10). This enzyme immobilizes
proteins within fully assembled cell walls, utilizing the cell wall
cross-bridge as a nucleophile
(11). Sortase C cuts LPNTA
sorting signals and anchors proteins to the peptidoglycan cross-bridges in
sporulating bacteria (12,
13). Finally, sortase D
catalyzes transpeptidation reactions in the assembly of pili
(14,
15). Sortase D recognizes the
amino group of lysine residues within the YPKN motif of pilin subunits as
nucleophiles (16). The
resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow
the pilus fiber (16,
17).Pili of Gram-positive bacteria comprised either two or three different
pilin subunits synthesized as cytoplasmic precursors with N-terminal signal
peptides and C-terminal sorting signals (P1 precursors)
(14,
18). After translocation
across the plasma membrane, P2 precursor species arise from removal of the
signal peptide from P1 precursors by a signal peptidase
(16). Bacillus cereus
pili are composed of two subunits; that is, the major pilin, BcpA, and the
minor pilin, BcpB (15). In
contrast to BcpA, which is deposited throughout the pilus, BcpB is found at
fiber tip (15). Sortase D
cleaves the BcpA LPXTG motif sorting signal between the threonine and
glycine residues to form an amide bond to the ε-amino group of the lysine
within the YPKN motif of adjacent BcpA subunits
(16). However, sortase A also
cleaves BcpA precursors, which are subsequently linked to the side chain amino
group of meso-diaminopimelic acid within lipid II
(19). The latter reaction
serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall
envelope (19).The conservation of sortase D, the YPKN motif, and C-terminal sorting
signal in major pilin subunits suggest a universal pilus assembly mechanism
among Gram-positive bacteria
(14,
20). However, the molecular
mechanism whereby bacilli deposit BcpB, the minor pilin, at the tip of BcpA
pili is not known. Although the BcpB precursor harbors an N-terminal signal
peptide and a C-terminal IPNTG sorting signal, it lacks the YPKN pilin motif
of the major subunit (15).
Furthermore, the substrate properties of the BcpB IPNTG sorting signal for the
four classes of sortases expressed by bacilli has yet to be established. 相似文献
16.
17.
18.
19.
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