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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
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Tatsuhiro Sato Akio Nakashima Lea Guo Fuyuhiko Tamanoi 《The Journal of biological chemistry》2009,284(19):12783-12791
Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway
by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully
reproduced in vitro by using mTORC1 immunoprecipitated by the use of
anti-raptor antibody from mammalian cells starved for nutrients. The low
in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is
dramatically increased by the addition of recombinant Rheb. On the other hand,
the addition of Rheb does not activate mTORC2 immunoprecipitated from
mammalian cells by the use of anti-rictor antibody. The activation of mTORC1
is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42
did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition,
the activation is dependent on the presence of bound GTP. We also find that
the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a
recently proposed mediator of Rheb action, appears not to be involved in the
Rheb-dependent activation of mTORC1 in vitro, because the preparation
of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of
Rheb results in a significant increase of binding of the substrate protein
4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that
competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation
of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated
by Rheb. Rheb does not induce autophosphorylation of mTOR. These results
suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to
regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins
(1). We have shown that Rheb
proteins are conserved and are found from yeast to human
(2). Although yeast and fruit
fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or
simply Rheb) and Rheb2 (RhebL1)
(2). Structurally, these
proteins contain G1-G5 boxes, short stretches of amino acids that define the
function of the Ras superfamily G-proteins including guanine nucleotide
binding (1,
3,
4). Rheb proteins have a
conserved arginine at residue 15 that corresponds to residue 12 of Ras
(1). The effector domain
required for the binding with downstream effectors encompasses the G2 box and
its adjacent sequences (1,
5). Structural analysis by
x-ray crystallography further shows that the effector domain is exposed to
solvent, is located close to the phosphates of GTP especially at residues
35–38, and undergoes conformational change during GTP/GDP exchange
(6). In addition, all Rheb
proteins end with the CAAX (C is cysteine, A is an aliphatic amino
acid, and X is the C-terminal amino acid) motif that signals
farnesylation. In fact, we as well as others have shown that these proteins
are farnesylated
(7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling
pathway that plays central roles in regulating protein synthesis and growth in
response to nutrient, energy, and growth conditions
(10–14).
Rheb is down-regulated by a TSC1·TSC2 complex that acts as a
GTPase-activating protein for Rheb
(15–19).
Recent studies established that the GAP domain of TSC2 defines the functional
domain for the down-regulation of Rheb
(20). Mutations in the
Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms
include the appearance of benign tumors called hamartomas at different parts
of the body as well as neurological symptoms
(21,
22). Overexpression of Rheb
results in constitutive activation of mTOR even in the absence of nutrients
(15,
16). Two mTOR complexes,
mTORC1 and mTORC2, have been identified
(23,
24). Whereas mTORC1 is
involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is
involved in the phosphorylation of Akt in response to insulin. It has been
suggested that Rheb is involved in the activation of mTORC1 but not mTORC2
(25).Although Rheb is clearly involved in the activation of mTOR, the mechanism
of activation has not been established. We as well as others have suggested a
model that involves the interaction of Rheb with the TOR complex
(26–28).
Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was
reported (29). Rheb has been
shown to interact with mTOR
(27,
30), and this may involve
direct interaction of Rheb with the kinase domain of mTOR
(27). However, this Rheb/mTOR
interaction is a weak interaction and is not dependent on the presence of GTP
bound to Rheb (27,
28). Recently, a different
model proposing that FKBP38 (FK506-binding protein
38) mediates the activation of
mTORC1 by Rheb was proposed
(31,
32). In this model, FKBP38
binds mTOR and negatively regulates mTOR activity, and this negative
regulation is blocked by the binding of Rheb to FKBP38. However, recent
reports dispute this idea
(33).To further characterize Rheb activation of mTOR, we have utilized an in
vitro system that reproduces activation of mTORC1 by the addition of
recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved
cells using anti-raptor antibody and have shown that its kinase activity
against 4E-BP1 is dramatically increased by the addition of recombinant Rheb.
Importantly, the activation of mTORC1 is specific to Rheb and is dependent on
the presence of bound GTP as well as an intact effector domain. FKBP38 is not
detected in our preparation and further investigation suggests that FKBP38 is
not an essential component for the activation of mTORC1 by Rheb. Our study
revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1
rather than increasing the kinase activity of mTOR. 相似文献
10.
Parmil K. Bansal Amanda Nourse Rashid Abdulle Katsumi Kitagawa 《The Journal of biological chemistry》2009,284(6):3586-3592
The kinetochore, which consists of DNA sequence elements and structural
proteins, is essential for high-fidelity chromosome transmission during cell
division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore
complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study,
we show that Sgt1 forms homodimers by performing in vitro and in
vivo immunoprecipitation and analytical ultracentrifugation analyses.
Analyses of the dimerization of Sgt1 deletion proteins showed that the
Skp1-binding domain (amino acids 1–211) contains the Sgt1
homodimerization domain. Also, the Sgt1 mutant proteins that were unable to
dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is
important for Sgt1-Skp1 binding. Restoring dimerization activity of a
dimerization-deficient sgt1 mutant (sgt1-L31P) by using the
CENP-B (centromere protein-B) dimerization
domain suppressed the temperature sensitivity, the benomyl sensitivity, and
the chromosome missegregation phenotype of sgt1-L31P. These results
strongly suggest that Sgt1 dimerization is required for kinetochore
assembly.Spindle microtubules are coupled to the centromeric region of the
chromosome by a structural protein complex called the kinetochore
(1,
2). The kinetochore is thought
to generate a signal that arrests cells during mitosis when it is not properly
attached to microtubules, thereby preventing aberrant chromosome transmission
to the daughter cells, which can lead to tumorigenesis
(3,
4). The kinetochore of the
budding yeast Saccharomyces cerevisiae has been characterized
thoroughly, genetically and biochemically; thus, its molecular structure is
the most well detailed to date. More than 70 different proteins comprise the
budding yeast kinetochore, and several of those are conserved in mammals
(2).The budding yeast centromere DNA is a 125-bp region that contains three
conserved regions, CDEI, CDEII, and CDEIII
(5,
6). CDEI is bound by Cbf1
(7–9).
CDEIII (25 bp) is essential for centromere function
(10) and is the site where
CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13
(11–18),
and Skp1 (17,
18), all of which are
essential for viability. Mutations in any of the four CBF3 proteins abolish
the ability of CDEIII to bind to CBF3
(19,
20). All of the described
kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores
dependent on the CBF3 complex
(2). Therefore, the CBF3
complex is the fundamental structure of the kinetochore, and the mechanism of
CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4
kinetochore-defective mutant dosage suppressor
(21). Sgt1 and Skp1 activate
Ctf13; thus, they are required for assembly of the CBF3 complex
(21). The molecular chaperone
Hsp90 is also required for the formation of the Skp1-Ctf13 complex
(22). Sgt1 has two highly
conserved motifs that are required for protein-protein interaction, the
tetratricopeptide repeat
(TPR)2
(21) and the CS
(CHORD protein- and Sgt1-specific) motif. We and others
(23–26)
have found that both domains are important for the interaction with Hsp90. The
Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore
complex; this interaction is an initial step in kinetochore assembly
(24,
26,
27) that is conserved between
yeast and humans (28,
29).In this study, we further characterized the molecular mechanism of this
assembly process. We found that Sgt1 forms dimers in vivo, and our
results strongly suggest that Sgt1 dimerization is required for kinetochore
assembly in budding yeast. 相似文献
11.
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14.
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
15.
Masataka Horiuchi Kosei Takeuchi Nobuo Noda Nobuyuki Muroya Toru Suzuki Takahisa Nakamura Junko Kawamura-Tsuzuku Kiyohiro Takahasi Tadashi Yamamoto Fuyuhiko Inagaki 《The Journal of biological chemistry》2009,284(19):13244-13255
The Tob/BTG family is a group of antiproliferative proteins containing two
highly homologous regions, Box A and Box B. These proteins all associate with
CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D)
family of deadenylases and is a component of the CCR4-Not deadenylase complex.
Here we determined the crystal structure of the complex of the N-terminal
region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas
hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human
poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was
mediated by both Box A and Box B of Tob. Cell growth assays using both
wild-type and mutant proteins revealed that deadenylase activity of Caf1 is
not critical but complex formation is crucial to cell growth inhibition. Caf1
tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers
several factors at its C-terminal region, such as poly(A)-binding proteins, to
exert antiproliferative activity.The Tob/BTG family (also called the APRO family) is a group of
antiproliferative proteins (1,
2) consisting of Tob
(3), Tob2
(4), BTG1
(5), BTG2/Tis21/PC3
(6-8),
PC3B (9), and ANA/BTG3
(10,
11) in mammalian cells,
in Drosophila, and FOG-3 in Caenorhabditis elegans
( AF17746412). A recent genome project
reported that the BTG/Tob family protein had already existed in
Choanoflagellida Monosiga brevicollis MX1. The N-terminal region of
the Tob/BTG family proteins is conserved and includes two highly homologous
regions, Box A and Box B. The Tob/BTG family proteins are involved in cell
cycle regulation in a variety of cells such as T lymphocytes, fibroblasts,
epithelial cells, and germ cells. In Tob-deficient mice, the incidence of
liver tumors is higher than in wild-type mice. Furthermore, because the levels
of tob expression are often repressed in human lung cancers,
suppression of its expression is thought to contribute to tumor progression
(13).The antiproliferative activities of the Tob/BTG family proteins are due to
their association with target proteins in cells. For example, Tob associates
with SMAD family proteins and acts as a negative regulator of SMAD signaling.
In osteoblasts, this negative regulation occurs via association with SMAD 1,
5, 6, and 8 (14,
15), and via association with
SMAD 2 and 4 in anergic quiescent T cells
(16). Tob/BTG family proteins
also bind to protein arginine methyltransferase, which regulates chromatin
assembly by histone methylation
(17). Much evidence has been
accumulated to suggest that CCR4-associated factor 1
(Caf1),2 also known as
Cnot7 and involved in the CCR4-Not deadenylase complex, is a common binding
partner of the Tob/BTG family proteins
(4,
18-21).
To reveal the functions of Caf1 in vivo, caf1-/- mice have
been generated in two groups. Male caf1-deficient mice are infertile
because of a malfunction of the testicular somatic cells that leads to a
defect in spermatogenesis (22,
23). Genetic analysis of the
nematode C. elegans also suggests that FOG3 (Tob orthologue)
interacts with CCF1, the C. elegans homologue of Caf1, and that this
interaction is essential for germ cells to initiate spermatogenesis
(24).Mouse and human Caf1 (mCaf1 and hCaf1) were found as homologues of yeast
Pop2, a component of the CCR4-Not complex
(18,
25). Yeast Pop2 displays weak
RNase activity but enhances the deadenylation of the poly(A) tail of mRNA by
the CCR4-Not deadenylase complex
(26-29).
The primary structure of mammalian Caf1 is related to that of the ribonuclease
D (RNase D) family, and all of the active site residues are well conserved
(30). Indeed, both mCaf1 and
hCaf1 have deadenylase activity
(31-33).Although the relationship between cell cycle repression and poly(A)
deadenylation is not well understood, mRNA degradation and synthesis are major
events in maintaining the cell cycle
(34). The mRNAs in a
eukaryotic cell have a wide range of half-lives. Degradation of mRNA is
initiated by shortening of the poly(A) tail. Thereafter, the 5′-cap
structure is removed and the remaining portion of the mRNA is rapidly
degraded. The degradation of eukaryotic mRNAs is regulated precisely at each
stage of the cell cycle. Tob was reported to associate with inducible
poly(A)-binding protein (iPABP) and to abrogate the translation of
interleukin-2 mRNA in vitro
(35). Recent reports also
showed that Tob and BTG2 interact with the CCR4-Not deadenylase complex using
the Tob/BTG2 domain and the cytoplasmic poly(A)-binding protein (PABPC1) using
the C-terminal tail and enhanced mRNA degradation
(36-38).To help elucidate the relationship between the antiproliferative activity
of Tob and the degradation of the poly(A) tail, we determined the crystal
structure of the Tob-hCaf1 complex. We found that hCaf1 has a structure
similar to yeast Pop2 and human PARN of deadenylases, exonuclease I, and the
Klenow fragment of DNA polymerase I from Escherichia coli. In
contrast, Tob has a novel structure. Specifically, Box A and Box B mediate the
interaction between Tob and hCaf1. Cell growth assays using the wild and
mutant proteins, together with the structural studies, revealed that the
complex formation is crucial to cell growth inhibition. 相似文献
16.
17.
Ivana I. Knezevic Sanda A. Predescu Radu F. Neamu Matvey S. Gorovoy Nebojsa M. Knezevic Cordus Easington Asrar B. Malik Dan N. Predescu 《The Journal of biological chemistry》2009,284(8):5381-5394
It is known that platelet-activating factor (PAF) induces severe
endothelial barrier leakiness, but the signaling mechanisms remain unclear.
Here, using a wide range of biochemical and morphological approaches applied
in both mouse models and cultured endothelial cells, we addressed the
mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and
of increased endothelial permeability. The formation of interendothelial gaps
filled with filopodia and lamellipodia is the cellular event responsible for
the disruption of endothelial barrier. We observed that PAF ligation of its
receptor induced the activation of the Rho GTPase Rac1. Following PAF
exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were
found associated with a membrane fraction from which they
co-immunoprecipitated with PAF receptor. In the same time frame with
Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were
relocated from the IEJs, and formation of numerous interendothelial gaps was
recorded. Notably, the response was independent of myosin light chain
phosphorylation and thus distinct from other mediators, such as histamine and
thrombin. The changes in actin status are driven by the PAF-induced localized
actin polymerization as a consequence of Rac1 translocation and activation.
Tiam1 was required for the activation of Rac1, actin polymerization,
relocation of junctional associated proteins, and disruption of IEJs. Thus,
PAF-induced IEJ disruption and increased endothelial permeability requires the
activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic
target against increased vascular permeability associated with inflammatory
diseases.The endothelial barrier is made up of endothelial cells
(ECs)4 connected to
each other by interendothelial junctions (IEJs) consisting of protein
complexes organized as tight junctions (TJs) and adherens junctions (AJs). In
addition, the focal adhesion complex located at the basal plasma membrane
enables firm contact of ECs with the underlying basement membrane and also
contributes to the barrier function
(1-3).
The glycocalyx, the endothelial monolayer, and the basement membrane all
together constitute the vascular barrier.The structural integrity of the ECs along with their proper functionality
are the two most important factors controlling the tightness of the
endothelial barrier. Changes affecting these factors cause loss of barrier
restrictiveness and leakiness. Therefore, defining and understanding the
cellular and molecular mechanisms controlling these processes is of paramount
importance. Increased width of IEJs in response to permeability-increasing
mediators (4) regulates the
magnitude of transendothelial exchange of fluid and solutes. Disruption of
IEJs and the resultant barrier leakiness contribute to the genesis of diverse
pathological conditions, such as inflammation
(5), metastasis
(6,
7), and uncontrolled
angiogenesis (8,
9).Accumulated evidence demonstrated that IEJs changes are responsible for
increased or decreased vascular permeability, and the generally accepted
mechanism responsible for them was the myosin light chain (MLC)-mediated
contraction of ECs (5,
10). However, published
evidence showed that an increase in vascular permeability could be obtained
without a direct involvement of any contractile mechanism
(11-16).The main component of the vascular barrier, the ECs, has more than 10% of
their total protein represented by actin
(17), which under
physiological salt concentrations subsists as monomers (G-actin) and assembled
into filaments (F-actin). A large number of actin-interacting proteins may
modulate the assembly, disassembly, and organization of G-actin and of actin
filaments within a given cell type. Similar to the complexity of
actin-interacting proteins found in other cell types, the ECs utilize their
actin binding proteins to stabilize the endothelial monolayer in order to
efficiently function as a selective barrier
(11). In undisturbed ECs, the
actin microfilaments are organized as different networks with distinctive
functional and morphological characteristics: the peripheral filaments also
known as peripheral dense band (PDB), the cytoplasmic fibers identified as
stress fibers (SF), and the actin from the membrane cytoskeleton
(18). The peripheral web,
localized immediately under the membrane, is associated with (i) the luminal
plasmalemma (on the apical side), (ii) the IEJ complexes on the lateral
surfaces, and (iii) the focal adhesion complexes on the abluminal side (the
basal part) of polarized ECs. The SF reside inside the endothelial cytoplasm
and are believed to be directly connected with the plasmalemma proper on the
luminal as well as on the abluminal side of the cell. As described, the
endothelial actin cytoskeleton (specifically the SF) seems to be a stable
structure helping the cells to remain flat under flow
(19). It is also established
that the actin fibers participate in correct localization of different
junctional complexes while keeping them in place
(20). However, it was
suggested that the dynamic equilibrium between F- and G-actin might modulate
the tightness of endothelial barrier in response to different challenges
(13).Mediators effective at nanomolar concentrations or less that disrupt the
endothelial barrier and increase vascular permeability include C2 toxin of
Clostridium botulinum, vascular permeability factor, better known as
vascular endothelial growth factor, and PAF
(21). C2 toxin increases
endothelial permeability by ribosylating monomeric G-actin at Arg-177
(22). This results in the
impairment of actin polymerization
(23), followed by rounding of
ECs (16) and the disruption of
junctional integrity. Vascular permeability factor was shown to open IEJs by
redistribution of junctional proteins
(24,
25) and by interfering with
the equilibrium of actin pools
(26). PAF
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocoline), a naturally
synthesized phospholipid is active at 10-10 m or less
(27). PAF is synthesized by
and acts on a variety of cell types, including platelets
(28), neutrophils
(29), monocytes
(30), and ECs
(31). PAF-mediated activation
of ECs induced cell migration
(32), angiogenesis
(7), and vascular
hyperpermeability (33)
secondary to disassembly of IEJs
(34). The effects of PAF on
the endothelium are initiated through a G protein-coupled receptor (PAF-R)
localized at the plasmalemma, in a large endosomal compartment inside the cell
(34), and also in the nuclear
membrane (35). In ECs, PAF-R
was shown to signal through Gαq and downstream activation of
phospholipase C isozymes (PLCβ3 and PLCγ1),
and via cSrc (32,
36). Studies have shown that
PAF challenge induced endothelial actin cytoskeletal rearrangement
(37) and marked vascular
leakiness (38); however, the
signaling pathways have not been elucidated.Therefore, in the present study, we carried out a systematic analysis of
PAF-induced morphological and biochemical changes of endothelial barrier
in vivo and in cultured ECs. We found that the opening of endothelial
barrier and the increased vascular leakiness induced by PAF are the result of
a shift in actin pools without involvement of EC contraction, followed by a
redistribution of tight junctional associated protein ZO-1 and adherens
junctional protein VE-cadherin. 相似文献
18.
Formin-homology (FH) 2 domains from formin proteins associate processively
with the barbed ends of actin filaments through many rounds of actin subunit
addition before dissociating completely. Interaction of the actin
monomer-binding protein profilin with the FH1 domain speeds processive barbed
end elongation by FH2 domains. In this study, we examined the energetic
requirements for fast processive elongation. In contrast to previous
proposals, direct microscopic observations of single molecules of the formin
Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed
that profilin is not required for formin-mediated processive elongation of
growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin
release the γ-phosphate of ATP on average >2.5 min after becoming
incorporated into filaments. Therefore, the release of γ-phosphate from
actin does not drive processive elongation. We compared experimentally
observed rates of processive elongation by a number of different FH2 domains
to kinetic computer simulations and found that actin subunit addition alone
likely provides the energy for fast processive elongation of filaments
mediated by FH1FH2-formin and profilin. We also studied the role of FH2
structure in processive elongation. We found that the flexible linker joining
the two halves of the FH2 dimer has a strong influence on dissociation of
formins from barbed ends but only a weak effect on elongation rates. Because
formins are most vulnerable to dissociation during translocation along the
growing barbed end, we propose that the flexible linker influences the
lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament
structures for diverse processes in eukaryotic cells (reviewed in Ref.
1). Formins stimulate
nucleation of actin filaments and, in the presence of the actin
monomer-binding protein profilin, speed elongation of the barbed ends of
filaments
(2-6).
The ability of formins to influence elongation depends on the ability of
single formin molecules to remain bound to a growing barbed end through
multiple rounds of actin subunit addition
(7,
8). To stay associated during
subunit addition, a formin molecule must translocate processively on the
barbed end as each actin subunit is added
(1,
9-12).
This processive elongation of a barbed end by a formin is terminated when the
formin dissociates stochastically from the growing end during translocation
(4,
10).The formin-homology
(FH)2 1 and
2 domains are the best conserved domains of formin proteins
(2,
13,
14). The FH2 domain is the
signature domain of formins, and in many cases, is sufficient for both
nucleation and processive elongation of barbed ends
(2-4,
7,
15). Head-to-tail homodimers
of FH2 domains (12,
16) encircle the barbed ends
of actin filaments (9). In
vitro, association of barbed ends with FH2 domains slows elongation by
limiting addition of free actin monomers. This “gating” behavior
is usually explained by a rapid equilibrium of the FH2-associated end between
an open state competent for actin monomer association and a closed state that
blocks monomer binding (4,
9,
17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for
profilin to stimulate formin-mediated elongation. Individual tracks of
polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer
the actin directly to the FH2-associated barbed end to increase processive
elongation rates
(4-6,
8,
10,
17).Rates of elongation and dissociation from growing barbed ends differ widely
for FH1FH2 fragments from different formin homologs
(4). We understand few aspects
of FH1FH2 domains that influence gating, elongation or dissociation. In this
study, we examined the source of energy for formin-mediated processive
elongation, and the influence of FH2 structure on elongation and dissociation
from growing ends. In contrast to previous proposals
(6,
18), we found that fast
processive elongation mediated by FH1FH2-formins is not driven by energy from
the release of the γ-phosphate from ATP-actin filaments. Instead, the
data show that the binding of an actin subunit to the barbed end provides the
energy for processive elongation. We found that in similar polymerizing
conditions, different natural FH2 domains dissociate from growing barbed ends
at substantially different rates. We further observed that the length of the
flexible linker between the subunits of a FH2 dimer influences dissociation
much more than elongation. 相似文献
19.
Eva Brombacher Simon Urwyler Curdin Ragaz Stefan S. Weber Keiichiro Kami Michael Overduin Hubert Hilbi 《The Journal of biological chemistry》2009,284(8):4846-4856
The causative agent of Legionnaires disease, Legionella
pneumophila, forms a replicative vacuole in phagocytes by means of the
intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV
secretion system and translocated effector proteins, some of which subvert
host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC
anchors to the membrane of Legionella-containing vacuoles (LCVs) by
specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a
nonbiased screen for novel L. pneumophila PI-binding proteins, we
identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the
predominant PtdIns(4)P-binding protein. Purified SidM specifically and
directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate
LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L.
pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding
domain of SidM was mapped to the 12-kDa C-terminal sequence, termed
“P4M” (PtdIns4P binding of
SidM/DrrA). The isolated P4M domain is largely helical and
displayed higher PtdIns(4)P binding activity in the context of the
α-helical, monomeric full-length protein. SidM constructs containing P4M
were translocated by Icm/Dot-proficient L. pneumophila and localized
to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via
its P4M domain. An L. pneumophila ΔsidM mutant strain
displayed significantly higher amounts of SidC on LCVs, suggesting that SidM
and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally,
RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by
host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits
PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC
and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative
agent of Legionnaires disease, but it evolved as a parasite of various species
of environmental predatory protozoa, including the social amoeba
Dictyostelium discoideum
(1,
2). The human disease is linked
to the inhalation of contaminated aerosols, followed by replication in
alveolar macrophages. To accommodate the transfer between host cells, L.
pneumophila alternates between replicative and transmissive phases, the
regulation of which includes an apparent quorum-sensing system
(3–5).In macrophages and amoebae, L. pneumophila forms a replicative
compartment, the Legionella-containing vacuole
(LCV).3 LCVs avoid
fusion with lysosomes (6),
intercept vesicular traffic at endoplasmic reticulum (ER) exit sites
(7), and fuse with the ER
(8–10).
The uptake of L. pneumophila and formation of LCVs in macrophages and
amoebae depends on the Icm/Dot type IV secretion system (T4SS)
(11–14).
Although more than 100 Icm/Dot substrates (“effector” proteins)
have been identified to date, only few are functionally characterized,
including effectors that interfere with host cell signal transduction, vesicle
trafficking, or apoptotic pathways
(15–18).Two Icm/Dot-translocated substrates, SidM/DrrA
(19,
20) and RalF
(21), have been characterized
as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small
GTPases. These bacterial GEFs are recruited to and activate their targets on
LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic
signal transduction pathways and in actin cytoskeleton regulation
(22). Inactive Rho GTPases
bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases
are activated by removal of the GDI and the exchange of GDP with GTP by GEFs,
which promotes the interaction with downstream effector proteins, such as
protein or lipid kinases and various adaptor proteins. The cycle is closed by
hydrolysis of the bound GTP, which is mediated by GTPase-activating
proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle
transport, and additionally, SidM acts as a GDI displacement factor (GDF) to
activate Rab1 (23,
24). The function of SidM is
assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA
preferentially binds to activated Rab1, thus supporting the recruitment of
early secretory vesicles by SidM
(19,
20,
23,
25,
26). Another Icm/Dot
substrate, LepB (27),
contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its
GTPase-activating protein function, thus acting as an antagonist of SidM
(24).The Icm/Dot substrate RalF recruits and activates the small GTPase
ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle
transport from Golgi to ER
(21). Dominant negative Arf1
(7,
28) or knockdown of Arf1 by
RNA interference (29) impairs
the formation of LCVs, as well as the recruitment of the Icm/Dot substrate
SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane
(31), where the proteins
specifically bind to the host cell lipid phosphatidylinositol 4-phosphate
(PtdIns(4)P) (32,
33). Phosphoinositides (PIs)
regulate eukaryotic receptor-mediated signal transduction, actin remodeling,
and membrane dynamics (34,
35). PtdIns(4)P is present on
the cytoplasmic membrane, but localizes preferentially to the
trans-Golgi network (TGN), where this PI is produced by an
Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ)
(36) to promote trafficking
along the secretory pathway. Recently, PtdIns(4)P was found to also mediate
the export of early secretory vesicles from ER exit sites
(37). At present, the L.
pneumophila effector proteins that mediate exploitation of host PI
signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using
different PIs coupled to agarose beads, we identified SidM as a major
PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a
novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs,
including the P4M domain, were found to be translocated and bind the LCV
membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ. 相似文献