共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
3.
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
4.
Susan R. Ferrari Jennifer Grubb Douglas K. Bishop 《The Journal of biological chemistry》2009,284(18):11766-11770
During homologous recombination, a number of proteins cooperate to catalyze
the loading of recombinases onto single-stranded DNA. Single-stranded
DNA-binding proteins stimulate recombination by coating single-stranded DNA
and keeping it free of secondary structure; however, in order for recombinases
to load on single-stranded-DNA-binding protein-coated DNA, the activity of a
class of proteins known as recombination mediators is required. Mediator
proteins coordinate the handoff of single-stranded DNA from single-stranded
DNA-binding protein to recombinase. Here we show that a complex of Mei5 and
Sae3 from Saccharomyces cerevisiae preferentially binds
single-stranded DNA and relieves the inhibition of the strand assimilation and
DNA binding abilities of the meiotic recombinase Dmc1 imposed by the
single-stranded DNA-binding protein replication protein A. Additionally, we
demonstrate the physical interaction of Mei5-Sae3 with replication protein A.
Our results, together with previous in vivo studies, indicate that
Mei5-Sae3 is a mediator of Dmc1 assembly during meiotic recombination in
S. cerevisiae.During meiosis, recombination between homologous chromosomes ensures proper
segregation into haploid products. Recombination events are initiated by the
formation of double strand breaks
(DSBs)2 in DNA
(1). This is followed by
resection of free DNA ends to yield 3′ single-stranded tails, upon which
recombinase assembles to form nucleoprotein filaments. Following recombinase
assembly, the nucleoprotein filament engages a donor chromatid, searches for
homologous DNA sequences on that chromatid, and promotes strand exchange to
yield a heteroduplex DNA intermediate often referred to as a joint molecule.
Although recombinase alone is capable of promoting homology search and strand
exchange in vitro, genetic and biochemical studies have demonstrated
that normal recombinase function in vivo requires the activity of a
number of accessory factors
(2). These factors enhance the
assembly of nucleoprotein filaments, target capture, homology search, and
dissociation of recombinase from duplex DNA.Most eukaryotes possess two recombinases, both homologues of the
Escherichia coli recombinase RecA: Rad51, which is the major
recombinase in mitotic cells and is also important during meiotic
recombination, and Dmc1, which functions only in meiosis. Dmc1 and Rad51 have
been shown to assemble at DSBs by immunofluorescence and chromatin
immunoprecipitation
(3–6),
and both proteins oligomerize on single-stranded DNA (ssDNA) to form
nucleofilaments that catalyze strand invasion
(7–9).A number of biochemical studies have defined the role of accessory factors
in stimulating the activity of Rad51
(10–12).
Replication protein A (RPA), the yeast ssDNA-binding protein (SSB), removes
secondary structure in ssDNA that otherwise prevents formation of fully
functional nucleoprotein filaments
(13). Both Rad52 protein
(11,
12) and the heterodimeric
protein Rad55/Rad57 (14) can
overcome the inhibitory effect of RPA on Rad51 nucleoprotein filament
formation in purified systems, mediating a handoff between RPA and Rad51. It
is thought that the mechanism for the mediator activity of Rad52 involves
Rad52 recognizing and binding to RPA-coated ssDNA, where it provides
nucleation sites for the recruitment of free molecules of Rad51
(15). The tumor suppressor
protein BRCA2 also serves as an assembly factor for Rad51 during mitosis in a
variety of species that encode orthologues of this protein, including mice
(16), corn smut
(17), and humans
(18).The meiosis-specific recombinase Dmc1 is stimulated by a distinct set of
accessory factors. Immunostaining studies suggest that the Rad51 mediators
Rad52 and Rad55/Rad57 are not required for assembly of Dmc1 foci in
vivo, although Rad51 itself promotes Dmc1 foci
(19–21).
More recently, immunostaining and chromatin immunoprecipitation experiments
demonstrated a role for the Mei5 and Sae3 proteins of Saccharomyces
cerevisiae in assembly of Dmc1 at sites of DSBs in vivo
(22,
23). Consistent with these
observations, mei5 and sae3 mutants display markedly similar
meiotic defects as compared with dmc1 mutants, including defects in
sporulation, spore viability, crossing over, DSB repair, progression through
meiosis, and synaptonemal complex formation
(19,
22–24).
Finally, the three proteins have been shown to physically interact; Mei5 and
Sae3 have been co-purified and co-immunoprecipitated, and an N-terminal
portion of Mei5 has been shown to interact with Dmc1 in a two-hybrid assay
(22).The fission yeast Schizosaccharomyces pombe encodes two proteins,
Swi5 and Sfr1, which share sequence homology with Sae3 and Mei5, respectively
(22). Swi5 and Sfr1 have been
shown to stimulate the strand exchange activity of Rhp51 (the S.
pombe Rad51 homologue) and Dmc1
(25). Although some results
indicate functional similarity of Swi5-Sfr1 and Mei5-Sae3, there are also
clear differences. The Mei5-Sae3 complex of budding yeast is expressed solely
during meiosis, and no mitotic phenotypes have been reported for mei5
or sae3 mutants (22,
24,
26). In contrast, the
Swi5-Sfr1 complex of fission yeast is expressed in mitotic and meiotic cells,
and mutations in SWI5 have been shown to cause defects in mitotic
recombination (27).
Furthermore, although mei5 and sae3 mutants are
phenotypically similar to dmc1 mutants, swi5 and
sfr1 mutants display more severe meiotic defects during fission yeast
meiosis than do dmc1 mutants
(27–29).
These data suggest that although Swi5-Sfr1 clearly contributes to Rad51
activity in fission yeast, it is possible that the activity of Mei5-Sae3 is
restricted to stimulating Dmc1 in budding yeast.In this study, a biochemical approach is used to test the budding yeast
Mei5-Sae3 complex for properties expected of a recombinase assembly mediator.
We show that Mei5-Sae3 binds both ssDNA and double-stranded DNA (dsDNA) but
binds ssDNA preferentially. We also show that Mei5-Sae3 can overcome the
inhibitory effects of RPA on the ssDNA binding and strand assimilation
activities of Dmc1. Finally, we show that Mei5-Sae3 and RPA bind one another
directly. These results indicate that Mei5-Sae3 acts directly as a mediator
protein for assembly of Dmc1. 相似文献
5.
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
6.
7.
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
8.
John W. Hardin Francis E. Reyes Robert T. Batey 《The Journal of biological chemistry》2009,284(22):15317-15324
In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are
responsible for 2′-O-methylation of tRNAs and rRNAs. The
archaeal box C/D small RNP complex requires a small RNA component (sRNA)
possessing Watson-Crick complementarity to the target RNA along with three
proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from
S-adenosylmethionine to the target RNA is performed by fibrillarin,
which by itself has no affinity for the sRNA-target duplex. Instead, it is
targeted to the site of methylation through association with Nop5p, which in
turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a
bridge between the targeting and catalytic functions of the box C/D small RNP
complex, we have employed alanine scanning to evaluate the interaction between
the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA
complex. From these data, we were able to construct an isolated RNA-binding
domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography
and binds to the L7Ae box C/D RNA complex with near wild type affinity. These
data demonstrate that the Nop-RBD is an autonomously folding and functional
module important for protein assembly in a number of complexes centered on the
L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full
functionality in the cell (1).
Currently there are over 100 known RNA modification types ranging from small
functional group substitutions to the addition of large multi-cyclic ring
structures (2). Transfer RNA,
one of many functional RNAs targeted for modification
(3-6),
possesses the greatest modification type diversity, many of which are
important for proper biological function
(7). Ribosomal RNA, on the
other hand, contains predominantly two types of modified nucleotides:
pseudouridine and 2′-O-methylribose
(8). The crystal structures of
the ribosome suggest that these modifications are important for proper folding
(9,
10) and structural
stabilization (11) in
vivo as evidenced by their strong tendency to localize to regions
associated with function (8,
12,
13). These roles have been
verified biochemically in a number of cases
(14), whereas newly emerging
functional modifications are continually being investigated.Box C/D ribonucleoprotein
(RNP)3 complexes serve
as RNA-guided site-specific 2′-O-methyltransferases in both
archaea and eukaryotes (15,
16) where they are referred to
as small RNP complexes and small nucleolar RNPs, respectively. Target RNA
pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl
group of the nucleotide five bases upstream of either the D or D′ box
motif of the sRNA (Fig. 1,
star) (17,
18). In archaea, the internal
C′ and D′ motifs generally conform to a box C/D consensus sequence
(19), and each sRNA contains
two guide regions ∼12 nucleotides in length
(20). The bipartite
architecture of the RNP potentially enables the complex to methylate two
distinct RNA targets (21) and
has been shown to be essential for site-specific methylation
(22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components
of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D
sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D,
C′, and D′ boxes are labeled. The target RNA binds the sRNA
through Watson-Crick pairing and is methylated by fibrillarin at the fifth
nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three
proteins for activity (23):
the ribosomal protein L7Ae
(24,
25), fibrillarin, and the
Nop56/Nop58 homolog Nop5p (Fig.
1). L7Ae binds to both box C/D and the C′/D′ motifs
(26), which respectively
comprise kink-turn (27) or
k-loop structures (28), to
initiate the assembly of the RNP
(29,
30). Fibrillarin performs the
methyl group transfer from the cofactor S-adenosylmethionine to the
target RNA
(31-33).
For this to occur, the active site of fibrillarin must be positioned precisely
over the specific 2′-hydroxyl group to be methylated. Although
fibrillarin methylates this functional group in the context of a Watson-Crick
base-paired helix (guide/target), it has little to no binding affinity for
double-stranded RNA or for the L7Ae-sRNA complex
(22,
26,
33,
34). Nop5p serves as an
intermediary protein bringing fibrillarin to the complex through its
association with both the L7Ae-sRNA complex and fibrillarin
(22). Along with its role as
an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses
other functions not yet fully understood. For example, Nop5p self-dimerizes
through a coiled-coil domain
(35) that in most archaea and
eukaryotic homologs includes a small insertion sequence of unknown function
(36,
37). However, dimerization and
fibrillarin binding have been shown to be mutually exclusive in
Methanocaldococcus jannaschii Nop5p, potentially because of the
presence of this insertion sequence
(36). Thus, whether Nop5p is a
monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate
its interaction with a L7Ae box C/D RNA complex because both the
fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal
structures (29,
35,
38). Individual residues on
the surface of a monomeric form of Nop5p (referred to as mNop5p)
(22) were mutated to alanine,
and the effect on binding affinity for a L7Ae box C/D motif RNA complex was
assessed through the use of electrophoretic mobility shift assays. These data
reveal that residues important for binding cluster within the highly conserved
NOP domain (39,
40). To demonstrate that this
domain is solely responsible for the affinity of Nop5p for the preassembled
L7Ae box C/D RNA complex, we expressed and purified it in isolation from the
full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA
complex with nearly wild type affinity, demonstrating that the Nop-RBD is
truly an autonomously folding and functional module. Comparison of our data
with the crystal structure of the homologous spliceosomal hPrp31-15.5K
protein-U4 snRNA complex (41)
suggests the adoption of a similar mode of binding, further supporting a
crucial role for the NOP domain in RNP complex assembly. 相似文献
9.
10.
11.
12.
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
13.
14.
15.
16.
Siying Wang Wen-Mei Yu Wanming Zhang Keith R. McCrae Benjamin G. Neel Cheng-Kui Qu 《The Journal of biological chemistry》2009,284(2):913-920
Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation
of its catalytic activity have been identified in Noonan syndrome and various
childhood leukemias. Recent studies suggest that the gain-of-function (GOF)
mutations of SHP-2 play a causal role in the pathogenesis of these diseases.
However, the molecular mechanisms by which GOF mutations of SHP-2 induce these
phenotypes are not fully understood. Here, we show that GOF mutations in
SHP-2, such as E76K and D61G, drastically increase spreading and migration of
various cell types, including hematopoietic cells, endothelial cells, and
fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G
knock-in mice is also enhanced. Mechanistic studies suggest that the increased
cell migration is attributed to the enhanced β1 integrin outside-in
signaling. In response to β1 integrin cross-linking or fibronectin
stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2
GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is
elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are
more sensitive than wild-type cells to the suppression of cell motility by
inhibition of these pathways. Collectively, these studies reaffirm the
positive role of SHP-2 phosphatase in cell motility and suggest a new
mechanism by which SHP-2 GOF mutations contribute to diseases.SHP-2, a multifunctional SH2 domain-containing protein-tyrosine phosphatase
implicated in diverse cell signaling processes
(1–3),
plays a critical role in cellular function. Homozygous deletion of Exon
2 (4) or Exon 3
(5) of the SHP-2 gene
(PTPN11) in mice leads to early embryonic lethality prior to and at
midgestation, respectively. SHP-2 null mutant mice die much earlier, at
peri-implantation (4). Exon
3 deletion mutation of SHP-2 blocks hematopoietic potential of embryonic
stem cells both in vitro and in vivo
(6–8),
whereas SHP-2 null mutation causes inner cell mass death and diminished
trophoblast stem cell survival
(4). Recent studies on SHP-2
conditional knock-out or tissue-specific knock-out mice have further revealed
an array of important functions of this phosphatase in various physiological
processes
(9–12).
The phenotypes demonstrated by loss of SHP-2 function are apparently
attributed to the role of SHP-2 in the cell signaling pathways induced by
growth factors/cytokines. SHP-2 generally promotes signal transmission in
growth factor/cytokine signaling in both catalytic-dependent and -independent
fashion
(1–3).
The positive role of SHP-2 in the intracellular signaling processes, in
particular, the ERK3
and PI3K/Akt kinase pathways, has been well established, although the
underlying mechanism remains elusive, in particular, the signaling function of
the catalytic activity of SHP-2 in these pathways is poorly understood.In addition to the role of SHP-2 in cell proliferation and differentiation,
the phenotypes induced by loss of SHP-2 function may be associated with its
role in cell migration. Indeed, dominant negative SHP-2 disrupts
Xenopus gastrulation, causing tail truncations
(13,
14). Targeted Exon 3
deletion mutation in SHP-2 results in decreased cell spreading, migration
(15,
16), and impaired limb
development in the chimeric mice
(7). The role of SHP-2 in cell
adhesion and migration has also been demonstrated by catalytically inactive
mutant SHP-2-overexpressing cells
(17–20).
The molecular mechanisms by which SHP-2 regulates these cellular processes,
however, have not been well defined. For example, the role of SHP-2 in the
activation of the Rho family small GTPases that is critical for cell motility
is still controversial. Both positive
(19,
21,
22) and negative roles
(18,
23) for SHP-2 in this context
have been reported. Part of the reason for this discrepancy might be due to
the difference in the cell models used. Catalytically inactive mutant SHP-2
was often used to determine the role of SHP-2 in cell signaling. In the
catalytically inactive mutant SHP-2-overexpressing cells, the catalytic
activity of endogenous SHP-2 is inhibited. However, as SHP-2 also functions
independent of its catalytic activity, overexpression of catalytically
deficient SHP-2 may also increase its scaffolding function, generating complex
effects.The critical role of SHP-2 in cellular function is further underscored by
the identification of SHP-2 mutations in human diseases. Genetic lesions in
PTPN11 that cause hyperactivation of SHP-2 catalytic activity have
been identified in the developmental disorder Noonan syndrome
(24) and various childhood
leukemias, including juvenile myelomonocytic leukemia (JMML), B cell acute
lymphoblastic leukemia, and acute myeloid leukemia
(25,
26). In addition, activating
mutations in SHP-2 have been identified in sporadic solid tumors
(27). The SHP-2 mutations
appear to play a causal role in the development of these diseases as SHP-2
mutations and other JMML-associated Ras or Neurofibromatosis 1 mutations are
mutually exclusive in the patients
(24–27).
Moreover, single SHP-2 gain-of-function (GOF) mutations are sufficient to
induce Noonan syndrome, cytokine hypersensitivity in hematopoietic progenitor
cells, and JMML-like myeloproliferative disease in mice
(28–32).
Gain-of-function cell models derived from the newly available SHP-2 GOF
mutation (D61G) knock-in mice
(28) now provide us with a
good opportunity to clarify the role of SHP-2 in cell motility. Unlike the
dominant negative approach in which overexpression of mutant forms of SHP-2
generates complex effects, the SHP-2 D61G knock-in model eliminates this
possibility as the mutant SHP-2 is expressed at the physiological level
(28). Additionally, defining
signaling functions of GOF mutant SHP-2 in cell movement can also help
elucidate the molecular mechanisms by which SHP-2 mutations contribute to the
relevant diseases. 相似文献
17.
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
18.
Michael A. Gitcho Jeffrey Strider Deborah Carter Lisa Taylor-Reinwald Mark S. Forman Alison M. Goate Nigel J. Cairns 《The Journal of biological chemistry》2009,284(18):12384-12398
Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and
Paget disease of bone is a rare, autosomal dominant disorder caused by
mutations in the VCP (valosin-containing protein) gene. The disease
is characterized neuropathologically by frontal and temporal lobar atrophy,
neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are
distinct from those seen in other sporadic and familial FTLD-U entities. The
major component of the ubiquitinated inclusions of FTLD with VCP
mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy
links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most
familial forms of FTLD-U. Understanding the relationship between individual
gene defects and pathologic TDP-43 will facilitate the characterization of the
mechanisms leading to neurodegeneration. Using cell culture models, we have
investigated the role of mutant VCP in intracellular trafficking,
proteasomal function, and cell death and demonstrate that mutations in the
VCP gene 1) alter localization of TDP-43 between the nucleus and
cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum
stress, 4) increase markers of apoptosis, and 5) impair cell viability. These
results suggest that VCP mutation-induced neurodegeneration is
mediated by several mechanisms.Frontotemporal lobar degeneration
(FTLD)2
accounts for 10% of all late onset dementias and is the third most frequent
neurodegenerative disease after Alzheimer disease and dementia with Lewy
bodies (1). FTLD with
ubiquitin-immunoreactive inclusions is genetically, clinically, and
neuropathologically heterogeneous
(2,
3). FTLD-U comprises several
distinct entities, including sporadic forms and familial cases caused by
mutations in the genes encoding VCP (valosin-containing protein), GRN
(progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR
DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9
(2,
3). Frontotemporal dementia
with inclusion body myopathy and Paget disease of bone is a rare, autosomal
dominant disorder caused by mutations in the VCP gene located on
chromosome 9p13-p12
(4-10)
(Fig. 1). This multisystem
disease is characterized by progressive muscle weakness and atrophy, increased
osteoclastic bone resorption, and early onset frontotemporal dementia, also
called FTLD (9,
11). Mutations in VCP
are also associated with dilatative cardiomyopathy with ubiquitin-positive
inclusions (12).
Neuropathologic features of FTLD with VCP mutation include frontal
and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive
inclusions (FTLD-U). The majority of aggregates are ubiquitin- and
TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion
is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal
cytoplasmic inclusions (NCIs). A small number of inclusions are
VCP-immunoreactive (5,
13). Pathologic TDP-43 in
inclusions links a spectrum of diseases in which TDP-43 pathology is a primary
feature, including FTLD-U, motor neuron disease, including amyotrophic lateral
sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and
Paget disease of bone, as well as an expanding spectrum of other disorders in
which TDP-43 pathology is secondary
(14,
15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing
protein. CDC48 (magenta), located within the N terminus (residues
22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1
(23-25,
28). There are two AAA-ATPase
domains (AAA; blue) at residues 240-283 and 516-569, which
are joined by two linker regions (L1 and L2;
red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical
signature similar to that seen in other sporadic and familial cases of FTLD-U,
including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease,
FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome
9p (3,
16). TDP-43 proteinopathy in
these disorders is characterized by hyperphosphorylation of TDP-43,
ubiquitination, and cleavage to form C-terminal fragments detected only in
insoluble brain extracts from affected brain regions
(16). Identification of TDP-43
as the major component of the ubiquitin-immunoreactive inclusions of FTLD with
VCP mutation supports the hypothesis that VCP gene mutations
cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase
associated with diverse cellular activities) superfamily. The N-terminal
domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell
division cycle protein 48)) and two AAA-ATPase domains that form a hexameric
complex (Fig. 1)
(17). Recently, it has been
shown that the N-terminal domain of VCP binds phosphoinositides
(18,
19). AKT (activated
serine-threonine protein kinase) phosphorylates VCP and is required for
constitutive VCP function (20,
21). AKT is activated through
phospholipid binding and phosphorylation via the phosphoinositide 3-kinase
signaling pathway, which is involved in cell survival
(22). The lipid binding domain
may recruit VCP to the cell membrane where it is phosphorylated by AKT
(19).The diversity of VCP functions is modulated, in part, by a variety of
intracellular cofactors, including p47, gp78, and Npl4-Ufd1
(23). Cofactor p47 has been
shown to play a role in the maintenance and biogenesis of both the endoplasmic
reticulum (ER) and Golgi apparatus
(24). The structure of p47
contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and
together they act as a chaperone to deliver membrane fusion machinery to the
site of adjacent membranes
(25). The function of the
p47-VCP complex is dependent upon cell division cycle 2 (CDC2)
serine-threonine kinase phosphorylation of p47
(26,
27). Also, VCP has been found
to interact with the cytosolic tail of gp78, an ER membrane-spanning E3
ubiquitin ligase that exclusively binds VCP and enhances ER-associated
degradation (ERAD) (28). The
Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting
of proteins through the ubiquitin-proteasome system
(29,
30). The cell survival
response of this complex has been found to be important in DNA damage repair
though activation by phosphorylation and its recruitment to double-stranded
breaks (20,
31). The Npl4-Ufd1-VCP
cytosolic complex is also recruited to the ER membrane, interacting with
Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At
the ER membrane, these misfolded proteins are targeted to the proteasome via
ERAD
(32-34).
VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome
system, implicating VCP in the cell survival pathway and neuroprotection
(21,
35-37).To investigate the mechanism of neurodegeneration caused by VCP
mutations, we first tested the hypothesis that VCP mutations decrease
cell viability in vitro using a neuroblastoma SHSY-5Y cell line and
then investigated cellular pathways that are known to lead to
neurodegeneration, including decrease in proteasome activity, caspase-mediated
degeneration, and a change in cellular localization of TDP-43. 相似文献
19.
20.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献