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Michael S. Friedman Sivan M. Oyserman Kurt D. Hankenson 《The Journal of biological chemistry》2009,284(21):14117-14125
Wnt11 signals through both canonical (β-catenin) and non-canonical
pathways and is up-regulated during osteoblast differentiation and fracture
healing. In these studies, we evaluated the role of Wnt11 during
osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases
β-catenin accumulation and promotes bone morphogenetic protein
(BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11
dramatically increases expression of the osteoblast-associated genes
Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating
endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also
increases expression of Rspo2 (R-spondin 2), a secreted factor known
to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing
Dmp1, Phex, and Bsp expression and promotes bone
morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates
Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor
(Tcf)/β-catenin signaling with dominant negative Tcf blocks
Wnt11-mediated expression of Dmp1, Phex, and Rspo2
and decreases mineralization. However, dominant negative Tcf fails to block
the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11
signals through β-catenin, activating Rspo2 expression, which is
then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and
maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b
enhances osteoblast differentiation
(1). Canonical Wnt signaling
through β-catenin has also been shown to enhance the chondroinductive and
osteoinductive properties of
BMP22
(2,
3). During BMP2-induced
osteoblast differentiation of mesenchymal stem cell lines, cross-talk between
BMP and Wnt pathways converges through the interaction of Smad4 with
β-catenin (2).Canonical Wnt signaling is also critical for skeletal development and
homeostasis. During limb development, expression of Wnt3a in the apical
ectodermal ridge of limb buds maintains cells in a highly proliferative and
undifferentiated state (4,
5). Disruption of canonical Wnt
signaling in Lrp5/Lrp6 compound knock-out mice results in limb- and
digit-patterning defects (6).
Wnt signaling is also involved in the maintenance of post-natal bone mass.
Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass,
whereas loss of Lrp5 function is associated with decreased bone mass and
osteoporosis pseudoglioma syndrome
(7,
8). Mice with increased Wnt10b
expression have increased trabecular bone, whereas Wnt10b-deficient mice have
reduced trabecular bone (9).
Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased
trabecular bone accrual throughout adulthood
(10).Although canonical Wnt signaling regulates osteoblastogenesis and bone
formation, the profile of endogenous Wnts that play a role in osteoblast
differentiation and maturation is not well described. During development,
Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum
(11). Wnt11 expression is
increased during glucocorticoid-induced osteogenesis
(12), indicating a potential
role for Wnt11 in osteoblast differentiation. Interestingly, Wnt11 activates
both β-catenin-dependent as well as β-catenin-independent signaling
pathways (13). Targeted
disruption of Wnt11 results in late embryonic/early post-natal death because
of cardiac dysfunction (14).
Although these mice have no reported skeletal developmental abnormalities,
early lethality obfuscates a detailed examination of post-natal skeletal
modeling and remodeling.In murine development, Wnt11 expression overlaps with the expression of
R-spondin 2 (Rspo2) in the apical ectodermal ridge
(11,
15). R-spondins are a novel
family of proteins that share structural features, including two conserved
cysteinerich furin-like domains and a thrombospondin type I repeat
(16). The four R-spondin
family members can activate canonical Wnt signaling
(15,
17–19).
Rspo3 interacts with Frizzled 8 and Lrp6 and enhances Wnt ligand signaling.
Rspo1 enhances Wnt signaling by interacting with Lrp6 and inhibiting
Dkk-mediated receptor internalization
(20). Rspo1 was also shown to
potentiate Wnt3a-mediated osteoblast differentiation
(21). Rspo2 knock-out
mice, which die at birth, have limb patterning defects associated with altered
β-catenin signaling
(22–24).
However, the role of Rspo2 in osteoblast differentiation and maturation
remains unclear.Herein we report that Wnt11 overexpression in MC3T3E1 pre-osteoblasts
activates β-catenin and augments BMP-induced osteoblast maturation and
mineralization. Wnt11 increases the expression of Rspo2.
Overexpression of Rspo2 in MC3T3E1 is sufficient for augmenting BMP-induced
osteoblast maturation and mineralization. Although antagonism of
Tcf/β-catenin signaling blocks the osteogenic effects of Wnt11, Rspo2
rescues this block, and knockdown of Rspo2 shows that it is required for
Wnt11-mediated osteoblast maturation and mineralization. These studies
identify both Wnt11 and Rspo2 as novel mediators of osteoblast maturation and
mineralization. 相似文献
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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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Denise A. Berti Cain Morano Lilian C. Russo Leandro M. Castro Fernanda M. Cunha Xin Zhang Juan Sironi Cl��cio F. Klitzke Emer S. Ferro Lloyd D. Fricker 《The Journal of biological chemistry》2009,284(21):14105-14116
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme
that has been proposed to metabolize peptides within cells, thereby affecting
antigen presentation and G protein-coupled receptor signal transduction.
However, only a small number of intracellular substrates of EP24.15 have been
reported previously. Here we have identified over 100 peptides in human
embryonic kidney 293 (HEK293) cells that are derived from intracellular
proteins; many but not all of these peptides are substrates or products of
EP24.15. First, cellular peptides were extracted from HEK293 cells and
incubated in vitro with purified EP24.15. Then the peptides were
labeled with isotopic tags and analyzed by mass spectrometry to obtain
quantitative data on the extent of cleavage. A related series of experiments
tested the effect of overexpression of EP24.15 on the cellular levels of
peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10
of the cellular peptides were incubated with purified EP24.15 in
vitro, and the cleavage was monitored by high pressure liquid
chromatography and mass spectrometry. Many of the EP24.15 substrates
identified by these approaches are 9–11 amino acids in length,
supporting the proposal that EP24.15 can function in the degradation of
peptides that could be used for antigen presentation. However, EP24.15 also
converts some peptides into products that are 8–10 amino acids, thus
contributing to the formation of peptides for antigen presentation. In
addition, the intracellular peptides described here are potential candidates
to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and
if this process is impaired, the elevated levels of aged proteins usually lead
to the formation of intracellular insoluble aggregates that can cause severe
pathologies (1). In mammalian
cells, most proteins destined for degradation are initially tagged with a
polyubiquitin chain in an energy-dependent process and then digested to small
peptides by the 26 S proteasome, a large proteolytic complex involved in the
regulation of cell division, gene expression, and other key processes
(2,
3). In eukaryotes, 30–90%
of newly synthesized proteins may be degraded by proteasomes within minutes of
synthesis (3,
4). In addition to proteasomes,
other extralysosomal proteolytic systems have been reported
(5,
6). The proteasome cleaves
proteins into peptides that are typically 2–20 amino acids in length
(7). In most cases, these
peptides are thought to be rapidly hydrolyzed into amino acids by
aminopeptidases
(8–10).
However, some intracellular peptides escape complete degradation and are
imported into the endoplasmic reticulum where they associate with major
histocompatibility complex class I
(MHC-I)3 molecules and
traffic to the cell surface for presentation to the immune system
(10–12).
Additionally, based on the fact that free peptides added to the intracellular
milieu can regulate cellular functions mediated by protein interactions such
as gene regulation, metabolism, cell signaling, and protein targeting
(13,
14), intracellular peptides
generated by proteasomes that escape degradation have been suggested to play a
role in regulating protein interactions
(15). Indeed, oligopeptides
isolated from rat brain tissue using the catalytically inactive EP24.15 (EC
3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and
were found capable of altering G protein-coupled receptor signal transduction
(16). Moreover, EP24.15
overexpression itself changed both angiotensin II and isoproterenol signal
transduction, suggesting a physiological function for its intracellular
substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family
that contains the HEXXH motif
(17). This enzyme was first
described as a neuropeptide-degrading enzyme present in the soluble fraction
of brain homogenates (18).
Whereas EP24.15 can be secreted
(19,
20), its predominant location
in the cytosol and nucleus suggests that the primary function of this enzyme
is not the extracellular degradation of neuropeptides and hormones
(21,
22). EP24.15 was shown in
vivo to participate in antigen presentation through MHC-I
(23–25)
and in vitro to bind
(26) or degrade
(27) some MHC-I associated
peptides. EP24.15 has also been shown in vitro to degrade peptides
containing 5–17 amino acids produced after proteasome digestion of
β-casein (28). EP24.15
shows substrate size restriction to peptides containing from 5 to 17 amino
acids because of its catalytic center that is located in a deep channel
(29). Despite the size
restriction, EP24.15 has a broad substrate specificity
(30), probably because a
significant portion of the enzyme-binding site is lined with potentially
flexible loops that allow reorganization of the active site following
substrate binding (29).
Recently, it has also been suggested that certain substrates may be cleaved by
an open form of EP24.15 (31).
This characteristic is supported by the ability of EP24.15 to accommodate
different amino acid residues at subsites S4 to S3′, which even includes
the uncommon post-proline cleavage
(30). Such biochemical and
structural features make EP24.15 a versatile enzyme to degrade structurally
unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15
were isolated and identified using mass spectrometry
(22). The majority of peptides
captured by the inactive enzyme were intracellular protein fragments that
efficiently interacted with EP24.15; the smallest peptide isolated in these
assays contained 5 and the largest 17 amino acids
(15,
16,
22,
32), which is within the size
range previously reported for natural and synthetic substrates of EP24.15
(18,
30,
33,
34). Interestingly, the
peptides released by the proteasome are in the same size range of EP24.15
competitive inhibitors/substrates
(7,
35,
36). Taken altogether, these
data suggest that in the intracellular environment EP24.15 could further
cleave proteasome-generated peptides unrelated to MHC-I antigen presentation
(15).Although the mutated inactive enzyme “capture” assay was
successful in identifying several cellular protein fragments that were
substrates for EP24.15, it also found some interacting peptides that were not
substrates. In this study, we used several approaches to directly screen for
cellular peptides that were cleaved by EP24.15. The first approach involved
the extraction of cellular peptides from the HEK293 cell line, incubation
in vitro with purified EP24.15, labeling with isotopic tags, and
analysis by mass spectrometry to obtain quantitative data on the extent of
cleavage. The second approach examined the effect of EP24.15 overexpression on
the cellular levels of peptides in the HEK293 cell line. The third set of
experiments tested synthetic peptides with purified EP24.15 in vitro,
and examined cleavage by high pressure liquid chromatography and mass
spectrometry. Collectively, these studies have identified a large number of
intracellular peptides, including those that likely represent the endogenous
substrates and products of EP24.15, and this original information contributes
to a better understanding of the function of this enzyme in vivo. 相似文献
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S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
14.
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
15.
Jonathan M. Budzik So-Young Oh Olaf Schneewind 《The Journal of biological chemistry》2009,284(19):12989-12997
Bacillus cereus and other Gram-positive bacteria elaborate pili
via a sortase D-catalyzed transpeptidation mechanism from major and minor
pilin precursor substrates. After cleavage of the LPXTG sorting
signal of the major pilin, BcpA, sortase D forms an amide bond between the
C-terminal threonine and the amino group of lysine within the YPKN motif of
another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the
BcpA sorting signal, resulting in a covalent bond between BcpA and the cell
wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the
minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal
threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby
positioning BcpB at the tip of pili. Thus, unique attributes of the sorting
signals of minor pilins provide Gram-positive bacteria with a universal
mechanism ordering assembly of pili.Sortases catalyze transpeptidation reactions to assemble proteins in the
envelope of Gram-positive bacteria
(1). Secreted proteins require
a C-terminal sorting signal for sortase recognition such that sortase cleaves
the substrate at a short peptide motif and forms a thioester-linked
intermediate to its active site cysteine
(2–4).
Nucleophilic attack by an amino group within the bacterial envelope resolves
the thioester intermediate, generating an amide bond tethering surface
proteins at their C terminus onto Gram-positive bacteria
(5). Four classes of sortases
can be distinguished on the basis of sequence homology and substrate
recognition (6,
7). Sortase A cleaves secreted
protein at LPXTG sorting signals and recognizes the amino group of
lipid II peptidoglycan precursors as a nucleophile
(8,
9). Sortase B cleaves protein
substrates at NPQTN sorting signals
(10). This enzyme immobilizes
proteins within fully assembled cell walls, utilizing the cell wall
cross-bridge as a nucleophile
(11). Sortase C cuts LPNTA
sorting signals and anchors proteins to the peptidoglycan cross-bridges in
sporulating bacteria (12,
13). Finally, sortase D
catalyzes transpeptidation reactions in the assembly of pili
(14,
15). Sortase D recognizes the
amino group of lysine residues within the YPKN motif of pilin subunits as
nucleophiles (16). The
resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow
the pilus fiber (16,
17).Pili of Gram-positive bacteria comprised either two or three different
pilin subunits synthesized as cytoplasmic precursors with N-terminal signal
peptides and C-terminal sorting signals (P1 precursors)
(14,
18). After translocation
across the plasma membrane, P2 precursor species arise from removal of the
signal peptide from P1 precursors by a signal peptidase
(16). Bacillus cereus
pili are composed of two subunits; that is, the major pilin, BcpA, and the
minor pilin, BcpB (15). In
contrast to BcpA, which is deposited throughout the pilus, BcpB is found at
fiber tip (15). Sortase D
cleaves the BcpA LPXTG motif sorting signal between the threonine and
glycine residues to form an amide bond to the ε-amino group of the lysine
within the YPKN motif of adjacent BcpA subunits
(16). However, sortase A also
cleaves BcpA precursors, which are subsequently linked to the side chain amino
group of meso-diaminopimelic acid within lipid II
(19). The latter reaction
serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall
envelope (19).The conservation of sortase D, the YPKN motif, and C-terminal sorting
signal in major pilin subunits suggest a universal pilus assembly mechanism
among Gram-positive bacteria
(14,
20). However, the molecular
mechanism whereby bacilli deposit BcpB, the minor pilin, at the tip of BcpA
pili is not known. Although the BcpB precursor harbors an N-terminal signal
peptide and a C-terminal IPNTG sorting signal, it lacks the YPKN pilin motif
of the major subunit (15).
Furthermore, the substrate properties of the BcpB IPNTG sorting signal for the
four classes of sortases expressed by bacilli has yet to be established. 相似文献
16.
17.
ATP-binding cassette (ABC) transporters transduce the free energy of ATP
hydrolysis to power the mechanical work of substrate translocation across cell
membranes. MsbA is an ABC transporter implicated in trafficking lipid A across
the inner membrane of Escherichia coli. It has sequence similarity
and overlapping substrate specificity with multidrug ABC transporters that
export cytotoxic molecules in humans and prokaryotes. Despite rapid advances
in structure determination of ABC efflux transporters, little is known
regarding the location of substrate-binding sites in the transmembrane segment
and the translocation pathway across the membrane. In this study, we have
mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using
site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin
labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster
at the cytoplasmic end of helices 3 and 6 at a site accessible from the
membrane/water interface and extending into an aqueous chamber formed at the
interface between the two transmembrane domains. Binding of a nonhydrolyzable
ATP analog inverts the transporter to an outward-facing conformation and
relieves DNR quenching by spin labels suggesting DNR exclusion from proximity
to the spin labels. The simplest model consistent with our data has DNR
entering near an elbow helix parallel to the water/membrane interface,
partitioning into the open chamber, and then translocating toward the
periplasm upon ATP binding.ATP-binding cassette
(ABC)2 transporters
transduce the energy of ATP hydrolysis to power the movement of a wide range
of substrates across the cell membranes
(1,
2). They constitute the largest
family of prokaryotic transporters, import essential cell nutrients, flip
lipids, and export toxic molecules
(3). Forty eight human ABC
transporters have been identified, including ABCB1, or P-glycoprotein, which
is implicated in cross-resistance to drugs and cytotoxic molecules
(4,
5). Inherited mutations in
these proteins are linked to diseases such as cystic fibrosis, persistent
hypoglycemia of infancy, and immune deficiency
(6).The functional unit of an ABC transporter consists of four modules. Two
highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze
ATP to supply the active energy for transport
(7). ABCs drive the mechanical
work of proteins with diverse functions ranging from membrane transport to DNA
repair (3,
5). Substrate specificity is
determined by two transmembrane domains (TMDs) that also provide the
translocation pathway across the bilayer
(7). Bacterial ABC exporters
are expressed as monomers, each consisting of one NBD and one TMD, that
dimerize to form the active transporter
(3). The number of
transmembrane helices and their organization differ significantly between ABC
importers and exporters reflecting the divergent structural and chemical
nature of their substrates (1,
8,
9). Furthermore, ABC exporters
bind substrates directly from the cytoplasm or bilayer inner leaflet and
release them to the periplasm or bilayer outer leaflet
(10,
11). In contrast, bacterial
importers have their substrates delivered to the TMD by a dedicated high
affinity substrate-binding protein
(12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on
the inner membrane to its final destination in the outer membrane requires the
ABC transporter MsbA (13).
Although MsbA has not been directly shown to transport lipid A, suppression of
MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits
bacterial growth strongly suggesting a role in translocation
(14-16).
In addition to this role in lipid A transport, MsbA shares sequence similarity
with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus
lactis, and Sav1866 of Staphylococcus aureus
(16-19).
ABCB1, a prototype of the ABC family, is a plasma membrane protein whose
overexpression provides resistance to chemotherapeutic agents in cancer cells
(1). LmrA and MsbA have
overlapping substrate specificity with ABCB1 suggesting that both proteins can
function as drug exporters
(18,
20). Indeed, cells expressing
MsbA confer resistance to erythromycin and ethidium bromide
(21). MsbA can be photolabeled
with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342
() across membrane vesicles in an energy-dependent manner
( H3334221).The structural mechanics of ABC exporters was revealed from comparison of
the MsbA crystal structures in the apo- and nucleotide-bound states as well as
from analysis by spin labeling EPR spectroscopy in liposomes
(17,
19,
22,
23). The energy harnessed from
ATP binding and hydrolysis drives a cycle of NBD association and dissociation
that is transmitted to induce reorientation of the TMD from an inward- to
outward-facing conformation
(17,
19,
22). Large amplitude motion
closes the cytoplasmic end of a chamber found at the interface between the two
TMDs and opens it to the periplasm
(23). These rearrangements
lead to significant changes in chamber hydration, which may drive substrate
translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile,
wasting the energy of ATP hydrolysis without substrate extrusion
(7). Consistent with this
model, ATP binding reduces ABCB1 substrate affinity, potentially through
binding site occlusion
(24-26).
Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP
hydrolysis increasing transport efficiency
(1,
27,
28). However, there is a
paucity of information regarding the location of substrate binding, the
transport pathway, and the structural basis of substrate recognition by ABC
exporters. In vitro studies of MsbA substrate specificity identify a
broad range of substrates that stimulate ATPase activity
(29). In addition to the
putative physiological substrates lipid A and lipopolysaccharide (LPS), the
ABCB1 substrates Ilmofosine, , and verapamil differentially enhance ATP
hydrolysis of MsbA ( H3334229,
30). Intrinsic MsbA tryptophan
(Trp) fluorescence quenching by these putative substrate molecules provides
further support of interaction
(29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model
of substrate binding to ABC efflux exporters. This so-called
“hydrophobic cleaner model” describes substrates binding from the
inner leaflet of the bilayer and then translocating through the TMD
(10,
31,
32). These studies also
identified a large number of residues involved in substrate binding and
selectivity (33). When these
crucial residues are mapped onto the crystal structures of MsbA, a subset of
homologous residues clusters to helices 3 and 6 lining the putative substrate
pathway (34). Consistent with
a role in substrate binding and specificity, simultaneous replacement of two
serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport
of ethidium and taxol, although and erythromycin interactions remain
unaffected ( H3334234).The tendency of lipophilic substrates to partition into membranes confounds
direct analysis of substrate interactions with ABC exporters
(35,
36). Such partitioning may
promote dynamic collisions with exposed Trp residues and nonspecific
cross-linking in photo-affinity labeling experiments. In this study, we
utilize a site-specific quenching approach to identify residues in the
vicinity of the daunorubicin (DNR)-binding site
(37). Although the data on DNR
stimulation of ATP hydrolysis is inconclusive
(20,
29,
30), the quenching of MsbA Trp
fluorescence suggests a specific interaction. Spin labels were introduced
along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent
quenching of DNR fluorescence. Residues that quench DNR cluster along the
cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR.
Furthermore, many of these residues are not lipid-exposed but face the
putative substrate chamber formed between the two TMDs. These residues are
proximal to two Trps, which likely explains the previously reported quenching
(29). Our results suggest DNR
partitions to the membrane and then binds MsbA in a manner consistent with the
hydrophobic cleaner model. Interpretation in the context of the crystal
structures of MsbA identifies a putative translocation pathway through the
transmembrane segment. 相似文献
18.
19.
Jaemin Lee Xiaofan Wang Bruno Di Jeso Peter Arvan 《The Journal of biological chemistry》2009,284(19):12752-12761
The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin
(Tg) has been identified as critically important in Tg export from the
endoplasmic reticulum. In a number of human kindreds suffering from congenital
hypothyroidism, and in the cog congenital goiter mouse and
rdw rat dwarf models, thyroid hormone synthesis is inhibited because
of mutations in the ChEL domain that block protein export from the endoplasmic
reticulum. We hypothesize that Tg forms homodimers through noncovalent
interactions involving two predicted α-helices in each ChEL domain that
are homologous to the dimerization helices of acetylcholinesterase. This has
been explored through selective epitope tagging of dimerization partners and
by inserting an extra, unpaired Cys residue to create an opportunity for
intermolecular disulfide pairing. We show that the ChEL domain is necessary
and sufficient for Tg dimerization; specifically, the isolated ChEL domain can
dimerize with full-length Tg or with itself. Insertion of an N-linked
glycan into the putative upstream dimerization helix inhibits homodimerization
of the isolated ChEL domain. However, interestingly, co-expression of upstream
Tg domains, either in cis or in trans, overrides the
dimerization defect of such a mutant. Thus, although the ChEL domain provides
a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL
domain actively stabilizes the Tg dimer complex for intracellular
transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of
thyroglobulin (Tg)2 to
the apical luminal cavity of thyroid follicles
(1). Once secreted, Tg is
iodinated via the activity of thyroid peroxidase
(2). A coupling reaction
involving a quinol-ether linkage especially engages di-iodinated tyrosyl
residues 5 and 130 to form thyroxine within the amino-terminal portion of the
Tg polypeptide (3,
4). Preferential iodination of
Tg hormonogenic sites is dependent not on the specificity of the peroxidase
(5) but upon the native
structure of Tg (6,
7). To date, no other thyroidal
proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746
amino acids) involves three regions called I-II-III comprised of
disulfide-rich repeat domains held together by intradomain disulfide bonds
(8,
9). The final 581 amino acids
of Tg are strongly homologous to acetylcholinesterase
(10–12).
Rate-limiting steps in the overall process of Tg secretion involve its
structural maturation within the endoplasmic reticulum (ER)
(13). Interactions between
regions I-II-III and the cholinesterase-like (ChEL) domain have recently been
suggested to be important in this process, with ChEL functioning as an
intramolecular chaperone and escort for I-II-III
(14). In addition, Tg
conformational maturation culminates in Tg homodimerization
(15,
16) with progression to a
cylindrical, and ultimately, a compact ovoid structure
(17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a
commonly affected site of mutation, including the recently described A2215D
(20,
21), R2223H
(22), G2300D, R2317Q
(23), G2355V, G2356R, and the
skipping of exon 45 (which normally encodes 36 amino acids), as well as the
Q2638stop mutant (24) (in
addition to polymorphisms including P2213L, W2482R, and R2511Q that may be
associated with thyroid overgrowth
(25)). As best as is currently
known, all of the congenital hypothyroidism-inducing Tg mutants are defective
for intracellular transport
(26). A homozygous G2300R
mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is
responsible for congenital hypothyroidism in rdw rats
(27,
28), whereas we identified the
Tg-L2263P point mutation as the cause of hypothyroidism in the cog
mouse (29). Such mutations
perturb intradomain structure
(30), and interestingly, block
homodimerization (31).
Acquisition of quaternary structure has long been thought to be required for
efficient export from the ER
(32) as exemplified by
authentic acetylcholinesterase
(33,
34) in which dimerization
enhances protein stability and export
(35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain)
is blocked within the ER (30),
whereas a secretory version of the isolated ChEL domain of Tg devoid of
I-II-III undergoes rapid and efficient intracellular transport and secretion
(14). A striking homology
positions two predicted α-helices of the ChEL domain to the identical
relative positions of the dimerization helices in acetylcholinesterase. This
raises the possibility that ChEL may serve as a homodimerization domain for
Tg, providing a critical function in maturation for Tg transport to the site
of thyroid hormone synthesis
(1).In this study, we provide unequivocal evidence for homodimerization of the
ChEL domain and “hetero”-dimerization of that domain with
full-length Tg, and we provide significant evidence that the predicted ChEL
dimerization helices provide a nidus for Tg assembly. On the other hand, our
data also suggest that upstream Tg regions known to interact with ChEL
(14) actively stabilize the Tg
dimer complex. Together, I-II-III and ChEL provide unique contributions to the
process of intracellular transport of Tg through the secretory pathway. 相似文献