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1.
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
2.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
3.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
4.
5.
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
6.
7.
8.
9.
Alexander Panov Peter Schonfeld Sergey Dikalov Richelle Hemendinger Herbert L. Bonkovsky Benjamin Rix Brooks 《The Journal of biological chemistry》2009,284(21):14448-14456
The finding that upon neuronal activation glutamate is transported
postsynaptically from synaptic clefts and increased lactate availability for
neurons suggest that brain mitochondria (BM) utilize a mixture of substrates,
namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We
studied how glutamate affected oxidative phosphorylation and reactive oxygen
species (ROS) production in rat BM oxidizing pyruvate + malate or succinate.
Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and
uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation
increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM
oxidizing glutamate + malate. Up to 70% of ROS generation was associated with
reverse electron transport. These effects of pyruvate + glutamate + malate
were observed only with BM and not with liver or heart mitochondria. The
effects of glutamate + pyruvate on succinate-supported respiration and ROS
generation were not organ-specific and depended only on whether mitochondria
were isolated with or without bovine serum albumin. With the non-bovine serum
albumin brain and heart mitochondria oxidizing succinate, the addition of
pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We
conclude that (i) during neuronal activation, simultaneous oxidation of
glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP
production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an
inherent mechanism that protects against ROS generation during reverse
electron transport.Recently, it has emerged that mitochondrial dysfunctions play an important
role in the pathogenesis of degenerative diseases of the central nervous
system
(1–3).
The processes underlying neuronal degeneration are complex, and some authors
suggest that several genetic alterations are involved
(4). However, another level of
complexity may be derived from the fact that virtually all cellular activities
depend upon energy metabolism in the cell
(5). Alterations in energy
metabolism processes within cells may also contribute to pathogenic mechanisms
underlying neurodegenerative disease.A large body of evidence suggests that increased oxidative stress is an
important pathogenic mechanism that promotes neurodegeneration
(6). Because neurons have a
long life span, and most neurodegenerative diseases have a clear association
with age (7), it is important
to understand mechanisms underlying reactive oxygen species
(ROS)2 production in
neurons. Recently, Kudin et al.
(8) analyzed the contribution
of mitochondria to the total ROS production in brain tissue. They concluded
that mitochondria are the major source of ROS and that at least 50% of ROS
generated by brain mitochondria was associated with succinate-supported
reverse electron transport (RET). Under conditions of normoxia, about 1% of
the respiratory chain electron flow was redirected to form superoxide
(8).Recently, we suggested that the organization of the respiratory chain
complexes into supercomplexes that occurs in brain mitochondria (BM)
(9) may represent one of the
intrinsic mechanisms to prevent excessive ROS generation
(10). In this paper, we put
forward the hypothesis that inhibition of Complex II by oxaloacetate (OAA)
represents another important intrinsic mechanism to prevent oxidative stress.
We provide evidence that glutamate and pyruvate specifically exert control
over the production of ROS at the level of Complex II. Below we present a
brief account of published theoretical and experimental evidence that underlie
our hypothesis.The neural processing of information is metabolically expensive
(11). More than 80% of energy
is spent postsynaptically to restore the ionic composition of neurons
(11). When neurons are
activated, reuptake of glutamate stimulates aerobic glycolysis in astroglial
cells (12), thereby making
lactate the major substrate for neuronal mitochondria
(4,
13). However, rapid conversion
of lactate to pyruvate in neurons requires activation of the malate-aspartate
shuttle (MAS). The shuttle is the major pathway for cytosolic reducing
equivalents from NADH to enter the mitochondria and be oxidized
(14,
15). The key component of MAS
is the mitochondrial aspartate/glutamate carrier (AGC)
(16), and recent data suggest
that the AGC is expressed mainly in neurons
(14). Absence of the AGC from
astrocytes in the brain implies a compartmentation of intermediary metabolism,
with glycolysis taking place in astrocytes and lactate oxidation in neurons
(13,
14,
17). Active operation of MAS
requires that a certain amount of glutamate must be transported from synaptic
clefts into activated neurons. In isolated BM, it has been shown that besides
pyruvate, glutamate is also a good respiratory substrate
(5,
18). In the presynaptic
elements, the concentration of cytosolic glutamate is ∼10 mm at
all times (19). Yudkoff et
al. (18) have shown that
synaptosomal mitochondria utilize glutamate and pyruvate as mitochondrial
respiratory substrates. Glutamate is also oxidized by the astroglial
mitochondria (13).Until recently, it was generally accepted that most of the glutamate is
rapidly removed from the synaptic cleft by glutamate transporters EAAT1 and
EAAT2 located on presynaptic termini and glial cells
(20–24).
However, recent data show that a significant fraction of glutamate is rapidly
bound and transported by the glutamate transporter isoform, EAAT4, located
juxtasynaptically in the membranes of spines and dendrites
(20,
25–28).
At the climbing fiber to Purkinje cell synapses in the cerebellum, about 17%
(28) or more than 50%
(29) of synaptically released
glutamate may be removed by postsynaptic transporters. Besides the cerebellum,
EAAT4 protein was found to be omnipresent throughout the fore- and midbrain
regions (30). Moreover, it was
shown that although most of the EAAT2 protein is astroglial, around 15% is
distributed in nerve terminals and axons in hippocampal slices and that this
protein may be responsible for more than half of the total uptake of glutamate
from synaptic clefts (24).
These data suggest that postsynaptic transport of glutamate into nerve
terminals where mitochondria are located
(31) may occur in all brain
regions. According to calculations of Brasnjo and Otis
(28), in a single synapse,
EAAT4 (excitatory amino acid transporter 4) binds and transports
postsynaptically about 1.3 ± 0.1 × 106 glutamate
molecules. In the brain, on average, 1 mm3 of tissue contains 1
× 108 synapses
(32,
33). Because of the high
density of synaptic contacts, the neuronal cells may be exposed to mediators
released from hundreds of firing synapses. Thus, in a narrow space of spines
and dendrites, several million glutamate molecules postsynaptically
transported from synaptic boutons may create local cytosolic concentration of
glutamate in the low millimolar range. Consequently, neuronal mitochondria,
particularly those located at the axonal or dendritic synaptic junctions, may,
in addition to metabolizing pyruvate, temporarily metabolize glutamate and
succinate formed during mitochondrial catabolism of γ-aminobutyric acid
in postsynaptic cells
(34).The purpose of this study was to examine how the neuromediator glutamate
affects respiratory activity and ROS generation in nonsynaptic BM when
combined with pyruvate and the tricarboxylic acid cycle intermediates
succinate and malate. We show that with pyruvate + glutamate + malate, the
rate of oxidative phosphorylation increased more than 50%, and in resting
mitochondria the rate of ROS generation associated with the reverse electron
transport increased severalfold. These effects were observed only with brain
and spinal cord mitochondria, not with liver or heart mitochondria, suggesting
that they may be restricted to neuronal cells.Taken together, the data presented support the hypothesis that in activated
neurons, the neuromediator glutamate stimulates mitochondrial ATP production
when energy demand is increased. However, in the absence of energy
consumption, glutamate + pyruvate may increase the generation of ROS
severalfold. We suggest that intrinsic inhibition of Complex II by
oxaloacetate is an important natural protective mechanism against ROS
associated with reverse electron transport. 相似文献
10.
11.
12.
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
13.
14.
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
15.
Yong Zhang Yong-Gang Wang Qi Zhang Xiu-Jie Liu Xuan Liu Li Jiao Wei Zhu Zhao-Huan Zhang Xiao-Lin Zhao Cheng He 《The Journal of biological chemistry》2009,284(18):12469-12479
TrkA receptor signaling is essential for nerve growth factor (NGF)-induced
survival and differentiation of sensory neurons. To identify possible
effectors or regulators of TrkA signaling, yeast two-hybrid screening was
performed using the intracellular domain of TrkA as bait. We identified
muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and
found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in
a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation
assays showed that endogenous TrkA interacted with Mint2 in rat tissue
homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA
colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2
overexpression inhibited NGF-induced neurite outgrowth in both PC12 and
cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression
by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2
was found to promote the retention of TrkA in the Golgi apparatus and inhibit
its surface sorting. Taken together, our data provide evidence that Mint2 is a
novel TrkA-regulating protein that affects NGF-induced neurite outgrowth,
possibly through a mechanism involving retention of TrkA in the Golgi
apparatus.The neurotrophin family member nerve growth factor
(NGF)3 is
essential for proper development, patterning, and maintenance of nervous
systems (1,
2). NGF has two known
receptors; TrkA, a single-pass transmembrane receptor-tyrosine kinase that
binds selectively to NGF, and p75, a transmembrane glycoprotein that binds all
members of the neurotrophin family
(3,
4). NGF binding activates the
kinase domain of TrkA, leading to autophosphorylation
(5). The resulting
phosphotyrosines become docking sites for adaptor proteins involved in signal
transduction pathways that lead to the activation of Ras, Rac,
phosphatidylinositol 3-kinase, phospholipase Cγ, and other effectors
(2,
6). Many of these
TrkA-interacting adaptor proteins have been identified and include, Grb2, APS,
SH2B, fibroblast growth factor receptor substrate 2 (FRS-2), Shc, and human
tumor imaginal disc 1 (TID1)
(7-10).
The identification of these binding partners has contributed greatly to our
understanding of the mechanisms underlying the functional diversity of
NGF-TrkA signaling.Studies have indicated that the transmission of NGF signaling in neurons
involves retrograde transport of NGF-TrkA complexes from the neurite tip to
the cell body
(11-14).
TrkA associates with components of cytoplasmic dynein, and it is thought that
vesicular trafficking of neurotrophins occurs via direct interaction of Trk
receptors with the dynein motor machinery
(14). Furthermore, the
atypical protein kinase C-interacting protein, p62, associates with TrkA and
plays a novel role in connecting receptor signals with the endosomal signaling
network required for mediating TrkA-induced differentiation
(15). Recently, the
membrane-trafficking protein Pincher has been found to mediate
macroendocytosis underlying retrograde signaling by TrkA
(16). Despite the progress
made to date in understanding Trk complex internalization and trafficking, the
mechanisms remain poorly understood.Mint2 (muc18-1-interacting protein 2) belongs to the Mint protein family,
which consists of three members, Mint1, Mint2, and Mint3. Mint proteins were
first identified as interacting proteins of the synaptic vesicle-docking
protein Munc18-1 (17,
18). Mint1 is also sometimes
referred to as mLIN-10, as it is the mammalian orthologue of the
Caenorhabditis elegans LIN-10
(19). Additionally, Mint1,
Mint2, and Mint3 are also referred to as X11α or X11, X11β or X11L
(X11-like), and X11γ or X11L2 (X11-like 2), respectively
(20). All Mint proteins
contain a conserved central phosphotyrosine binding (PTB) domain and two
contiguous C-terminal PDZ domains (repeated sequences in the brain-specific
protein PSD-95, the Drosophila septate junction protein Discs large,
and the epithelial tight junction protein ZO-1)
(17,
18,
21). Mint1 and Mint2 are
expressed only in neuronal tissue
(17), whereas Mint3 is
ubiquitously expressed (18).
Although the function of Mints proteins is not fully clear, their interactions
with the docking and exocytosis factors Mun18 -1 and CASK, ADP-ribosylation
factor (Arf) GTPases involved in vesicle budding
(22), and other synaptic
adaptor proteins, such as neurabin-II/spinophilin
(23), tamalin
(24), and kalirin-7
(25), all suggest possible
roles for Mints in synaptic vesicle docking and exocytosis. Mint proteins have
also been implicated in the trafficking and/or processing of β-amyloid
precursor protein (β-APP). Through their PTB domains, all three Mints
bind to a motif within the cytoplasmic domain of β-APP
(21,
26-29),
and Mint1 and Mint2 can stabilize β-APP, affect β-APP processing,
and inhibit the production and secretion of Aβ
(28,
30-32).
Although the mechanisms by which Mints inhibit β-APP processing are not
yet well known, Mints and their binding partners have emerged as potential
therapeutic targets for the treatment of Alzheimer disease.To uncover new TrkA-interacting factors and gain insight into the
mechanisms that guide TrkA intracellular trafficking and other aspects of TrkA
signaling, we conducted a yeast two-hybrid screen of a brain cDNA library
using the intracellular domain of TrkA as bait. The screen identified several
candidate TrkA-interacting proteins, one of which was Mint2. Follow-up binding
assays showed that the PTB domain of Mint2 alone was necessary and sufficient
for mediating the interaction with TrkA. Endogenous Mint2 was also
coimmunoprecipitated and colocalized with TrkA in rat DRG tissue.
Overexpression and knockdown studies showed that Mint2 could significantly
inhibit NGF-induced neurite outgrowth in both TrkA-expressing PC12 cells and
DRG neurons. Moreover, Mint2 was found to induce the retention of TrkA in the
Golgi apparatus and inhibit its surface sorting. Our results suggest that
Mint2 is a novel regulator of TrkA receptor signaling. 相似文献
16.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
17.
18.
19.
Siddhartha Mitra Andrey S. Tsvetkov Steven Finkbeiner 《The Journal of biological chemistry》2009,284(7):4398-4403
The accumulation of mutant protein in intracellular aggregates is a common
feature of neurodegenerative disease. In Huntington disease, mutant huntingtin
leads to inclusion body (IB) formation and neuronal toxicity. Impairment of
the ubiquitin-proteasome system (UPS) has been implicated in IB formation and
Huntington disease pathogenesis. However, IBs form asynchronously in only a
subset of cells with mutant huntingtin, and the relationship between IB
formation and UPS function has been difficult to elucidate. Here, we applied
single-cell longitudinal acquisition and analysis to monitor mutant huntingtin
IB formation, UPS function, and neuronal toxicity. We found that proteasome
inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB
formation, the UPS is more impaired in neurons that go on to form IBs than in
those that do not. After forming IBs, impairment is lower in neurons with IBs
than in those without. These findings suggest IBs are a protective cellular
response to mutant protein mediated in part by improving intracellular protein
degradation.Huntington disease
(HD)4 is a progressive
incurable neurodegenerative disorder caused by the expansion of a
polyglutamine (polyQ) stretch in the N-terminal end of the huntingtin (htt)
protein above a threshold length of ∼36
(1). The deposition of
polyQ-expanded aggregated mutant htt in inclusion bodies (IBs) is a hallmark
of HD, and IBs are found in human post-mortem samples, transgenic mouse brain,
and cell-culture models (2).
The accumulation of ubiquitinated proteins in IBs has implicated the
ubiquitin-proteasome system (UPS) in the pathogenesis of HD, amyotrophic
lateral sclerosis, Parkinson disease, and polyQ-mediated disorders
(3).The UPS is a major pathway of intracellular protein degradation. After a
series of three reactions, each catalyzed by a different set of enzymes,
ubiquitin, a 76-amino acid polypeptide, forms an isopeptide bond with the
amino group of lysine residues on substrate proteins. Several lysine residues
within ubiquitin are sites for more ubiquitin additions. Once a protein
accumulates four or more ubiquitins, it is efficiently targeted to the
proteasome for degradation. The proteasome binds polyubiquitinated substrates
and hydrolyzes ubiquitin isopeptide bonds, releasing ubiquitin moieties before
degrading substrate proteins through chymotrypsin-like, trypsin-like, and
post-glutamyl peptidase activities
(3).Increased polyubiquitin levels and changes in ubiquitin linkages accompany
the accumulation of UPS substrates in the brains of HD patients and transgenic
mice and in cellular HD models
(4). UPS substrates accumulate
throughout the cell in polyQ models, even before IB formation
(5,
6). This has added to the
confusion over whether polyQ expansion leads to toxicity through direct
impairment of proteasomal degradation. Proteasomes have been reported to
cleave polyQ stretches efficiently
(7), inefficiently
(8), or essentially not at all
(9). In vivo,
polyQ-dependent degeneration occurs with no detectable proteasome inhibition
(10,
11) or is tightly linked to it
(12,
13). The inability of some
studies to detect UPS impairment in HD models may be due to the limited
sensitivity of conventional approaches to identify cell-to-cell variations in
UPS function.The relationship between IB formation and UPS function has been difficult
to determine. Protein turnover in cells with IBs is evidently reduced and
accompanied by the accumulation of cellular proteins
(14–16);
HEK293 cells containing mutant htt IBs have a greater degree of UPS impairment
than those without IBs (5).
Proteasome subunits and heat shock proteins colocalize with IBs, but it is
unclear if this colocalization facilitates protein delivery or unfolding at
the mouth of active proteasomes, or if it harms proteasome function by
sequestering essential cellular machinery
(18). Some IBs are relatively
static (8,
25), but the proteins in
others are dynamically exchanged with cytoplasmic and nuclear pools
(19,
20).UPS function is critical to cellular homeostasis. Deletion of one of the
two inducible polyubiquitin genes in mice leads to lower intracellular
ubiquitin levels in germ cells and hypothalamic neurons. These same
populations undergo cell-cycle arrest and hypothalamic neurodegeneration,
respectively (22,
23). Cell lines expressing
mutant huntingtin accumulate ubiquitinated proteins and undergo cell-cycle
arrest in G2/M (5). In neurons,
UPS impairment may lead to cell death through an accumulation of signals for
apoptosis, a decrease in NF-κB signaling, sensitization to other toxic
stimuli, remodeling of synapses, retraction of neurites, or other unidentified
mechanisms (24). The effect of
UPS impairment depends on cell type and cell cycle, and the relationship
between UPS impairment and striatal neuronal survival is largely unknown.Diffuse species of mutant htt induce IB formation and neuronal death in a
protein concentration-dependent manner
(2). IB formation delays
neuronal death, suggesting that IB formation helps neurons cope with toxic
diffuse mutant htt. Whether the effect of IB formation on survival is mediated
through UPS function has been difficult to determine. IB formation and
neuronal death occur asynchronously in overlapping but distinct subsets of
neurons that express mutant htt. The observation that IB formation is not
required for UPS impairment also complicates population analysis
(6,
26).To explore this problem, we applied single-cell analysis. We tracked single
neurons over their entire lifetimes, gaining spatial and temporal resolution
while simultaneously monitoring IB formation, UPS inhibition, and neuronal
toxicity. 相似文献
20.
Lina Du Robert W. Hickey H��lya Bayir Simon C. Watkins Vladimir A. Tyurin Fengli Guo Patrick M. Kochanek Larry W. Jenkins Jin Ren Greg Gibson Charleen T. Chu Valerian E. Kagan Robert S. B. Clark 《The Journal of biological chemistry》2009,284(4):2383-2396
Sex-dependent differences in adaptation to famine have long been
appreciated, thought to hinge on female versus male preferences for
fat versus protein sources, respectively. However, whether these
differences can be reduced to neurons, independent of typical nutrient depots,
such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A
vital adaptation to starvation is autophagy, a mechanism for recycling amino
acids from organelles and proteins. Here we show that segregated neurons from
males in culture are more vulnerable to starvation than neurons from females.
Nutrient deprivation decreased mitochondrial respiration, increased
autophagosome formation, and produced cell death more profoundly in neurons
from males versus females. Starvation-induced neuronal death was
attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7
knockdown using small interfering RNA; or l-carnitine, essential
for transport of fatty acids into mitochondria, all more effective in neurons
from males versus females. Relative tolerance to nutrient deprivation
in neurons from females was associated with a marked increase in triglyceride
and free fatty acid content and a cytosolic phospholipase A2-dependent
increase in formation of lipid droplets. Similar sex differences in
sensitivity to nutrient deprivation were seen in fibroblasts. However,
although inhibition of autophagy using Atg7 small interfering RNA
inhibited cell death during starvation in neurons, it increased cell death in
fibroblasts, implying that the role of autophagy during starvation is both
sex- and tissue-dependent. Thus, during starvation, neurons from males more
readily undergo autophagy and die, whereas neurons from females mobilize fatty
acids, accumulate triglycerides, form lipid droplets, and survive longer.Sex-dependent differences in adaptation to famine have long been
appreciated (1,
2), thought to hinge on a
female preference for fat sources, in contrast to a male preference for
protein sources (3). Fatty acid
metabolism is different between sexes normally
(4) and under conditions of
starvation (1,
2). During exercise, in
addition to increases in carbohydrate requirement, men increase their need for
amino acids, whereas women increase mobilization of fat
(5). Furthermore, sex-dependent
responses to nutritional stress associated with either self-induced weight
loss or illness-related cachexia also exist
(6,
7).An important adaptation to starvation is autophagy (autophagy-associated
proteins, abbreviated ATG). Classic, starvation-induced autophagy is initiated
by nutrient and amino acid deprivation, glucagon, and cAMP
(8,
9). ATG7, a ubiquitin E1-like
enzyme, is essential for autophagy, with phosphorylation of preautophagosomal
membranes, formation of ATG12-ATG5 complexes, and processing of ATG8/LC3
(microtubule-associated protein light chain-3) as other crucial steps in this
process (10).
Starvation-induced autophagy is regulated by class III phosphatidylinositol
3-kinase and the Bcl-2-interacting partner, Beclin-1
(11). The autophagosomes then
engulf cytoplasmic material and/or organelles, such as mitochondria, the
latter sometimes referred to as “mitophagy,” disassembling large
proteins and organelles to recycle amino acids and other nutrients, an
important response to starvation
(12).It is unknown whether starvation can induce autophagy in the brain;
however, there is evidence that critical starvation can result in brain
atrophy in humans. It has been reported that ∼30% of people during a
prolonged hunger strike (mean of 199 days) will show brain tissue loss
(13), and brain shrinkage in
patients with anorexia nervosa is well documented
(14,
15). Although 48 h of food
deprivation does not produce detectable autophagy in brains from mice
(16), the aforementioned
reports are consistent with long durations of starvation as a bona
fide stimulus for autophagy in brain. There are recent studies suggesting
that other stimuli can induce autophagy in the brain, such as trauma
(17) and ischemia
(18), and that autophagy may
contribute to neuronal death. There is also evidence for autophagy in the
human brain after trauma and critical illness
(19), which probably includes
both elements of malnutrition and systemic stress. A potential role for brain
atrophy as a contributor to neurological morbidity in the critically ill and
injured is an emerging topic
(20). 相似文献