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1.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
2.
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
3.
Sophie Pattingre Chantal Bauvy St��phane Carpentier Thierry Levade Beth Levine Patrice Codogno 《The Journal of biological chemistry》2009,284(5):2719-2728
Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated
during periods of nutrient starvation to preserve cell integrity. Ceramide is
a bioactive sphingolipid associated with a large range of cell processes. Here
we show that short-chain ceramides (C2-ceramide and
C6-ceramide) and stimulation of the de novo ceramide
synthesis by tamoxifen induce the dissociation of the complex formed between
the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This
dissociation is required for macroautophagy to be induced either in response
to ceramide or to starvation. Three potential phosphorylation sites,
Thr69, Ser70, and Ser87, located in the
non-structural N-terminal loop of Bcl-2, play major roles in the dissociation
of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal
protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to
stimulate macroautophagy. These findings reveal a new aspect of sphingolipid
signaling in up-regulating a major cell process involved in cell adaptation to
stress.Macroautophagy (referred to below as “autophagy”) is a
vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is
conserved in eukaryotic cells
(1–3).
Autophagy is initiated by the formation of a multimembrane-bound autophagosome
that engulfs cytoplasmic proteins and organelles. The last stage in the
process results in fusion with the lysosomal compartments, where the
autophagic cargo undergoes degradation. Basal autophagy is important in
controlling the quality of the cytoplasm by removing damaged organelles and
protein aggregates. Inhibition of basal autophagy in the brain is deleterious,
and leads to neurodegeneration in mouse models
(4,
5). Stimulation of autophagy
during periods of nutrient starvation is a physiological response present at
birth and has been shown to provide energy in various tissues of newborn pups
(6). In cultured cells,
starvation-induced autophagy is an autonomous cell survival mechanism, which
provides nutrients to maintain a metabolic rate and level of ATP compatible
with cell survival (7). In
addition, starvation-induced autophagy blocks the induction of apoptosis
(8). In other contexts, such as
drug treatment and a hypoxic environment, autophagy has also been shown to be
cytoprotective in cancer cells
(9,
10). However, autophagy is
also part of cell death pathways in certain situations
(11). Autophagy can be a
player in apoptosis-independent type-2 cell death (type-1 cell death is
apoptosis), also known as autophagic cell death. This situation has been shown
to occur when the apoptotic machinery is crippled in mammalian cells
(12,
13). Autophagy can also be
part of the apoptotic program, for instance in tumor necrosis
factor-α-induced cell death when NF-κB is inhibited
(14), or in human
immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T
cells (15). Moreover autophagy
has recently been shown to be required for the externalization of
phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of
apoptotic cells (16).The complex relationship between autophagy and apoptosis reflects the
intertwined regulation of these processes
(17,
18). Many signaling pathways
involved in the regulation of autophagy also regulate apoptosis. This
intertwining has recently been shown to occur at the level of the molecular
machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been
shown to inhibit starvation-induced autophagy by interacting with the
autophagy protein Beclin 1
(19). Beclin 1 is one of the
Atg proteins conserved from yeast to humans (it is the mammalian orthologue of
yeast Atg6) and is involved in autophagosome formation
(20). Beclin 1 is a platform
protein that interacts with several different partners, including hVps34
(class III phosphatidylinositol 3-kinase), which is responsible for the
synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is
important for events associated with the nucleation of the isolation membrane
before it elongates and closes to form autophagosomes in response to other Atg
proteins, including the Atg12 and
LC32
(microtubule-associated protein light chain 3 is the mammalian orthologue of
the yeast Atg8) ubiquitin-like conjugation systems
(3,
21). Various partners
associated with the Beclin 1 complex modulate the activity of hVps34. For
instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1,
and Bif-1 all up-regulate it
(22,
23).In view of the intertwining between autophagy and apoptosis, it is
noteworthy that Beclin 1 belongs to the BH3-only family of proteins
(24–26).
However, and unlike most of the proteins in this family, Beclin 1 is not able
to trigger apoptosis when its expression is forced in cells
(27). A BH3-mimetic drug,
ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger
autophagy by mirroring the effect of starvation
(25).The sphingolipids constitute a family of bioactive lipids
(28–32)
of which several members, such as ceramide and sphingosine 1-phosphate, are
signaling molecules. These molecules constitute a “sphingolipid
rheostat” that determines the fate of the cell, because in many settings
ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic
effect (31,
32). However, ceramide is also
engaged in a wide variety of other cell processes, such as the formation of
exosomes (33),
differentiation, cell proliferation, and senescence
(34). Recently we showed that
both ceramide and sphingosine 1-phosphate are able to stimulate autophagy
(35,
36). It has also been shown
that ceramide triggers autophagy in a large panel of mammalian cells
(37–39).
However, elucidation of the mechanism by which ceramide stimulates autophagy
is still in its infancy. We have previously demonstrated that ceramide induces
autophagy in breast and colon cancer cells by inhibiting the Class I
phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central
role in inhibiting autophagy
(36). Inhibition of mTOR is
another hallmark of starvation-induced autophagy
(17). This finding led us to
investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results
presented here show that ceramide is more potent than starvation in
dissociating the Beclin 1-Bcl-2 complex (see Ref.
40). This dissociation is
dependent on three phosphorylation sites (Thr69, Ser70,
and Ser87) located in a non-structural loop of Bcl-2. Ceramide
induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2.
Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation,
and thus the induction of autophagy by ceramide. These findings help to
explain how autophagy is regulated by a major lipid second messenger. 相似文献
4.
5.
Dejiang Feng Andrzej Witkowski Stuart Smith 《The Journal of biological chemistry》2009,284(17):11436-11445
The objective of this study was to evaluate the physiological importance of
the mitochondrial fatty acid synthesis pathway in mammalian cells using the
RNA interference strategy. Transfection of HEK293T cells with small
interfering RNAs targeting the acyl carrier protein (ACP) component reduced
ACP mRNA and protein levels by >85% within 24 h. The earliest phenotypic
changes observed were a marked decrease in the proportion of
post-translationally lipoylated mitochondrial proteins recognized by
anti-lipoate antibodies and a reduction in their catalytic activity, and a
slowing of the cell growth rate. Later effects observed included a reduction
in the specific activity of respiratory complex I, lowered mitochondrial
membrane potential, the development of cytoplasmic membrane blebs containing
high levels of reactive oxygen species and ultimately, cell death.
Supplementation of the culture medium with lipoic acid offered some protection
against oxidative damage but did not reverse the protein lipoylation defect.
These observations are consistent with a dual role for ACP in mammalian
mitochondrial function. First, as a key component of the mitochondrial fatty
acid biosynthetic pathway, ACP plays an essential role in providing the
octanoyl-ACP precursor required for the protein lipoylation pathway. Second,
as one of the subunits of complex I, ACP is required for the efficient
functioning of the electron transport chain and maintenance of normal
mitochondrial membrane potential.Eukaryotes employ two distinct systems for the synthesis of fatty acids
de novo. The bulk of fatty acids destined for membrane biogenesis and
energy storage are synthesized in the cytosolic compartment by megasynthases
in which the component enzymes are covalently linked in very large
polypeptides; this system is referred to as the type I fatty acid synthase
(FAS)2
(1,
2). A second system localized
in mitochondria is composed of a suite of discrete, freestanding enzymes that
closely resemble their counterparts in prokaryotes
(3–10),
which are characterized as type II FASs
(11). Most of the constituent
enzymes of the mitochondrial fatty acid biosynthetic system have been
identified and characterized in fungi and animals; all are nuclear-encoded
proteins that are transported to the matrix compartment of mitochondria. Fungi
with deleted mitochondrial FAS genes fail to grow on non-fermentable carbon
sources, have low levels of lipoic acid and elevated levels of mitochondrial
lysophospholipids (12,
13). These observations
indicate that the mitochondrial FAS may serve to provide the octanoyl
precursor required for the biosynthesis of lipoyl moieties de novo,
as well as providing fatty acids that are utilized in remodeling of
mitochondrial membrane phospholipids
(14). The mitochondrial FAS
system in animals is less well characterized. However, kinetic analysis of the
β-ketoacyl synthase enzyme responsible for catalysis of the chain
extension reaction in human mitochondria suggested that this system is
uniquely engineered to produce mainly octanoyl moieties and has limited
ability to form long-chain products
(9). Indeed, studies with a
reconstituted system from bovine heart mitochondrial matrix extracts confirmed
that octanoyl moieties are the main product and are utilized for the synthesis
of lipoyl moieties (15). One
of the key components of the prokaryotic and mitochondrial FAS systems is a
small molecular mass, freestanding protein, the ACP, that shuttles substrates
and pathway intermediates to each of the component enzymes. The mitochondrial
ACP is localized primarily in the matrix compartment
(16), but a small fraction is
integrated into complex I of the electron transport chain
(17–23).
As is the case with many of the other 45 subunits of complex I, the role of
the ACP subunit is unclear
(24). To clarify the
physiological importance of the mitochondrial FAS, and the mitochondrial ACP
in particular, in mammalian mitochondrial function we have utilized an RNA
interference strategy to knockdown the mitochondrial ACP in cultured HEK293T
cells. 相似文献
6.
Ian G. Ganley Du H. Lam Junru Wang Xiaojun Ding She Chen Xuejun Jiang 《The Journal of biological chemistry》2009,284(18):12297-12305
Autophagy is a degradative process that recycles long-lived and faulty
cellular components. It is linked to many diseases and is required for normal
development. ULK1, a mammalian serine/threonine protein kinase, plays a key
role in the initial stages of autophagy, though the exact molecular mechanism
is unknown. Here we report identification of a novel protein complex
containing ULK1 and two additional protein factors, FIP200 and ATG13, all of
which are essential for starvation-induced autophagy. Both FIP200 and ATG13
are critical for correct localization of ULK1 to the pre-autophagosome and
stability of ULK1 protein. Additionally, we demonstrate by using both cellular
experiments and a de novo in vitro reconstituted reaction that FIP200
and ATG13 can enhance ULK1 kinase activity individually but both are required
for maximal stimulation. Further, we show that ATG13 and ULK1 are
phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner,
indicating that the ULK1·ATG13·FIP200 complex acts as a node for
integrating incoming autophagy signals into autophagosome biogenesis.Macroautophagy (herein referred to as autophagy) is a catabolic process
whereby long-lived proteins and damaged organelles are shuttled to lysosomes
for degradation. This process is conserved in all eukaryotes. Under normal
growth conditions a housekeeping level of autophagy exists. Under stress, such
as nutrient starvation, autophagy is strongly induced resulting in the
engulfment of cytosolic components and organelles in specialized
double-membrane structures termed autophagosomes. Following fusion of the
outer autophagosomal membrane with lysosomes, the inner membrane and its
cytoplasmic cargo are degraded and recycled
(1–3).
Recent work has implicated autophagy in many disease pathologies, including
cancer, neurodegeneration, as well as in eliminating intracellular pathogens
(4–8).The morphology of autophagy was first described in mammalian cells over 50
years ago (9). However, it is
only recently through yeast genetic screens, that multiple autophagy-related
(ATG) genes have been identified
(10–12).
The yeast ATG proteins have been classified into four major groups: the Atg1
protein kinase complex, the Vps34 phosphatidylinositol 3-phosphate kinase
complex, the Atg8/Atg12 conjugation systems, and the Atg9 recycling complex
(13). Even though many ATG
genes are now known, most of which have functional homologs in mammalian cells
(14,
15), the molecular mechanism
by which they sense the initial triggers and subsequently dictate
autophagy-specific intracellular membrane events is far from understood.In yeast, one of the earliest autophagy-specific events is believed to
involve the Atg1 protein kinase complex. Atg1 is a serine/threonine protein
kinase and a key autophagy-regulator
(16). Atg1 is complexed to at
least two other proteins during autophagy, Atg13 and Atg17, both of which are
required for normal Atg1 function and autophagosome generation
(17–19).
Classical signaling pathways such as the cAMP-dependent kinase (PKA) pathway
or the Tor kinase pathway appear to converge upon this complex, placing Atg1
at an early stage during autophagosome biogenesis
(20–22).
Atg1 phosphorylation by PKA blocks its association with the forming
autophagosome (21), while the
Tor pathway hyperphosphorylates Atg13 causing a reduced affinity of Atg13 for
Atg1, resulting in repression of autophagy
(17,
19). In contrast, nutrient
starvation or inhibition of Tor leads to dephosphorylation of Atg13 thus
increased Atg1 complex formation and kinase activity, resulting in stimulation
of autophagy (19).
Surprisingly, the physiological substrates of Atg1 kinase have not been
identified; thus how Atg1 transduces upstream autophagic signaling is
undefined. Recently, mammalian homologs of Atg1 have been identified as ULK1
and ULK2 (Unc-51-like
kinase)2
(23–25).
ULK1 and ULK2 are ubiquitously expressed and localize to the isolation
membrane, or forming autophagosome, upon nutrient starvation
(25); RNAi-mediated depletion
of ULK1 in HEK293 cells compromises autophagy
(23,
24). The exact role of ULK1
versus ULK2 in autophagy is unclear, and it is possible some
redundancy exists between the two isoforms
(26).Given the conservation of autophagy from yeast to man, it is interesting to
note that no mammalian counterpart to yeast Atg13 or Atg17 had been identified
until very recently. The protein FIP200 (focal adhesion kinase
family-interacting protein of 200 kDa) was
identified as an autophagy-essential binding partner of both ULK1 and ULK2
(25), and it has been
speculated that FIP200 might be the equivalent of yeast Atg17, despite low
sequence similarity (25,
27).In this study, we delve deeper into the molecular regulation of ULK1 to
gain a better insight into how mammalian signaling pathways affect autophagy
initiation. We describe here the identification of a triple complex consisting
of ULK1, FIP200, and the mammalian equivalent of Atg13. This complex is
required not only for localization of ULK1 to the isolation membrane but also
for maximal kinase activity. In addition, both ATG13 and ULK1 are kinase
substrates in the mTOR pathway and thus might function to sense nutrient
starvation. Therefore, this study defines the role of mammalian
ULK1-ATG13-FIP200 complex in mediating the initial autophagic triggers and to
transduce the signal to the core autophagic machinery. 相似文献
7.
Daniel Lingwood Sebastian Schuck Charles Ferguson Mathias J. Gerl Kai Simons 《The Journal of biological chemistry》2009,284(18):12041-12048
Cell membranes predominantly consist of lamellar lipid bilayers. When
studied in vitro, however, many membrane lipids can exhibit
non-lamellar morphologies, often with cubic symmetries. An open issue is how
lipid polymorphisms influence organelle and cell shape. Here, we used
controlled dimerization of artificial membrane proteins in mammalian tissue
culture cells to induce an expansion of the endoplasmic reticulum (ER) with
cubic symmetry. Although this observation emphasizes ER architectural
plasticity, we found that the changed ER membrane became sequestered into
large autophagic vacuoles, positive for the autophagy protein LC3. Autophagy
may be targeting irregular membrane shapes and/or aggregated protein. We
suggest that membrane morphology can be controlled in cells.The observation that simple mixtures of amphiphilic (polar) lipids and
water yield a rich flora of phase structures has opened a long-standing debate
as to whether such membrane polymorphisms are relevant for living organisms
(1–7).
Lipid bilayers with planar geometry, termed lamellar symmetry, dominate the
membrane structure of cells. However, this architecture comprises only a
fraction of the structures seen with in vitro lipid-water systems
(7–11).
The propensity to form lamellar bilayers (a property exclusive to
cylindrically shaped lipids) is flanked by a continuum of lipid structures
that occur in a number of exotic and probably non-physiological
non-bilayer configurations
(3,
12). However, certain lipids,
particularly those with smaller head groups and more bulky hydrocarbon chains,
can adopt bilayered non-lamellar phases called cubic phases. Here the
bilayer is curved everywhere in the form of saddle shapes corresponding to an
energetically favorable minimal surface of zero mean curvature
(1,
7). Because a substantial
number of the lipids present in biological membranes, when studied as
individual pure lipids, form cubic phases
(13), cubic membranes have
received particular interest in cell biology.Since the application of electron microscopy
(EM)3 to the study of
cell ultrastructure, unusual membrane morphologies have been reported for
virtually every organelle (14,
15). However, interpretation
of three-dimensional structures from two-dimensional electron micrographs is
not easy (16). In seminal
work, Landh (17) developed the
method of direct template correlative matching, a technique that unequivocally
assesses the presence of cubic membranes in biological specimens
(16). Cubic phases adopt
mathematically well defined three-dimensional configurations whose
two-dimensional analogs have been derived
(4,
17). In direct template
correlative matching, electron micrographs are matched to these analogs. Cubic
cell membrane geometries and in vitro cubic phases of purified lipid
mixtures do differ in their lattice parameters; however, such deviations are
thought to relate to differences in water activity and lipid to protein ratios
(10,
14,
18). Direct template
correlative matching has revealed thousands of examples of cellular cubic
membranes in a broad survey of electron micrographs ranging from protozoa to
human cells (14,
17) and, more recently, in the
mitochondria of amoeba (19)
and in subcellular membrane compartments associated with severe acute
respiratory syndrome virus
(20). Analysis of cellular
cubic membranes has also been furthered by the development of EM tomography
that confirmed the presence of cubic bilayers in the mitochondrial membranes
of amoeba (21,
22).Although it is now clear that cubic membranes can exist in living cells,
the generation of such architecture would appear tightly regulated, as
evidenced by the dominance of lamellar bilayers in biology. In this light, we
examined the capability and implications of generating cubic membranes in the
endoplasmic reticulum (ER) of mammalian tissue culture cells. The ER is a
spatially interconnected complex consisting of two domains, the nuclear
envelope and the peripheral ER
(23–26).
The nuclear envelope surrounds the nucleus and is composed of two continuous
sheets of membranes, an inner and outer nuclear membrane connected to each
other at nuclear pores. The peripheral ER constitutes a network of branching
trijunctional tubules that are continuous with membrane sheet regions that
occur in closer proximity to the nucleus. Recently it has been suggested that
the classical morphological definition of rough ER (ribosome-studded) and
smooth ER (ribosome-free) may correspond to sheet-like and tubular ER domains,
respectively (27). The ER has
a strong potential for cubic architectures, as demonstrated by the fact that
the majority of cubic cell membranes in the EM record come from ER-derived
structures (14,
17). Furthermore, ER cubic
symmetries are an inducible class of organized smooth ER (OSER), a definition
collectively referring to ordered smooth ER membranes (=stacked cisternae on
the outer nuclear membrane, also called Karmelle
(28–30),
packed sinusoidal ER (31),
concentric membrane whorls
(30,
32–34),
and arrays of crystalloid ER
(35–37)).
Specifically, weak homotypic interactions between membrane proteins produce
both a whorled and a sinusoidal OSER phenotype
(38), the latter exhibiting a
cubic symmetry (16,
39).We were able to produce OSER with cubic membrane morphology via induction
of homo-dimerization of artificial membrane proteins. Interestingly, the
resultant cubic membrane architecture was removed from the ER system by
incorporation into large autophagic vacuoles. To assess whether these cubic
symmetries were favored in the absence of cellular energy, we depleted ATP. To
our surprise, the cells responded by forming large domains of tubulated
membrane, suggesting that a cubic symmetry was not the preferred conformation
of the system. Our results suggest that whereas the endoplasmic reticulum is
capable of adopting cubic symmetries, both the inherent properties of the ER
system and active cellular mechanisms, such as autophagy, can tightly control
their appearance. 相似文献
8.
9.
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
10.
11.
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
12.
Increased Enzymatic O-GlcNAcylation of Mitochondrial Proteins Impairs
Mitochondrial Function in Cardiac Myocytes Exposed to High
Glucose 总被引:1,自引:0,他引:1
Yong Hu Jorge Suarez Eduardo Fricovsky Hong Wang Brian T. Scott Sunia A. Trauger Wenlong Han Ying Hu Mary O. Oyeleye Wolfgang H. Dillmann 《The Journal of biological chemistry》2009,284(1):547-555
13.
14.
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
15.
16.
Kristel Vercauteren Natalie Gleyzer Richard C. Scarpulla 《The Journal of biological chemistry》2009,284(4):2307-2319
17.
18.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
19.
Scot J. Stone Malin C. Levin Ping Zhou Jiayi Han Tobias C. Walther Robert V. Farese Jr. 《The Journal of biological chemistry》2009,284(8):5352-5361
The synthesis and storage of neutral lipids in lipid droplets is a
fundamental property of eukaryotic cells, but the spatial organization of this
process is poorly understood. Here we examined the intracellular localization
of acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), an enzyme that catalyzes
the final step of triacylglycerol (TG) synthesis in eukaryotes. We found that
DGAT2 expressed in cultured cells localizes to the endoplasmic reticulum (ER)
under basal conditions. After providing oleate to drive TG synthesis, DGAT2
also localized to near the surface of lipid droplets, where it co-localized
with mitochondria. Biochemical fractionation revealed that DGAT2 is present in
mitochondria-associated membranes, specialized domains of the ER that are
highly enriched in lipid synthetic enzymes and interact tightly with
mitochondria. The interaction of DGAT2 with mitochondria depended on 67
N-terminal amino acids of DGAT2, which are not conserved in family members
that have different catalytic functions. This targeting signal was sufficient
to localize a red fluorescent protein to mitochondria. A highly conserved,
positively charged, putative mitochondrial targeting signal was identified in
murine DGAT2 between amino acids 61 and 66. Thus, DGAT2, an ER-resident
transmembrane domain-containing enzyme, is also found in
mitochondria-associated membranes, where its N terminus may promote its
association with mitochondria.Most eukaryotic cells can synthesize neutral lipids, such as
triacylglycerols
(TGs)2 and sterol
esters, and store them in cytosolic lipid droplets. Yet, a molecular
understanding of this process and how it is spatially organized is lacking.
For example, lipid substrates for TG synthesis (fatty acids and glycerolipid
precursors) are found in the cytoplasm and membranes, energy for activating
fatty acids (by converting to fatty acyl-CoA) comes from mitochondria, and the
enzymes that catalyze TG formation are primarily found in the mitochondria and
endoplasmic reticulum (ER). How the cell orchestrates this complex anabolic
process to maximize lipid synthesis and storage during times of substrate
excess is poorly understood.In most cells, TG synthesis occurs via the glycerol 3-phosphate (Kennedy)
pathway and involves multiple enzymatic reactions in different subcellular
compartments (1). The fatty
acids for TG synthesis must first be “activated” by acyl-CoA
synthases, a family of enzymes that localize to membranes of different
compartments, including the ER, mitochondria, and plasma membrane
(2), and utilize ATP to ligate
CoA to the fatty acyl chain. Next, these fatty acids enter the Kennedy pathway
of glycerolipid synthesis, in which the first two reactions occur in both the
ER and mitochondria. In the first reaction, glycerol 3-phosphate and a fatty
acyl-CoA are combined to yield lysophosphatidic acid through the actions of
glycerol-3-phosphate acyltransferase enzymes
(1,
3). In the second reaction,
1-acylglycerol-3-phosphate O-acyltransferase enzymes catalyze the
esterification of lysophosphatidic acid with fatty acyl-CoA to form
phosphatidic acid (1,
4). Next, phosphatidic acid is
dephosphorylated at membrane surfaces by phosphatidate phosphatase to yield
diacylglycerol (1,
5,
6). All these steps are highly
organized spatially, which is likely to be important for the efficiency of the
pathway.The final reaction of TG synthesis is catalyzed by acyl-CoA: diacylglycerol
acyltransferase (DGAT) enzymes
(7-9).
The two mammalian DGATs, DGAT1 and DGAT2
(10,
11), which are encoded by
genes of different families, have distinct roles in TG synthesis
(12). DGAT2 is the major TG
biosynthetic enzyme in eukaryotes. Dgat2-deficient mice die shortly
after birth and are almost completely devoid of TG
(13), indicating an essential
requirement for DGAT2. Catalysis of TG synthesis is conserved in the DGAT2
gene family, with functional orthologs in many species, including Dga1p in
Saccharomyces cerevisiae, which contributes to a major portion of TG
synthesis
(14-16).Little is known about the intracellular localization of DGAT enzymes. DGAT
activity is present in microsomes
(7,
17,
18), but in vitro
assays do not distinguish between DGAT1 and DGAT2. A DGAT2-green fluorescent
fusion protein expressed in HeLa cells localized to the ER
(19), and Dga1p activity in
S. cerevisiae localizes to the ER and lipid droplets
(16). DGAT1 and DGAT2
expressed in COS-7 cells localized primarily to the ER
(20). A recent study of the
subcellular localizations of tung tree DGAT1 and DGAT2 in tobacco BY-2 cells
revealed that the enzymes are located in distinct, non-overlapping regions of
the ER (21). Most recently,
DGAT2 was reported to co-localize with lipid droplets in cultured adipocytes
(22). As a step toward a
better understanding of the cellular organization of processes that contribute
to TG synthesis and storage, we determined the subcellular localization of
murine DGAT2 in mammalian cells. 相似文献