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Jenny Erales Sabrina Lignon Brigitte Gontero 《The Journal of biological chemistry》2009,284(19):12735-12744
A new role is reported for CP12, a highly unfolded and flexible protein,
mainly known for its redox function with A4
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized
CP12 can prevent the in vitro thermal inactivation and aggregation of
GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not
redox-dependent. The protection is specific to CP12, because other proteins,
such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do
not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific
chaperone, since it does not protect other proteins, such as catalase, alcohol
dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is
necessary to prevent the aggregation and inactivation, since the mutant C66S
that does not form any complex with GAPDH cannot accomplish this protection.
Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is
partially able to protect and to slow down the inactivation and aggregation.
Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of
GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox
function but also behaves as a specific “chaperone-like protein”
for GAPDH, although a stable and not transitory interaction is observed. This
new function of CP12 may explain why it is also present in complexes involving
A2B2 GAPDHs that possess a regulatory C-terminal
extension (GapB subunit) and therefore do not require CP12 to be
redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most
photosynthetic organisms, including cyanobacteria
(1,
2), higher plants
(3), the diatom
Asterionella formosa
(4,
5), and green
(1) and red algae
(6). It allows the formation of
a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH),3 two key
enzymes of the Calvin cycle pathway, and was recently shown to interact with
fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway
(7). The
phosphoribulokinase·GAPDH·CP12 complex has been extensively
studied in Chlamydomonas reinhardtii
(8,
9) and in Arabidopsis
thaliana (10,
11). In the green alga C.
reinhardtii, the interaction between CP12 and GAPDH is strong
(8). GAPDH may exist as a
homotetramer composed of four GapA subunits (A4) in higher plants,
cyanobacteria, and green and red algae
(6,
12), but in higher plants, it
can also exist as a heterotetramer (A2B2), composed of
two subunits, GapA and GapB
(13,
14). GapB, up to now, has
exclusively been found in Streptophyta, but recently two
prasinophycean green algae, Ostreococcus tauri and Ostreococcus
lucimarinus, were also shown to possess a GapB gene, whereas
CP12 is missing (15).
The GapB subunit is similar to the GapA subunit but has a C-terminal extension
containing two redox-regulated cysteine residues
(16). Thus, although the
A4 GAPDHs lack these regulatory cysteine residues
(13,
14,
17–20),
they are also redox-regulated through its interaction with CP12, since the C
terminus of this small protein resembles the C-terminal extension of the GapB
subunit. The regulatory cysteine residues for GapA are thus supplied by CP12,
as is well documented in the literature
(1,
8,
11,
16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs)
(21–26).
The amino acid composition of these proteins causes them to have no or few
secondary structures. Their total or partial lack of structure and their high
flexibility allow them to be molecular adaptors
(27,
28). They are often able to
bind to several partners and are involved in most cellular functions
(29,
30). Recently, some IUPs have
been described in photosynthetic organisms
(31,
32).There are many functional categories of IUPs
(22,
33). They can be, for
instance, involved in permanent binding and have (i) a scavenger role,
neutralizing or storing small ligands; (ii) an assembler role by forming
complexes; and (iii) an effector role by modulating the activity of a partner
molecule (33). These functions
are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating
its redox properties (8,
34,
35), and can also bind a metal
ion (36,
37). IUPs can also bind
transiently to partners, and some of them have been found to possess a
chaperone activity (31,
38). This chaperone function
was first shown for α-synuclein
(39) and for α-casein
(40), which are fully
disordered. The amino acid composition of IUPs is less hydrophobic than those
of soluble proteins; hence, they lack hydrophobic cores and do not become
insoluble when heated. Since CP12 belongs to this family, we tested if it was
resistant to heat treatment and finally, since it is tightly bound to GAPDH,
if it could prevent aggregation of its partner, GAPDH, an enzyme well known
for its tendency to aggregate
(41–44)
and consequently a substrate commonly used in chaperone studies
(45,
46).Unlike chaperones, which form transient, dynamic complexes with their
protein substrates through hydrophobic interactions
(47,
48), CP12 forms a stable
complex with GAPDH. The interaction involves the C-terminal part of the
protein and the presence of negatively charged residues on CP12
(35). However, only a
site-directed mutagenesis has been performed to characterize the interaction
site on GAPDH. Although the mutation could have an indirect effect, the
residue Arg-197 was shown to be a good candidate for the interaction site
(49).In this report, we accordingly used proteolysis experiments coupled with
mass spectrometry to detect which regions of GAPDH are protected by its
association with CP12. To conclude, the aim of this report was to characterize
a chaperone function of CP12 that had never been described before and to map
the interaction site on GAPDH using an approach that does not involve
site-directed mutagenesis. 相似文献
6.
Kaliyamurthi Venkatachalam Balachandar Venkatesan Anthony J. Valente Peter C. Melby Sailesh Nandish Jane E. B. Reusch Robert A. Clark Bysani Chandrasekar 《The Journal of biological chemistry》2009,284(21):14414-14427
WNT1-inducible signaling pathway protein-1 (WISP1), a member of the
CYR61/CTGF/Nov family of growth factors, can mediate cell growth,
transformation, and survival. Previously we demonstrated that WISP1 is
up-regulated in post-infarct heart, stimulates cardiac fibroblast
proliferation, and is induced by the proinflammatory cytokine tumor necrosis
factor-α (TNF-α). Here we investigated (i) the localization of
TNF-α and WISP1 in post-infarct heart, (ii) the mechanism of
TNF-α-mediated WISP1 induction in primary human cardiac fibroblasts
(CF), (iii) the role of WISP1 in TNF-α-mediated CF proliferation and
collagen production, and (iv) the effects of WISP1 on TNF-α-mediated
cardiomyocyte death. TNF-α and WISP1 expressions were increased in the
border zones and non-ischemic remote regions of the post-ischemic heart. In
CF, TNF-α potently induced WISP1 expression in cyclic AMP response
element-binding protein (CREB)-dependent manner. TNF-α induced CREB
phosphorylation in vitro and DNA binding and reporter gene activities
in vivo. TNF-α induced CREB activation via ERK1/2, and
inhibition of ERK1/2 and CREB blunted TNF-α-mediated WISP1 induction.
Most importantly, WISP1 knockdown attenuated TNF-α stimulated collagen
production and CF proliferation. Furthermore, WISP1 attenuated
TNF-α-mediated cardiomyocyte death, thus demonstrating pro-mitogenic and
pro-survival effects for WISP1 in myocardial constituent cells. Our results
suggest that a TNF-α/WISP1 signaling pathway may contribute to
post-infarct cardiac remodeling, a condition characterized by fibrosis and
progressive cardiomyocyte loss.Acute myocardial infarction
(MI)2 is a major cause
of morbidity and mortality worldwide. After acute MI, the two principal
determinants of patient outcome are myocardial infarct size and the extent of
left ventricular (LV) remodeling
(1–3).
Infarct size is determined during the acute phase immediately post-MI. LV
remodeling on the other hand is a continuous and maladaptive process
characterized by progressive left ventricular hypertrophy, fibrosis,
ventricular dilatation, and by the gradual deterioration of cardiac
performance, leading ultimately to congestive heart failure
(1–3).Numerically, fibroblasts are the major cell type in the heart, but they
constitute a smaller total volume compared with cardiomyocytes
(4). Fibroblasts are associated
primarily with modulation of extracellular matrix and tissue healing/repair
(4–6).
Fibroblasts secrete collagens, fibrillins, fibronectin, laminin, and matrix
metalloproteinases and, thus, are responsible for the maintenance of
connective tissue homeostasis
(4–7).
We and others have shown that primary cardiac fibroblasts also produce various
cytokines, chemokines, and growth factors
(4–6,
8,
9), which tightly regulate the
physiological function of the cells. Under pathological conditions, however,
where the expression of cytokines and growth factors is significantly altered,
the fibroblasts can undergo differentiation, migration, and proliferation,
resulting in pathological fibrosis because of excessive accumulation of
collagens and other ECM proteins
(4–6).The six members of the CCN (CYR61/CTGF/Nov) family of
growth factors (Cyr61 (cysteine-rich 61, CCN1), CTGF (connective tissue growth
factor, CCN2), Nov (nephroblastoma-overexpressed, CCN3), WISP1 (WNT1-inducible
signaling pathway protein-1, CCN4), WISP2 (CCN5), and WISP3 (CCN6)) are
involved in a number of cellular processes, including adhesion, migration, and
proliferation
(10–12).
Although their roles in angiogenesis and oncogenesis are well characterized,
with the exception of CTGF, their precise contribution to myocardial
remodeling is unknown. Recently we demonstrated that whereas WISP1 is
expressed in the heart at low basal levels, permanent occlusion of the left
anterior descending coronary artery significantly up-regulates its expression
in the non-ischemic myocardium
(13). Furthermore, we have
found that WISP1 exerts both pro-hypertrophic and pro-mitogenic effects in
vitro, stimulating Akt-dependent cardiomyocyte growth, as well as
collagen synthesis and fibroblast proliferation
(13). Because cardiomyocyte
hypertrophy and fibroblast proliferation play central roles in remodeling and
WISP1 regulates these two critical processes, it is plausible that WISP1 also
mediates cardiac remodeling after myocardial ischemia, infarction, and
inflammation. However, the mechanisms involved in the induction and regulation
of WISP1 under these conditions have not been well characterized.The proinflammatory cytokine tumor necrosis factor (TNF)-α is
expressed at low basal levels in the normal myocardium but is significantly
up-regulated after infection, inflammation, and injury
(14,
15). Although low levels are
considered cytoprotective (16,
17), aberrant expression of
TNF-α during myocardial ischemic injury and inflammation induces
cardiomyocyte death, hypertrophy of surviving cardiomyocytes, contractile
dysfunction, fibroblast proliferation, fibrosis, and adverse remodeling
(14–17).
TNF-α exerts its biological effects via two cell surface receptors,
TNFR1 (55 kDa) and TNFR2 (75 kDa)
(18). TNFR1, which is more
abundantly expressed in the heart, appears to be the main signaling receptor
for TNF-α and is implicated in transmitting its deleterious effects
(18). On the other hand,
signaling through TNFR2 appears to exert a protective effect in the heart
(18,
19). Of note, all myocardial
constituent cells, including fibroblasts, express both TNFR1 and TNFR2 and,
therefore, are targets of TNF-α.We have recently demonstrated that TNF-α induces WISP1 expression in
cardiomyocytes (13). However,
neither its localization in post-infarct myocardium, its role in
TNF-α-mediated cardiac fibroblast (CF) proliferation and collagen
production, nor its effects on TNF-α-mediated cardiomyocyte death are
known. Our results show that both TNF-α and WISP1 are localized in the
border zone and the non-infarct remote region in post-infarct myocardium.
Furthermore, TNF-α induces WISP1 expression in CF via a
TNFR2/MEK1/ERK1/2/CREB pathway and stimulates fibroblast collagen production
in part via WISP1. In addition, WISP1 exerts pro-survival effects in
cardiomyocytes, antagonizing TNF-α-mediated cardiomyocyte death.
Collectively, these results indicate that WISP1 exerts pro-mitogenic and
pro-survival effects in cardiac cell type-specific manner, and
TNF-α/WISP1 signaling may be an important contributing mechanism in
post-infarct cardiac remodeling. 相似文献
7.
Jason D. Hoffert Chung-Lin Chou Mark A. Knepper 《The Journal of biological chemistry》2009,284(22):14683-14687
Vasopressin controls renal water excretion largely through actions to
regulate the water channel aquaporin-2 in collecting duct principal cells. Our
knowledge of the mechanisms involved has increased markedly in recent years
with the advent of methods for large-scale systems-level profiling such as
protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide
microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible
aspects of everyday physiology. An outdoor tennis game on a hot summer day can
result in substantial water losses by sweating, and the kidneys respond by
reducing water excretion. In contrast, excessive intake of water, a frequent
occurrence in everyday life, results in excretion of copious amounts of clear
urine. These responses serve to exact tight control on the tonicity of body
fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg
of H2O through the regulated return of water from the pro-urine in
the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails.
For example, polyuria (rapid uncontrolled excretion of water) is a sometimes
devastating consequence of lithium therapy for bipolar disorder. On the other
side of the coin are water balance disorders that result from excessive renal
water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia
due to excessive water retention can be seen with severe congestive heart
failure, hepatic cirrhosis, and the syndrome of inappropriate
antidiuresis.The chief regulator of water excretion is the peptide hormone
AVP,2 whereas the
chief molecular target for regulation is the water channel AQP2. In this
minireview, we describe new progress in the understanding of the molecular
mechanisms involved in regulation of AQP2 by AVP in collecting duct cells,
with emphasis on new information derived from “systems-level”
approaches involving large-scale profiling and screening techniques such as
oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid
analysis. Most of the progress with these techniques is in the identification
of individual molecules involved in AVP signaling and binding interactions
with AQP2. Additional related issues are addressed in several recent reviews
(1–4). 相似文献
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Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
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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
17.
18.
Volker Rudolph Francisco J. Schopfer Nicholas K. H. Khoo Tanja K. Rudolph Marsha P. Cole Steven R. Woodcock Gustavo Bonacci Alison L. Groeger Franca Golin-Bisello Chen-Shan Chen Paul R. S. Baker Bruce A. Freeman 《The Journal of biological chemistry》2009,284(3):1461-1473
Nitrated derivatives of fatty acids (NO2-FA) are pluripotent
cell-signaling mediators that display anti-inflammatory properties. Current
understanding of NO2-FA signal transduction lacks insight into how
or if NO2-FA are modified or metabolized upon formation or
administration in vivo. Here the disposition and metabolism of
nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated
in plasma and liver after intravenous injection in mice. High performance
liquid chromatography-tandem mass spectrometry analysis showed that no
18:1-NO2 or metabolites were detected under basal conditions,
whereas administered 18:1-NO2 is rapidly adducted to plasma
thiol-containing proteins and glutathione. NO2-FA are also
metabolized via β-oxidation, with high performance liquid
chromatography-tandem mass spectrometry analysis of liver lipid extracts of
treated mice revealing nitro-7-cis-hexadecenoic acid,
nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic
acid and corresponding coenzyme A derivatives of 18:1-NO2 as
metabolites. Additionally, a significant proportion of 18:1-NO2 and
its metabolites are converted to nitroalkane derivatives by saturation of the
double bond, and to a lesser extent are desaturated to diene derivatives.
There was no evidence of the formation of nitrohydroxyl or conjugated ketone
derivatives in organs of interest, metabolites expected upon
18:1-NO2 hydration or nitric oxide (•NO) release. Plasma
samples from treated mice had significant extents of protein-adducted
18:1-NO2 detected by exchange to added β-mercaptoethanol.
This, coupled with the observation of 18:1-NO2 release from
glutathione-18:1-NO2 adducts, supports that reversible and
exchangeable NO2-FA-thiol adducts occur under biological
conditions. After administration of [3H]18:1-NO2, 64% of
net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%),
kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue
compartments, and may include metabolites not yet identified. In aggregate,
these findings show that electrophilic FA nitroalkene derivatives (a)
acquire an extended half-life by undergoing reversible and exchangeable
electrophilic reactions with nucleophilic targets and (b) are
metabolized predominantly via saturation of the double bond and
β-oxidation reactions that terminate at the site of acyl-chain
nitration.The reaction of unsaturated fatty acids with nitric oxide (•NO)- and
nitrite species,
including nitrogen dioxide (•NO2), peroxynitrite
(ONOO–), and nitrous acid (HNO2), yields a complex
array of oxidized and nitrated products
(1–4).
The mechanisms of biological fatty acid nitration, the structural isomer
distribution of nitrated fatty acids
(NO2-FAs)2
and the signaling actions of specific NO2-FA regioisomers remain
incompletely characterized. Current data reveal that, during fatty acid
oxidation and nitration, vinyl nitro regioisomers represent a component of
these products that display distinctive chemical reactivities and
receptor-dependent signaling actions. Here, we investigate the metabolic fate
of the nitroalkene derivative of oleic acid
(1,
2).Unsaturated fatty acid nitration was first described in model studies of
air-pollutant-induced lipid oxidation where lipids were exposed to high
concentrations of •NO2
(5,
6). More recently nitrated
unsaturated fatty acids have been reported as products of acidic reactions of
, radical chain termination
reactions induced by •NO
(7–10),
and the oxidation of to
•NO2 by the leukocyte-derived enzyme myeloperoxidase
(1). Various mechanisms can
mediate the formation of nitroalkene derivatives of unsaturated fatty acids
(11), including homolytic
attack of •NO2
(12), reaction of
•NO2 with a pre-existing fatty acid carbon-centered radical
(2,
13), and the protonation of
nitrite under acidic conditions
(pH 5.5 and lower) to yield an array of HNO2-derived nitrating
species (3,
14). The conditions promoting
fatty acid nitration by •NO and
species (low
oxygen tension, radical formation, and low pH) are not expected to be broadly
distributed systemically (e.g. in plasma or extracellular fluids).
Rather, nitration reactions will preferably occur during inflammatory or
metabolic stress in microenvironments such as the intermembrane space of
mitochondria, the low pH environment of the digestive tract, and activated
macrophage and neutrophil-rich compartments. Moreover, the acidic,
replete and low O2
tension conditions that promote nitration reactions are characteristic of
inflammatory loci. Although multiple reactions leading to accelerated
formation of nitrating species occur at specific anatomic sites, plasma levels
of nitrated fatty acids are expected to be low due to events described
herein.Robust electrophilic reactivity and avid nuclear lipid receptor ligand
activity have conferred to the class of fatty acid nitroalkene derivatives
potent anti-inflammatory properties that occur predominantly via
non-cGMP-dependent mechanisms. Nitro derivatives of oleic and linoleic acid
inhibit leukocyte and platelet activation
(15), vascular smooth muscle
proliferation (16),
lipopolysaccharide-stimulated macrophage cytokine secretion
(17), activate peroxisome
proliferator-activated receptor-γ
(1,
18), and induce endothelial
heme oxygenase 1 expression
(19). NO2-FA also
potently modulate nuclear factor-erythroid 2-related factor 2/Kelch-like
ECH-associating protein 1 (Nrf2/Keap1)
(16,
17) and nuclear factor
κB (NFκB)-regulated inflammatory signaling
(17). Previous observations of
the •NO-mediated, cGMP-dependent vessel relaxation induced by
NO2-FA were made under serum- and lipid-free conditions. More
recently, it has been appreciated that micellar and membrane stabilization of
NO2-FA prevents Nef-like aqueous decay reactions and consequent
•NO release, supporting that the predominant signaling actions mediated
by NO2-FA are •NO and cGMP-independent
(20,
21).Current data indicate that electrophilic adduction of biological targets
primarily accounts for NO2-FA signal transduction. The high
electronegativity of NO2 substituents, when bound to an alkenyl
carbon of fatty acids, confers an electrophilic nature to the adjacent
β-carbon and enables Michael addition reaction with nucleophiles such as
protein His and Cys residues. This process, termed nitroalkylation
(22), results in the
clinically detectable and reversible adduction of the nucleophilic thiol of
glutathione (GSH) and both cysteine and histidine residues of
glyceraldehyde-3-phosphate dehydrogenase
(23). Furthermore, inhibition
of NFκB signaling occurs via nitroalkylation of p65 subunit thiols
(17), and recent findings
reveal that NO2-FA activation of peroxisome proliferator-activated
receptor-γ is uniquely induced by covalent nitroalkylation of the ligand
binding domain
Cys-285.3Multiple reports support the endogenous generation and presence of nitrated
fatty acids (1,
24), first observed in bovine
papillary muscles as a vicinal nitrohydroxyeicosatetraenoic acid
(25). Nitrolinoleate has been
detected in human blood plasma and cholesteryl nitrolinoleate in human plasma
and lipoproteins (4,
26), with hyperlipidemic and
post-prandial conditions elevating plasma levels of NO2-FA. Further
support for the inflammatory generation of NO2-FA comes from
lipopolysaccharide and interferon-γ-activated murine J774.1 macrophages,
where increased nitration of the acyl chain of cholesteryl linoleate was
paralleled by increased macrophage expression and activity of nitric-oxide
synthase 2 (27).To date, insight into the mechanisms of nitroalkene signaling actions
overshadows knowledge of the generation, trafficking, and metabolism of
nitroalkenes in vivo. Appreciating that NO2-FA derivatives
are detectable clinically, and that their levels increase following
•NO-dependent oxidative reactions
(4,
28), challenges still exist in
their routine detection. Because the in vivo administration of
NO2-FA may exert anti-inflammatory benefit, the disposition and
metabolite profiles of these species in vivo is of relevance. Here we
report that only 2.4% of nitrooctadecenoic acid (18:1-NO2) is
immediately detectable in the vascular compartment as native
18:1-NO2 upon intravenous injection in mice, with the remaining
pool of 18:1-NO2 (a) reversibly bound to plasma and tissue
thiols via Michael addition; (b) metabolized to nitro-octadecanoic
acid (18:0-NO2) and nitro-octadecadienoic acid
(18:2-NO2); and (c) catabolized by hepatic
β-oxidation following thioester formation with coenzyme A. 相似文献
19.
Tomoya Isaji Yuya Sato Tomohiko Fukuda Jianguo Gu 《The Journal of biological chemistry》2009,284(18):12207-12216
N-Glycosylation of integrin α5β1 plays a crucial role
in cell spreading, cell migration, ligand binding, and dimer formation, but
the detailed mechanisms by which N-glycosylation mediates these
functions remain unclear. In a previous study, we showed that three potential
N-glycosylation sites (α5S3–5) on the β-propeller of
the α5 subunit are essential to the functional expression of the
subunit. In particular, site 5 (α5S5) is the most important for its
expression on the cell surface. In this study, the function of the
N-glycans on the integrin β1 subunit was investigated using
sequential site-directed mutagenesis to remove the combined putative
N-glycosylation sites. Removal of the N-glycosylation sites
on the I-like domain of the β1 subunit (i.e. the Δ4-6
mutant) decreased both the level of expression and heterodimeric formation,
resulting in inhibition of cell spreading. Interestingly, cell spreading was
observed only when the β1 subunit possessed these three
N-glycosylation sites (i.e. the S4-6 mutant). Furthermore,
the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5
mutant of the α5 subunit. Taken together, the results of the present
study reveal for the first time that N-glycosylation of the I-like
domain of the β1 subunit is essential to both the heterodimer formation
and biological function of the subunit. Moreover, because the
α5S3-5/β1S4-6 mutant represents the minimal
N-glycosylation required for functional expression of the β1
subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α
and a β subunit (1). The
interaction between integrin and the extracellular matrix is essential to both
physiologic and pathologic events, such as cell migration, development, cell
viability, immune homeostasis, and tumorigenesis
(2,
3). Among the integrin
superfamily, β1 integrin can combine with 12 distinct α subunits
(α1–11, αv) to form heterodimers, thereby acquiring a wide
variety of ligand specificity
(1,
4). Integrins are thought to be
regulated by inside-out signaling mechanisms that provoke conformational
changes, which modulate the affinity of integrin for the ligand
(5). However, an increasing
body of evidence suggests that cell-surface carbohydrates mediate a variety of
interactions between integrin and its extracellular environment, thereby
affecting integrin activity and possibly tumor metastasis as well
(6–8).Guo et al. (9)
reported that an increase in β1–6-GlcNAc sugar chains on the
integrin β1 subunit stimulated cell migration. In addition, elevated
sialylation of the β1 subunit, because of Ras-induced STGal-I transferase
activity, also induced cell migration
(10,
11). Conversely, cell
migration and spreading were reduced by the addition of a bisecting GlcNAc,
which is a product of N-acetylglucosaminyltransferase III
(GnT-III),2 to the
α5β1 and α3β1 integrins
(12,
13). Alterations of
N-glycans on integrins might also regulate their cis interactions
with membrane-associated proteins, including the epidermal growth factor
receptor, the galectin family, and the tetraspanin family of proteins
(14–19).In addition to the positive and negative regulatory effects of
N-glycan, several research groups have reported that
N-glycans must be present on integrin α5β1 for the
αβ heterodimer formation and proper integrin-matrix interactions.
Consistent with this hypothesis, in the presence of the glycosylation
inhibitor, tunicamycin, normal integrin-substrate binding and transport to the
cell surface are inhibited
(20). Moreover, treatment of
purified integrin with N-glycosidase F blocked both the inherent
association of the subunits and the interaction between integrin and
fibronectin (FN) (21). These
results suggest that N-glycosylation is essential to the functional
expression of α5β1. However, because integrin α5β1
contains 26 potential N-linked glycosylation sites, 14 in the α
subunit and 12 in the β subunit, identification of the sites that are
essential to its biological functions is key to understanding the molecular
mechanisms by which N-glycans alter integrin function. Recently, our
group determined that N-glycosylation of the β-propeller domain
on the α5 subunit is essential to both heterodimerization and biological
functions of the subunit. Furthermore, we determined that sites 3–5 are
the most important sites for α5 subunit-mediated cell spreading and
migration on FN (22). The
purpose of this study was to clarify the roles of N-glycosylation of
the β1 subunit. Therefore, we performed combined substitutions in the
putative N-glycosylation sites by replacement of asparagine residues
with glutamine residues. We subsequently introduced these mutated genes into
β1-deficient epithelial cells (GE11). The results of these mutation
experiments revealed that the N-glycosylation sites on the I-like
domain of the β1 subunit, sites number 4–6 (S4-6), are essential to
both heterodimer formation and biological functions, such as cell
spreading. 相似文献
20.
Haihong Zong Claire C. Bastie Jun Xu Reinhard Fassler Kevin P. Campbell Irwin J. Kurland Jeffrey E. Pessin 《The Journal of biological chemistry》2009,284(7):4679-4688
Integrin receptor plays key roles in mediating both inside-out and
outside-in signaling between cells and the extracellular matrix. We have
observed that the tissue-specific loss of the integrin β1 subunit in
striated muscle results in a near complete loss of integrin β1 subunit
protein expression concomitant with a loss of talin and to a lesser extent, a
reduction in F-actin content. Muscle-specific integrin β1-deficient mice
had no significant difference in food intake, weight gain, fasting glucose,
and insulin levels with their littermate controls. However, dynamic analysis
of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a
44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose
clearance, respectively. The whole body insulin resistance resulted from a
specific inhibition of skeletal muscle glucose uptake and glycogen synthesis
without any significant effect on the insulin suppression of hepatic glucose
output or insulin-stimulated glucose uptake in adipose tissue. The reduction
in skeletal muscle insulin responsiveness occurred without any change in GLUT4
protein expression levels but was associated with an impairment of the
insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not
threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation
occurred concomitantly with a decrease in integrin-linked kinase expression
but with no change in the mTOR·Rictor·LST8 complex (mTORC2).
These data demonstrate an in vivo crucial role of integrin β1
signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins
composed of a single α and β subunit assembled into a heterodimeric
complex. There are 19 α and 8 β mammalian subunit isoforms that
combine to form 25 distinct α,β heterodimeric receptors
(1-5).
These receptors play multiple critical roles in conveying extracellular
signals to intracellular responses (outside-in signaling) as well as altering
extracellular matrix interactions based upon intracellular changes (inside-out
signaling). Despite the large overall number of integrin receptor complexes,
skeletal muscle integrin receptors are limited to seven α subunit
subtypes (α1, α3, α4, α5, α6, α7, and
αν subunits), all associated with the β1 integrin subunit
(6,
7).Several studies have suggested an important cross-talk between
extracellular matrix and insulin signaling. For example, engagement of β1
subunit containing integrin receptors was observed to increase
insulin-stimulated insulin receptor substrate
(IRS)2
phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation
of protein kinase B/Akt
(8-11).
Integrin receptor regulation of focal adhesion kinase was reported to modulate
insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton
organization in cultured hepatocytes and myoblasts
(12,
13). Similarly, the
integrin-linked kinase (ILK) was suggested to function as one of several
potential upstream kinases that phosphorylate and activate Akt
(14-18).
In this regard small interfering RNA gene silencing of ILK in fibroblasts and
conditional ILK gene knockouts in macrophages resulted in a near complete
inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation
concomitant with an inhibition of Akt activity and phosphorylation of Akt
downstream targets (19).
However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2)
has been identified in several other studies as the Akt Ser-473 kinase
(20,
21). In addition to Ser-473,
Akt protein kinase activation also requires phosphorylation on threonine 308
Thr-30 by phosphoinositide-dependent protein kinase, PDK1
(22-24).In vivo, skeletal muscle is the primary tissue responsible for
postprandial (insulin-stimulated) glucose disposal that results from the
activation of signaling pathways leading to the translocation of the
insulin-responsive glucose transporter, GLUT4, from intracellular sites to the
cell surface membranes (25,
26). Dysregulation of any step
of this process in skeletal muscle results in a state of insulin resistance,
thereby predisposing an individual for the development of diabetes
(27-33).
Although studies described above have utilized a variety of tissue culture
cell systems to address the potential involvement of integrin receptor
signaling in insulin action, to date there has not been any investigation of
integrin function on insulin action or glucose homeostasis in vivo.
To address this issue, we have taken advantage of Cre-LoxP technology to
inactivate the β1 integrin receptor subunit gene in striated muscle. We
have observed that muscle creatine kinase-specific integrin β1 knock-out
(MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose
infusion rate and glucose clearance. The impairment of insulin-stimulated
skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease
in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK
expression. Together, these data demonstrate an important cross-talk between
integrin receptor function and insulin action and suggests that ILK may
function as an Akt Ser-473 kinase in skeletal muscle. 相似文献