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1.
Michael A. Gitcho Jeffrey Strider Deborah Carter Lisa Taylor-Reinwald Mark S. Forman Alison M. Goate Nigel J. Cairns 《The Journal of biological chemistry》2009,284(18):12384-12398
Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and
Paget disease of bone is a rare, autosomal dominant disorder caused by
mutations in the VCP (valosin-containing protein) gene. The disease
is characterized neuropathologically by frontal and temporal lobar atrophy,
neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are
distinct from those seen in other sporadic and familial FTLD-U entities. The
major component of the ubiquitinated inclusions of FTLD with VCP
mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy
links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most
familial forms of FTLD-U. Understanding the relationship between individual
gene defects and pathologic TDP-43 will facilitate the characterization of the
mechanisms leading to neurodegeneration. Using cell culture models, we have
investigated the role of mutant VCP in intracellular trafficking,
proteasomal function, and cell death and demonstrate that mutations in the
VCP gene 1) alter localization of TDP-43 between the nucleus and
cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum
stress, 4) increase markers of apoptosis, and 5) impair cell viability. These
results suggest that VCP mutation-induced neurodegeneration is
mediated by several mechanisms.Frontotemporal lobar degeneration
(FTLD)2
accounts for 10% of all late onset dementias and is the third most frequent
neurodegenerative disease after Alzheimer disease and dementia with Lewy
bodies (1). FTLD with
ubiquitin-immunoreactive inclusions is genetically, clinically, and
neuropathologically heterogeneous
(2,
3). FTLD-U comprises several
distinct entities, including sporadic forms and familial cases caused by
mutations in the genes encoding VCP (valosin-containing protein), GRN
(progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR
DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9
(2,
3). Frontotemporal dementia
with inclusion body myopathy and Paget disease of bone is a rare, autosomal
dominant disorder caused by mutations in the VCP gene located on
chromosome 9p13-p12
(4-10)
(Fig. 1). This multisystem
disease is characterized by progressive muscle weakness and atrophy, increased
osteoclastic bone resorption, and early onset frontotemporal dementia, also
called FTLD (9,
11). Mutations in VCP
are also associated with dilatative cardiomyopathy with ubiquitin-positive
inclusions (12).
Neuropathologic features of FTLD with VCP mutation include frontal
and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive
inclusions (FTLD-U). The majority of aggregates are ubiquitin- and
TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion
is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal
cytoplasmic inclusions (NCIs). A small number of inclusions are
VCP-immunoreactive (5,
13). Pathologic TDP-43 in
inclusions links a spectrum of diseases in which TDP-43 pathology is a primary
feature, including FTLD-U, motor neuron disease, including amyotrophic lateral
sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and
Paget disease of bone, as well as an expanding spectrum of other disorders in
which TDP-43 pathology is secondary
(14,
15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing
protein. CDC48 (magenta), located within the N terminus (residues
22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1
(23-25,
28). There are two AAA-ATPase
domains (AAA; blue) at residues 240-283 and 516-569, which
are joined by two linker regions (L1 and L2;
red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical
signature similar to that seen in other sporadic and familial cases of FTLD-U,
including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease,
FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome
9p (3,
16). TDP-43 proteinopathy in
these disorders is characterized by hyperphosphorylation of TDP-43,
ubiquitination, and cleavage to form C-terminal fragments detected only in
insoluble brain extracts from affected brain regions
(16). Identification of TDP-43
as the major component of the ubiquitin-immunoreactive inclusions of FTLD with
VCP mutation supports the hypothesis that VCP gene mutations
cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase
associated with diverse cellular activities) superfamily. The N-terminal
domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell
division cycle protein 48)) and two AAA-ATPase domains that form a hexameric
complex (Fig. 1)
(17). Recently, it has been
shown that the N-terminal domain of VCP binds phosphoinositides
(18,
19). AKT (activated
serine-threonine protein kinase) phosphorylates VCP and is required for
constitutive VCP function (20,
21). AKT is activated through
phospholipid binding and phosphorylation via the phosphoinositide 3-kinase
signaling pathway, which is involved in cell survival
(22). The lipid binding domain
may recruit VCP to the cell membrane where it is phosphorylated by AKT
(19).The diversity of VCP functions is modulated, in part, by a variety of
intracellular cofactors, including p47, gp78, and Npl4-Ufd1
(23). Cofactor p47 has been
shown to play a role in the maintenance and biogenesis of both the endoplasmic
reticulum (ER) and Golgi apparatus
(24). The structure of p47
contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and
together they act as a chaperone to deliver membrane fusion machinery to the
site of adjacent membranes
(25). The function of the
p47-VCP complex is dependent upon cell division cycle 2 (CDC2)
serine-threonine kinase phosphorylation of p47
(26,
27). Also, VCP has been found
to interact with the cytosolic tail of gp78, an ER membrane-spanning E3
ubiquitin ligase that exclusively binds VCP and enhances ER-associated
degradation (ERAD) (28). The
Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting
of proteins through the ubiquitin-proteasome system
(29,
30). The cell survival
response of this complex has been found to be important in DNA damage repair
though activation by phosphorylation and its recruitment to double-stranded
breaks (20,
31). The Npl4-Ufd1-VCP
cytosolic complex is also recruited to the ER membrane, interacting with
Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At
the ER membrane, these misfolded proteins are targeted to the proteasome via
ERAD
(32-34).
VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome
system, implicating VCP in the cell survival pathway and neuroprotection
(21,
35-37).To investigate the mechanism of neurodegeneration caused by VCP
mutations, we first tested the hypothesis that VCP mutations decrease
cell viability in vitro using a neuroblastoma SHSY-5Y cell line and
then investigated cellular pathways that are known to lead to
neurodegeneration, including decrease in proteasome activity, caspase-mediated
degeneration, and a change in cellular localization of TDP-43. 相似文献
2.
Joseph T. Roland Lynne A. Lapierre James R. Goldenring 《The Journal of biological chemistry》2009,284(2):1213-1223
Rab proteins influence vesicle trafficking pathways through the assembly of
regulatory protein complexes. Previous investigations have documented that
Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate
distinct trafficking pathways. We have now determined that a related Rab
protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10
localized to a system of tubules and vesicles that have partially overlapping
localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the
expression of dominant-negative myosin V tails. Interaction with Rab10 was
dependent on the presence of the alternatively spliced exon D in myosin Va and
myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and
fluorescence resonance energy transfer studies confirmed that Rab10 binding to
myosin V tails in vivo required the alternatively spliced exon D. In
contrast to our previous work, we found that Rab11a can interact with both
myosin Va and myosin Vb tails independent of their splice isoform. These
results indicate that Rab GTPases regulate diverse endocytic trafficking
pathways through recruitment of multiple myosin V isoforms.Eukaryotic cells are comprised of networks of highly organized membranous
structures that require the efficient and timely movement of diverse
intracellular proteins for proper function. Molecular motors provide the
physical force needed to move these materials along microtubules and actin
microfilaments. Unconventional myosin motors, such as those belonging to
classes V, VI, and VII, have roles in the trafficking and recycling of
membrane-bound structures in eukaryotic cells
(1) and are recruited to
discrete vesicle populations. Myosin VI is involved in clathrin-mediated
endocytosis (2), whereas myosin
VIIa participates in the proper development of stereocilia of inner ear hair
cells and the transport of pigment granules in retinal pigmented epithelial
cells (3,
4). Similarly, the three
members of vertebrate class V myosins, myosin Va, myosin Vb, and myosin Vc,
are required for the proper transport of a wide array of membrane cargoes,
such as the melanosomes of pigment cells, synaptic vesicles in neurons, apical
recycling endosomes in polarized epithelial cells, and bulk recycling vesicles
in non-polarized cells (5).Members of the Rab family of small GTPases regulate many cellular systems,
including membrane trafficking
(6,
7). Certain Rab proteins
associate with and regulate the function of class V myosins. Rab27a, in a
complex with the adaptor protein melanophilin/Slac2-a, is required to localize
myosin Va to the surface of melanin-filled pigment granules in vertebrates
(8-10),
whereas Rab27a and Slac2-c/MyRIP associate with both Myosin Va and myosin VIIa
(3,
11). Rab11a, in a complex with
its adaptor protein Rab11-FIP2, associates with myosin Vb on recycling
endosomes
(12-14)
where the tripartite complex regulates the recycling of a variety of cargoes
(15-19).
In addition, Rab8a associates with both myosin Vb
(20) and myosin Vc
(21) as part of the
non-clathrin-mediated tubular recycling system
(20). Recently, Rab11a has
also been shown to associate with myosin Va in the transport of AMPA receptors
in dendritic spines (22),
contributing to the model of myosin V regulation by multiple Rab proteins.Previous investigations have documented alternative splicing of myosin Va
in a tissue-specific manner
(23-28).
Alternate splicing occurs in a region lying between the coiled-coil region of
the neck of the motor and the globular tail region. Three exons in particular
are subject to alternative splicing: exons B, D, and F
(23-25).
Exon F is critical for association with melanophilin/Slac2 and Rab27a
(8,
9,
29,
30). Additionally, exon B is
required for the interaction of myosin Va with dynein light chain 2 (DLC2)
(27,
28). Currently no function for
the alternatively spliced exon D has been reported. Similar to myosin Va,
myosin Vb contains exons A, B, C, D, and E, whereas no exon F has yet been
identified in myosin Vb (Fig.
1A). In addition, exon B in myosin Vb does not resemble
the dynein light chain 2 (DLC2) binding region in myosin Va
(27,
28), and therefore, it likely
does not interact with DLC2. On the other hand, exon D is highly conserved
among Myosin Va, myosin Vb, and myosin Vc, suggesting a common function in
these molecular motors.Open in a separate windowFIGURE 1.Tissue distribution of human myosin Va and myosin Vb splice
isoforms. A, schematic of the alternative exon organization in
the tails of myosin Va and myosin Vb. It is known that exons B, D, and F are
subject to alternative splicing in myosin Va, whereas there is only evidence
that exon D is alternatively spliced in myosin Vb, which does not contain exon
F. B, alignment of exon D sequences from mouse and human myosin V''s.
myosin Va and myosin Vb both contain exon D (amino acids 1320-1346 of myosin
Va and 1315-1340 of myosin Vb), whereas myosin Vc contains an exon D-like
region (amino acids 1124-1147 of human myosin Vc) that is not known to be
alternatively spliced. Alignment of the exon D regions from all three motors
reveals a high degree of homology, especially in the center of the exon.
Asterisks indicate amino acid identities. C, PCR-based
analysis of human tissue panels reveals the alternative splicing pattern of
exon D in myosin Va and myosin Vb. Primers flanking the region encoding exon D
for both motors were used to amplify cDNA from human MTC™ panels
(Clontech). cDNA amplified from HeLa cell RNA as well as myosin Va and myosin
Vb tail constructs were used as positive controls. Variants expressing exon D
(upper bands) and lacking exon D (lower bands) were visible.
Per., peripheral; Pos., positive.Here we report that Rab10, a protein related to Rab8a and thought to have
similar function
(31-35),
localizes to a system of tubules and vesicles overlapping in distribution with
Rab8a in HeLa cells. Utilizing dominant-negative myosin V tail constructs, we
show that Rab8a and Rab10 can interact with Myosin Va, myosin Vb, and myosin
Vc in vivo. In addition, we have determined that the alternatively
spliced exon D in both myosin Va and myosin Vb is required for interaction
with Rab10. In contrast to our previous findings, we demonstrate that Rab11a
is able to interact with both myosin Va and myosin Vb tails in an exon
independent-manner. These results reveal that multiple Rab proteins
potentially regulate all three class V myosin motors. 相似文献
3.
4.
Tsuneo Ferguson Jitesh A. Soares Tanja Lienard Gerhard Gottschalk Joseph A. Krzycki 《The Journal of biological chemistry》2009,284(4):2285-2295
Archaeal methane formation from methylamines is initiated by distinct
methyltransferases with specificity for monomethylamine, dimethylamine, or
trimethylamine. Each methylamine methyltransferase methylates a cognate
corrinoid protein, which is subsequently demethylated by a second
methyltransferase to form methyl-coenzyme M, the direct methane precursor.
Methylation of the corrinoid protein requires reduction of the central cobalt
to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric
iron-sulfur protein, was isolated from Methanosarcina barkeri and is
required for in vitro ATP-dependent reductive activation of
methylamine:CoM methyl transfer from all three methylamines. In the absence of
the methyltransferases, highly purified RamA was shown to mediate the
ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for
the monomethylamine corrinoid protein, MtmC. The ramA gene is located
near a cluster of genes required for monomethylamine methyltransferase
activity, including MtbA, the methylamine-specific CoM methylase and the
pyl operon required for co-translational insertion of pyrrolysine
into the active site of methylamine methyltransferases. RamA possesses a
C-terminal ferredoxin-like domain capable of binding two tetranuclear
iron-sulfur proteins. Mutliple ramA homologs were identified in
genomes of methanogenic Archaea, often encoded near methyltrophic
methyltransferase genes. RamA homologs are also encoded in a diverse selection
of bacterial genomes, often located near genes for corrinoid-dependent
methyltransferases. These results suggest that RamA mediates reductive
activation of corrinoid proteins and that it is the first functional archetype
of COG3894, a family of redox proteins of unknown function.Most methanogenic Archaea are capable of producing methane only from carbon
dioxide. The Methanosarcinaceae are a notable exception as representatives are
capable of methylotrophic methanogenesis from methylated amines, methylated
thiols, or methanol. Methanogenesis from these substrates requires methylation
of 2-mercaptoethanesulfonic acid (coenzyme M or CoM) that is subsequently used
by methylreductase to generate methane and a mixed disulfide whose reduction
leads to energy conservation
(1–4).Methylation of CoM with trimethylamine
(TMA),4 dimethylamine
(DMA), or monomethylamine (MMA) is initiated by three distinct
methyltransferases that methylate cognate corrinoid-binding proteins
(3). MtmB, the MMA
methyltransferase, specifically methylates cognate corrinoid protein, MtmC,
with MMA (see Fig. 1)
(5,
6). The DMA methyltransferase,
MtbB, and its cognate corrinoid protein, MtbC, interact specifically to
demethylate DMA (7,
8). TMA is demethylated by the
TMA methyltransferase (MttB) in conjunction with the TMA corrinoid protein
(MttC) (8,
9). Each of the methylated
corrinoid proteins is a substrate for a methylcobamide:CoM methyltransferase,
MtbA, which produces methyl-CoM
(10–12).Open in a separate windowFIGURE 1.MMA:CoM methyl transfer. A schematic of the reactions catalyzed by
MtmB, MtmC, and MtbA is shown that emphasizes the key role of MtmC in the
catalytic cycle of both methyltransferases. Oxidation to Co(II)-MtmC of the
supernucleophilic Co(I)-MtmC catalytic intermediate inactivates methyl
transfer from MMA to the thiolate of coenzyme M (HSCoM). In
vitro reduction of the Co(II)-MtmC with either methyl viologen reduced to
the neutral species or with RamA in an ATP-dependent reaction can regenerate
the Co(I) species. In either case in vitro Ti(III)-citrate is the
ultimate source of reducing power.CoM methylation with methanol requires the methyltransferase MtaB and the
corrinoid protein MtaC, which is then demethylated by another
methylcobamide:CoM methyltransferase, MtaA
(13–15).
The methylation of CoM with methylated thiols such as dimethyl sulfide in
Methanosarcina barkeri is catalyzed by a corrinoid protein that is
methylated by dimethyl sulfide and demethylated by CoM, but in this case an
associated CoM methylase carries out both methylation reactions
(16).In bacteria, analogous methyltransferase systems relying on small corrinoid
proteins are used to achieve methylation of tetrahydrofolate. In
Methylobacterium spp., CmuA, a single methyltransferase with a
corrinoid binding domain, along with a separate pterin methylase, effect the
methylation of tetrahydrofolate with chloromethane
(17,
18). In Acetobacterium
dehalogenans and Moorella thermoacetica various three-component
systems exist for specific demethylation of different phenylmethyl ethers,
such as vanillate (19) and
veratrol (20), again for the
methylation of tetrahydrofolate. Sequencing of the genes encoding the
corrinoid proteins central to the archaeal and bacterial methylotrophic
pathways revealed they are close homologs. Furthermore, genes predicted to
encode such corrinoid proteins and pterin methyltransferases are widespread in
bacterial genomes, often without demonstrated metabolic function. All of these
corrinoid proteins are similar to the well characterized cobalamin binding
domain of methionine synthase
(21,
22).In contrast, the TMA, DMA, MMA, and methanol methyltransferases are not
homologous proteins. The methylamine methyltransferases do share the common
distinction of having in-frame amber codons
(6,
8) within their encoding genes
that corresponds to the genetically encoded amino acid pyrrolysine
(23–25).
Pyrrolysine has been proposed to act in presenting a methylammonium adduct to
the central cobalt ion of the corrinoid protein for methyl transfer
(3,
23,
26). However, nucleophilic
attack on a methyl donor requires the central cobalt ion of a corrinoid
cofactor is in the nucleophilic Co(I) state rather than the inactive Co(II)
state (27). Subsequent
demethylation of the methyl-Co(III) corrinoid cofactor regenerates the
nucleophilic Co(I) cofactor. The Co(I)/Co(II) in the cobalamin binding domain
of methionine synthase has an Em value of -525 mV at pH 7.5
(28). It is likely to be
similarly low in the homologous methyltrophic corrinoid proteins. These low
redox potentials make the corrinoid cofactor subject to adventitious oxidation
to the inactive Co(II) state (Fig.
1).During isolation, these corrinoid proteins are usually recovered in a
mixture of Co(II) or hydroxy-Co(III) states. For in vitro studies,
chemical reduction can maintain the corrinoid protein in the active Co(I)
form. The methanol:CoM or the phenylmethyl ether:tetrahydrofolate
methyltransferase systems can be activated in vitro by the addition
of Ti(III) alone as an artificial reductant
(14,
19). In contrast, activation
of the methylamine corrinoid proteins further requires the addition of methyl
viologen as a redox mediator. Ti(III) reduces methyl viologen to the extremely
low potential neutral species. In vitro activation with these agents
does not require ATP (5,
7,
9).Cellular mechanisms also exist to achieve the reductive activation of
corrinoid cofactors in methyltransferase systems. Activation of human
methionine synthase involves reduction of the co(II)balamin by methionine
synthase reductase (29),
whereas the Escherichia coli enzyme requires flavodoxin
(30). The endergonic reduction
is coupled with the exergonic methylation of the corrinoid with
S-adenosylmethionine
(27). An activation system
exists in cellular extracts of A. dehalogenans that can activate the
veratrol:tetrahydrofolate three-component system and catalyze the direct
reduction of the veratrol-specific corrinoid protein to the Co(I) state;
however, the activating protein has not been purified
(31).For the methanogen methylamine and methanol methyltransferase systems, an
activation process is readily detectable in cell extracts that is ATP- and
hydrogen-dependent (32,
33). Daas et al.
(34,
35) examined the activation of
the methanol methyltransferase system in M. barkeri and purified in
low yield a methyltransferase activation protein (MAP) which in the presence
of a preparation of hydrogenase and uncharacterized proteins was required for
ATP-dependent reductive activation of methanol:CoM methyl transfer. MAP was
found to be a heterodimeric protein without a UV-visible detectable prosthetic
group. Unfortunately, no protein sequence has been reported for MAP, leaving
the identity of the gene in question. The same MAP protein was also suggested
to activate methylamine:CoM methyl transfer, but this suggestion was based on
results with crude protein fractions containing many cellular proteins other
than MAP (36).Here we report of the identification and purification to near-homogeneity
of RamA (reductive activation of
methyltransfer, amines), a protein mediating activation
of methylamine:CoM methyl transfer in a highly purified system
(Fig. 1). Quite unlike MAP,
which was reported to lack prosthetic groups, RamA is an iron-sulfur protein
that can catalyze reduction of a corrinoid protein such as MtmC to the Co(I)
state in an ATP-dependent reaction (Fig.
1). Peptide mapping of RamA allowed identification of the gene
encoding RamA and its homologs in the genomes of Methanosarcina spp.
RamA belongs to COG3894, a group of uncharacterized metal-binding proteins
found in a number of genomes. RamA, thus, provides a functional example for a
family of proteins widespread among bacteria and Archaea whose physiological
role had been largely unknown. 相似文献
5.
Bryson W. Katona Shrikant Anant Douglas F. Covey William F. Stenson 《The Journal of biological chemistry》2009,284(5):3354-3364
Bile acids are steroid detergents that are toxic to mammalian cells at high
concentrations; increased exposure to these steroids is pertinent in the
pathogenesis of cholestatic disease and colon cancer. Understanding the
mechanisms of bile acid toxicity and apoptosis, which could include
nonspecific detergent effects and/or specific receptor activation, has
potential therapeutic significance. In this report we investigate the ability
of synthetic enantiomers of lithocholic acid (ent-LCA),
chenodeoxycholic acid (ent-CDCA), and deoxycholic acid
(ent-DCA) to induce toxicity and apoptosis in HT-29 and HCT-116
cells. Natural bile acids were found to induce more apoptotic nuclear
morphology, cause increased cellular detachment, and lead to greater capase-3
and -9 cleavage compared with enantiomeric bile acids in both cell lines. In
contrast, natural and enantiomeric bile acids showed similar effects on
cellular proliferation. These data show that bile acid-induced apoptosis in
HT-29 and HCT-116 cells is enantiospecific, hence correlated with the absolute
configuration of the bile steroid rather than its detergent properties. The
mechanism of LCA- and ent-LCA-induced apoptosis was also investigated
in HT-29 and HCT-116 cells. These bile acids differentially activate initiator
caspases-2 and -8 and induce cleavage of full-length Bid. LCA and
ent-LCA mediated apoptosis was inhibited by both pan-caspase and
selective caspase-8 inhibitors, whereas a selective caspase-2 inhibitor
provided no protection. LCA also induced increased CD95 localization to the
plasma membrane and generated increased reactive oxygen species compared with
ent-LCA. This suggests that LCA/ent-LCA induce apoptosis
enantioselectively through CD95 activation, likely because of increased
reactive oxygen species generation, with resulting procaspase-8 cleavage.Bile acids are physiologic steroids that are necessary for the proper
absorption of fats and fat-soluble vitamins. Their ability to aid in these
processes is largely due to their amphipathic nature and thus their ability to
act as detergents. Despite the beneficial effects, high concentrations of bile
acids are toxic to cells
(1-11).
High fat western diets induce extensive recirculation of the bile acid pool,
resulting in increased exposure of the colonic epithelial cells to these toxic
steroids (12,
13). A high fat diet is also a
risk factor for colon carcinogenesis; increased bile acid exposure is
responsible for some of this risk. Bile acids can contribute to both colon
cancer formation and progression, and their effects on colonic proliferation
and apoptosis aid this process by disrupting the balance between cell growth
and cell death, as well as helping to select for bile acid-resistant cells
(14,
15).In colonocyte-derived cell lines bile acid-induced apoptosis is thought to
proceed through mitochondrial destabilization with resulting mitochondrial
permeability transition formation and cytochrome c release as well as
generation of oxidative stress
(1,
9-11).
Bile acid-induced apoptosis has also been extensively explored in hepatocyte
derived cell lines with mechanisms including mitochondria dysfunction
(16-23),
endoplasmic reticulum stress
(24), ligand-independent
activation of death receptor pathways
(18,
25-28),
and modulation of cellular apoptotic and anti-apoptotic Bcl-2 family proteins
(29).Although ample evidence exists for multiple mechanisms of bile acid-induced
apoptosis, the precise interactions responsible for initiating these apoptotic
pathways are still unclear. Bile acids have been shown to interact directly
with specific receptors (30,
31). These steroids can also
initiate cellular signaling through nonspecific membrane perturbations
(32), and evidence exists
showing that other simple detergents (i.e. Triton X-100) are capable
of inducing caspase cleavage nonspecifically with resultant apoptosis
(33). Therefore, hydrophobic
bile acids may interact nonspecifically with cell membranes to alter their
physical properties, bind to receptors specific for these steroids, or utilize
a combination of both specific and nonspecific interactions to induce
apoptosis.Bile acid enantiomers could be useful tools for elucidating mechanisms of
bile acid toxicity and apoptosis. These enantiomers, known as
ent-bile acids, are synthetic nonsuperimposable mirror images of
natural bile acids with identical physical properties except for optical
rotation. Because bile acids are only made in one absolute configuration
naturally, ent-bile acids must be constructed using a total synthetic
approach. Recently we reported the first synthesis of three enantiomeric bile
acids: ent-lithocholic acid
(ent-LCA),2
ent-chenodeoxycholic acid (ent-CDCA), and
ent-deoxycholic acid (ent-DCA)
(Fig. 1)
(34,
35). Enantiomeric bile acids
have unique farnesoid X receptor, vitamin D receptor, pregnane X receptor, and
TGR5 receptor activation profiles compared with the corresponding natural bile
acids (34). This illustrates
that natural and enantiomeric bile acids interact differently within chiral
environments because of their distinct three-dimensional configurations
(Fig. 1). Despite these
differences in chiral interactions, ent-bile acids have physical
properties identical to those of their natural counterparts including
solubility and critical micelle concentrations
(34,
35). With different receptor
interaction profiles and identical physical properties compared with natural
bile acids, ent-bile acids are ideal compounds to differentiate
between the receptor-mediated and the non-receptor-mediated functions of
natural bile acids.Open in a separate windowFIGURE 1.Natural and enantiomeric bile acids. Structures and
three-dimensional projection views of natural LCA, CDCA, DCA, and their
enantiomers (ent-LCA, ent-CDCA, and ent-DCA). The
three-dimensional ent-steroid structure is rotated 180° around
the long axis for easier comparison with the natural steroid.In this study we explore the enantioselectivity of LCA-, CDCA-, and
DCA-mediated toxicity and apoptosis in two human colon adenocarcinoma cell
lines, HT-29 and HCT-116. Because the mechanism of natural LCA induced
apoptosis has never been characterized, we then examined in more detail LCA-
and ent-LCA-mediated apoptosis in colon cancer cells. These studies
will not only explore the LCA apoptotic mechanism but will also determine
whether ent-LCA signals through similar cellular pathways. 相似文献
6.
Heather B. Claxton David L. Akey Monica K. Silver Suzanne J. Admiraal Janet L. Smith 《The Journal of biological chemistry》2009,284(8):5021-5029
Two thioesterases are commonly found in natural product biosynthetic
clusters, a type I thioesterase that is responsible for removing the final
product from the biosynthetic complex and a type II thioesterase that is
believed to perform housekeeping functions such as removing aberrant units
from carrier domains. We present the crystal structure and the kinetic
analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide
synthetases/polyketide synthase rifamycin biosynthetic cluster of
Amycolatopsis mediterranei. Steady-state kinetics show that RifR has
a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm
of the acyl carrier domain over the hydrolysis of acyl units from the
phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the
decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA
and methylmalonyl-CoA. Multiple RifR conformations and structural similarities
to other thioesterases suggest that movement of a helical lid controls access
of substrates to the active site of RifR.Assembly line complexes, which include modular polyketide synthases
(PKS)3 and
nonribosomal peptide synthetases (NRPS), are multifunctional proteins composed
of modules that work in succession to synthesize secondary metabolites, many
of which are precursors of potent antibiotics, immunosuppressants, anti-tumor
agents, and other bioactive compounds. Rifamycin, the precursor to the
anti-tuberculosis drug rifampicin, is produced by the rifamycin assembly line
complex, which is an NRPS/PKS hybrid system composed of one NRPS-like and 10
PKS modules (1). Each module in
an assembly line complex extends and modifies the intermediate compound before
passing it on to the next module in the series
(Fig. 1A). The
intermediate compounds are covalently attached through a thioester linkage to
the phosphopantetheine arm (Ppant) of carrier domains, one associated with
each module, until they are released from the synthase, usually by a type I
thioesterase (TEI) (2,
3).Open in a separate windowFIGURE 1.Proposed functions of thioesterase II proteins. A, chain
elongation by a PKS module. The chain elongation intermediate is transferred
from the ACP of the upstream module to the ketosynthase (KS) domain.
The acyltransferase (AT) domain transfers an acyl group building
block from CoA to the ACP within the module. The KS domain catalyzes
condensation of the new building block with the intermediate, releasing
CO2. B, production of a decarboxylated acyl unit by the
ketosynthase domain and the subsequent hydrolysis by a TEII. C,
mispriming of a PKS by transfer of an acyl-phosphopantetheine arm by a
promiscuous phosphopantetheinyl transferase (Pptase) and the
subsequent hydrolysis by a TEII. D, hydrolysis of an amino acid
derivative by a TEII from an NRPS module comprising an adenylation domain
(A) and a peptide carrier protein (PCP) domain.TEIs are usually integrated into the final module of the assembly line
complex and remove the final product through macrocyclization or hydrolysis.
Occasionally, tandem type I thioesterases are integrated at the C terminus of
the final module of NRPS pathways
(4).Although TEIs are covalently attached to the terminal module and generally
process only the final product of an assembly line complex, type II
thioesterases (TEIIs) are discrete proteins that can remove intermediates from
any module in the complex. A variety of functions have been attributed to
TEIIs, the most prevalent of which is a “housekeeping function,”
the removal of aberrant acyl units from carrier domains. These aberrant acyl
units may be due to premature decarboxylation by a PKS ketosynthase domain
(5)
(Fig. 1B) or to
mispriming of the carrier domain by a promiscuous phosphopantetheinyl
transferase
(6–8)
(Fig. 1C). Other
proposed functions for TEIIs include the removal of intermediates from the
synthase as in the case of the mammary gland rat fatty acid synthase (FAS)
TEII in lactating rats, which removes medium chain
C8-C12 fatty acids from the ACP domain
(9) and the removal of amino
acid derivatives from a carrier domain
(10–13),
allowing these derivatives to be incorporated into the natural product by a
later module in the assembly line complex
(Fig. 1D).Disruption of the TEI function results in a complete loss of product,
whereas disruption of TEII function results in a significant decrease in
product yield (30–95%)
(4,
14–24).
Removal of the TEII from the rifamycin assembly line resulted in a 60%
decrease in product yield
(25). Neither TEIs nor TEIIs
may rescue the disrupted function of the other
(6), but a TEII from another
pathway may rescue the function of a disrupted TEII
(26).Two models have been proposed for the TEII housekeeping function
(5). In the high specificity
model, the TEII scans the complex and efficiently removes only aberrant acyl
units. In the low specificity model, the TEII removes both correct and
incorrect acyl units from the Ppant arm at an inefficient rate. Correct acyl
units are quickly incorporated into the growing intermediate compound. In
contrast, incorrect acyl units stall the assembly line, providing a longer
window of opportunity for removal by a TEII. Thus a slow, low specificity
enzyme can be effective.TEIIs from different pathways have differing specificities, but general
trends include a preference for decarboxylated acyl units over carboxylated
acyl units (5,
6,
27), substrates linked to a
carrier domain over substrates linked to CoA or the phosphopantetheine mimic
N-acetylcysteamine (7,
28), and single amino acids
over di- or tri-peptides (6,
7). TEIIs are able to hydrolyze
substrates attached to carrier domains from their native pathway as well as
other pathways (6,
20,
28).PKS/NRPS/FAS thioesterases belong to the α/β hydrolase family.
Structures are reported for seven PKS/NRPS/FAS thioesterases: crystal
structures for the TEIs from the pikromycin (PikTE) PKS
(29), 6-deoxyerythronolide B
(DEBSTE) PKS (30), surfactin
NRPS (SrfTE) (31), fengycin
NRPS (FenTE) (32), and human
fatty acid synthase (hFasTE)
(33) systems, and NMR
structures for enterobactin TEI
(34), and surfactin TEII
(35). Like PKS modules, PKS
TEIs are dimers. The dimer interface comprises two N-terminal helices that are
unique to the PKS TEIs. NRPS TEIs are monomeric, like NRPS modules. The NRPS
TEII of surfactin is also monomeric
(31). Although the FAS complex
is dimeric, the FAS TEI is a monomer
(33). All of the TEs have an
α-helical insertion after strand β5 that forms a lid over the
active site. Additionally, in the PKS TEIs, the N-terminal dimer-forming
helices contribute to the lid structure, forming a fixed channel that runs the
length of the TE and contains the active site. In contrast, the active site
pocket of monomeric NRPS TEIs and TEIIs is flexible; two conformations of the
lid and active site pocket were observed in the surfactin TEI (SrfTEI) crystal
structure (7), and chemical
shift observations suggested greater flexibility for residues of the lid
region in the surfactin TEII (SrfTEII) solution structure
(35). These movements seem to
be of functional importance, because a movement of a linker peptide in SrfTEI
determines the shape of the active site pocket and a movement of the first lid
helix appears to modulate access to the active site
(31).We report the structure and activity of recombinant RifR, the TEII of the
rifamycin biosynthetic cluster. Steady-state kinetic analysis of the
hydrolytic activity of RifR on a wide range of acyl-CoA and acyl-ACP
substrates demonstrates that acyl-ACP substrates are preferred over the
acyl-CoAs. Aberrant, decarboxylated acyl units are processed more efficiently
than are the natural rifamycin building blocks. We report the crystal
structure of RifR, the first for any hybrid PKS/NRPS TEII. The size and shape
of the substrate chamber are variable, because one of the elements forming the
chamber, an extended linker segment, is highly flexible, and different crystal
forms reveal different shapes for the substrate binding site. Access to the
active site is severely restricted, and structural comparisons with other
thioesterases suggest that a conformational change in the lid and the flexible
linker region is required for access to the substrate pocket. 相似文献
7.
Mait�� Montero-Hadjadje Salah Elias Laurence Chevalier Magalie Benard Yannick Tanguy Val��rie Turquier Ludovic Galas Laurent Yon Maria M. Malagon Azeddine Driouich St��phane Gasman Youssef Anouar 《The Journal of biological chemistry》2009,284(18):12420-12431
Chromogranin A (CgA) has been proposed to play a major role in the
formation of dense-core secretory granules (DCGs) in neuroendocrine cells.
Here, we took advantage of unique features of the frog CgA (fCgA) to assess
the role of this granin and its potential functional determinants in hormone
sorting during DCG biogenesis. Expression of fCgA in the constitutively
secreting COS-7 cells induced the formation of mobile vesicular structures,
which contained cotransfected peptide hormones. The fCgA and the hormones
coexpressed in the newly formed vesicles could be released in a regulated
manner. The N- and C-terminal regions of fCgA, which exhibit remarkable
sequence conservation with their mammalian counterparts were found to be
essential for the formation of the mobile DCG-like structures in COS-7 cells.
Expression of fCgA in the corticotrope AtT20 cells increased
pro-opiomelanocortin levels in DCGs, whereas the expression of N- and
C-terminal deletion mutants provoked retention of the hormone in the Golgi
area. Furthermore, fCgA, but not its truncated forms, promoted
pro-opiomelanocortin sorting to the regulated secretory pathway. These data
demonstrate that CgA has the intrinsic capacity to induce the formation of
mobile secretory granules and to promote the sorting and release of peptide
hormones. The conserved terminal peptides are instrumental for these
activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the
constitutive secretory pathway. The fundamental feature of neuroendocrine and
endocrine cells is the occurrence of dense-core secretory granules
(DCGs),3
which are key cytoplasmic organelles responsible for secretion of hormones,
neuropeptides, and neurotransmitters through the regulated secretory pathway
(RSP). Storage at high concentrations of these secretory products is required
for their finely tuned release in response to extracellular stimulation
(1,
2). DCG biogenesis starts with
the budding of immature secretory granules (ISGs) from the
trans-Golgi network (TGN) through interactions between lipid rafts
and protein components, in a similar manner to constitutive vesicle budding
(2,
3). The ISG budding is followed
by a multistep maturation process to form the mature secretory granules,
including removal of the constitutive secretory proteins and lysosomal enzymes
inadvertently packaged into ISGs
(4).Despite increasing knowledge of the various steps of DCG formation, the
nature of the sorting signals for entry of proteins into the DCGs and the
molecular machinery required to generate secretory granules are not fully
elucidated (5,
6). Several recent studies
highlighted the role of members of the granin family, which may represent the
driving force for granulogenesis in the TGN
(2), although this notion has
been a matter of debate (7).
Granins are soluble acidic proteins widely distributed in endocrine and
neuroendocrine cells, which are characterized by the ability to aggregate at
acidic pH and a high Ca2+ environment
(8,
9). These conditions are found
in the lumen of the TGN allowing granins to aggregate in this compartment and
to be segregated from constitutively secreted proteins
(10,
11). The granin aggregates are
believed to associate directly or indirectly with lipid rafts at the TGN to
induce budding and formation of the ISGs. A prominent role of chromogranin A
(CgA) in the regulation of DCG formation in endocrine and neuroendocrine cells
has been proposed. Thus, depletion of CgA in PC12 cells led to a dramatic
decrease in the number of DCGs
(12), and exogenously
expressed CgA in these depleted PC12 cells, as in DCG-deficient endocrine A35C
and 6T3 cells, restored DCG biogenesis
(12,
13). Besides, expression of
granins in non-endocrine, constitutively secreting cells such as CV-1, NIH3T3,
or COS-7 cells provoked the formation of DCG-like structures that release
their content in response to Ca2+ influx
(12,
14,
15). Further investigations
performed in CgA null mice and transgenic mice expressing antisense RNA
against CgA also revealed a reduction in the number of DCGs in chromaffin
cells that was associated with an impairment of catecholamine storage, thus
demonstrating the crucial role of CgA in normal DCG biogenesis
(16,
17). In CgA knockout mice, the
introduction of the gene expressing human CgA restored the regulated secretory
phenotype (16). A different
CgA null mice strain exhibited no discernable effect on DCG formation, but
elevated catecholamine secretion
(18), proving that CgA
deficiency is associated with hormone storage impairment in neuroendocrine
cells in vivo, a finding that was confirmed in vitro
(19). The CgA-/-
mice strain generated by Hendy et al.
(18) exhibited a compensatory
overexpression of other granins, pointing to a possible overlap in granin
function in secretory granule biogenesis.We reported previously that the frog CgA (fCgA) gene is coordinately
regulated with the pro-opiomelanocortin (POMC) gene in the pituitary pars
intermedia during the neuroendocrine reflex of skin color change, which allows
amphibia to adapt to their environment through the release of POMC-derived
melanotropic peptides (20,
21). Sequence comparison of
fCgA with its mammalian orthologs revealed a high conservation of the N- and
C-terminal domains, and far less conservation of the central part of the
protein (Fig. 1A),
suggesting that these domains may play a role in DCG formation and hormone
release in various species (9,
20,
21). To assess the role of
fCgA and its conserved N- and C-terminal regions in hormone sorting, storage,
and secretion, we engineered different constructs that produce the native
unmodified (no tag added) protein and truncated forms lacking the conserved N-
and C-terminal domains, and we developed an antibody that specifically
recognizes the central region of fCgA. Using the constitutively secreting
COS-7 cells, which are devoid of DCGs, we could demonstrate for the first time
that CgA is essential for targeting peptide hormones to newly formed mobile
DCG-like structures. In the CgA-expressing AtT20 cells, which exhibit an only
moderate capacity to sort secretory proteins to the regulated pathway
(22), the granin plays a
pivotal role in the sorting and release of POMC. The conserved terminal
peptides of CgA are instrumental for these activities.Open in a separate windowFIGURE 1.Specificity of the antibody directed against frog CgA. A,
scheme depicting the structure of fCgA and showing the high conservation of
the terminal regions and the percentages of amino acid identity between frog
and human CgA sequences. The highly conserved peptide WE14 and dibasic
cleavage sites are also indicated. B, Western blot showing that the
antibody developed against fCgA recognized the protein and several processing
intermediates in frog but not rat pituitary extracts, whereas an antibody,
directed against the WE14 conserved peptide, detected CgA and its processing
products in both rat and frog pituitary extracts. C,
immunofluorescence analysis of frog pituitary and adrenal glands, and rat
adrenal gland using the antibodies against fCgA and WE14. cx, cortex;
DL, distal lobe; IL, intermediate lobe; and m,
medulla. Scale bars equal 10 μm. 相似文献
8.
Hans Bakker Takuji Oka Angel Ashikov Ajit Yadav Monika Berger Nadia A. Rana Xiaomei Bai Yoshifumi Jigami Robert S. Haltiwanger Jeffrey D. Esko Rita Gerardy-Schahn 《The Journal of biological chemistry》2009,284(4):2576-2583
In mammals, xylose is found as the first sugar residue of the
tetrasaccharide
GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the
formation of the glycosaminoglycans heparin/heparan sulfate and
chondroitin/dermatan sulfate. It is also found in the trisaccharide
Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor
repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes
the formation of the UDP-xylose substrate for the different
xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in
the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place
in these organelles, no obvious requirement exists for membrane transport of
UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has
been documented, and we recently succeeded with the cloning of a human
UDP-xylose transporter (SLC25B4). Here we provide new evidence for a
functional role of UDP-xylose transport by characterization of a new Chinese
hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The
mutant fails to initiate glycosaminoglycan synthesis and is not capable of
xylosylating Notch. Complementation was achieved by expression of a
cytoplasmic variant of UXS, which proves the existence of a functional Golgi
UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid
occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated
control of the cytoplasmically localized UDP-glucose dehydrogenase in the
mutant. The data presented in this study suggest the bidirectional transport
of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in
controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.Xylose is only known to occur in two different mammalian glycans. First,
xylose is the starting sugar residue of the common tetrasaccharide,
GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, attached to
proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans
(GAGs)2
(1). Second, xylose is found in
the trisaccharide Xylα1,3Xylα1,3Glcβ1-O-Ser in
epidermal growth factor (EGF)-like repeats of proteins, such as blood
coagulation factors VII and IX
(2) and Notch
(3)
(Fig. 1). Two variants of
O-xylosyltransferases (XylT1 and XylT2) are responsible for the
initiation of glycosaminoglycan biosynthesis, which differ in terms of
acceptor specificity and tissue distribution
(4-7),
and two different enzymatic activities have been identified that catalyze
xylosylation of O-glucose residues added to EGF repeats
(8-10).
On Notch, O-glucose occurs on EGF repeats in a similar fashion as
O-fucose, which modifications have been shown to influence
ligand-mediated Notch signaling
(11-16).
Recently, rumi, the gene encoding the Notch
O-glucosyltransferase in Drosophila, has been identified,
and inactivation of the gene was found to cause a temperature-sensitive
Notch phenotype (17).
Although this finding clearly demonstrated that O-glucosylation is
essential for Notch signaling, the importance of xylosylation for Notch
functions remains ambiguous.Open in a separate windowFIGURE 1.UDP-xylose metabolism in mammalian cells. A, UDP-Xyl is
synthesized in two steps from UDP-Glc by the enzymes UGDH, forming UDP-GlcA,
and UXS, also referred to as UDP-glucuronic acid decarboxylase. UGDH is
inhibited by the product of the second enzyme, UDP-Xyl
(42). B, in mammals,
UDP-Xyl is synthesized within the lumen of the ER/Golgi, where it is substrate
for different xylosyltransferases incorporating xylose in the
glycosaminoglycan core (XylT1 and XylT2) or in O-glucose-linked
glycans. The nucleotide sugar transporter SLC35D1
(52) has been shown to
transport UDP-GlcA over the ER membrane and SLC35B4
(29) to transport UDP-Xyl over
the Golgi membrane. The function of this latter transporter is unclear.Several different Chinese hamster ovary (CHO) cell lines with defects in
GAG biosynthesis have been isolated by screening for reduced incorporation of
sulfate (18) and reduced
binding of fibroblast growth factor 2 (FGF-2)
(19,
20) and by direct selection
with FGF-2 conjugated to the plant cytotoxin saporin
(21). Isolated cells (called
pgs, for proteoglycan synthesis mutants)
(21) exhibited defects in
various stages of GAG biosynthesis, ranging from the initiating
xylosyltransferase to specific sulfation reactions
(18,
19,
21-25).
Mutants that affect overall GAG biosynthesis were shown to have a defect in
the assembly of the common core tetrasaccharide. Interestingly, these latter
mutants could be separated into clones in which GAG biosynthesis can be
restored by the external addition of xylosides as artificial primers and those
that cannot (18). The two
mutants belonging to the first group are pgsA-745 and pgsB-761. Although
pgs-745 is defective in XylT2
(4-6,
18), pgsB-761 exhibits a
defect in galactosyltransferase I (B4GalT7), the enzyme that catalyzes the
first step in the elongation of the xylosylated protein (25 (see
Fig. 1B). Restoration
of GAG biosynthesis in the latter mutant presumably occurs through a second
β1-4-galactosyltransferase, able to act on xylosides when provided at
high concentration but not on the endogenous protein-linked xylose.Here we describe the isolation of a third CHO cell line (pgsI-208) with the
xyloside-correctable phenotype. The mutant is deficient in UDP-xylose synthase
(UXS), also known as UDP-glucuronic acid decarboxylase. This enzyme catalyzes
the synthesis of UDP-Xyl, the common donor substrate for the different
xylosyltransferases, by decarboxylation of UDP-glucuronic acid. Importantly,
UXS in the animal cell is localized in the lumen of the ER and/or Golgi
(26-28),
superseding at first sight the need for the Golgi UDP-xylose transporter,
which has been recently cloned and characterized
(29). Using this cell variant,
experiments were designed that establish the functional significance of
UDP-Xyl transport with respect to UDP-glucuronic acid production and
xylosylation. 相似文献
9.
Wenli Li Jianhua Ju Scott R. Rajski Hiroyuki Osada Ben Shen 《The Journal of biological chemistry》2008,283(42):28607-28617
Tautomycin (TTM) is a highly potent and specific protein phosphatase
inhibitor isolated from Streptomyces spiroverticillatus. The
biological activity of TTM makes it an important lead for drug discovery,
whereas its spiroketal-containing polyketide chain and rare dialkylmaleic
anhydride moiety draw attention to novel biosynthetic chemistries responsible
for its production. To elucidate the biosynthetic machinery associated with
these novel molecular features, the ttm biosynthetic gene cluster
from S. spiroverticillatus was isolated and characterized, and its
involvement in TTM biosynthesis was confirmed by gene inactivation and
complementation experiments. The ttm cluster was localized to a 86-kb
DNA region, consisting of 20 open reading frames that encode three modular
type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five
proteins for methoxymalonyl-S-acyl carrier protein biosynthesis
(Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and
regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF
and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is
proposed based on functional assignments from sequence analysis, which agrees
well with previous feeding experiments, and has been further supported by
in vivo gene inactivation experiments. These findings set the stage
to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM
analogs.Tautomycin (TTM)2
is a polyketide natural product first isolated in 1987 from Streptomyces
spiroverticillatus (1).
The structure and stereochemistry of TTM were established on the basis of
chemical degradation and spectroscopic evidence
(2-4).
TTM contains several features not common to polyketide natural products,
including a spiroketal group, a methoxymalonate-derived unit, and an acyl
chain bearing a dialkylmaleic anhydride moiety. Structurally related to TTM is
tautomycetin (TTN), which was first isolated in 1989 from Streptomyces
griseochromogenes following the discovery of TTM
(5,
6). The structure of TTN was
deduced by chemical degradation and spectroscopic analysis
(6), and its stereochemistry
was established by comparison of spectral data with those of TTN degradation
products and synthetic fragments
(7). Both TTM and TTN exist as
tautomeric mixtures composed of two interconverting anhydride and diacid forms
in approximately a 5:4 ratio under neutral conditions
(Fig. 1A)
(1,
2).Open in a separate windowFIGURE 1.A, structures of TTM and TTN in anhydride or diacid forms, and
biosynthetic origin of the dialkylmaleic anhydride by feeding experiments
using 13C-labeled acetate and propionate. The
methoxymalonate-derived unit in TTM is highlighted by the dotted oval.
R, polyketide moiety of TTM or TTN. B, selected natural product
inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric
anhydride moiety. C, selected natural products containing a
dialkylmaleic anhydride moiety.Early studies of TTM revealed its ability to induce morphological changes
in leukemia cells (8). However,
it was later realized that TTM is a potent and specific inhibitor of protein
phosphatases (PPs) PP-1 and PP-2A
(9). PP-1 and PP-2A are two of
the four major serine/threonine protein phosphatases that regulate diverse
cellular events such as cell division, gene expression, muscle contraction,
glycogen metabolism, and neuronal signaling in eukaryotic cells
(10-12).
Many natural product PP-1 and PP-2A inhibitors are known, including okadaic
acid (13), calyculin-A
(14), phoslactomycin,
spirastrellolide, and cantharidin
(15)
(Fig. 1B), as well as
TTM (16,
17), and TTN
(18). They have served as
useful tools to study PP-involved intracellular events in vivo and as
novel leads for drug discovery
(10-12).
Among these PP inhibitors, TTM and TTN are unique because of their PP-1
selectivity. Despite their structural similarities, TTM exhibits potent
specific inhibition of PP-1 and PP-2A with IC50 values of 22-32
nm and only a slight preference for PP-1
(18). Conversely, TTN shows
nearly a 40-fold higher binding affinity to PP-1 (IC50 = 1.6
nm) than to PP-2A (IC50 = 62 nm)
(18). Because the major
structural differences between TTM and TTN reside in the region distal to the
dialkylmaleic anhydride moiety (Fig.
1A), it has been proposed that differences in these
moieties might be responsible for the PP-1 selectivity
(17-19).
Finally, TTN also has an impressive immuno-suppressive activity
(20,
21), which is apparently
devoid for TTM. Clearly, the structural differences between these two
polyketides translate into large, exploitable differences in bio-activities,
yet an understanding of the biosynthetic origins of these differences remains
elusive.The spiroketal and dialkylmaleic anhydride features of TTM are uncommon for
polyketide natural products, as is the methoxymalonate-derived unit
(Fig. 1A). Few studies
have been carried out for spiroketal biosynthesis, yet it is reasonably common
among the phosphatase inhibitors such as calyculin A, okadaic acid, and a few
others (Fig. 1B). Less
common, but still found in the phosphatase inhibitor cantharidin, as well as
TTM and TTN, is the dialkylmaleic anhydride moiety
(Fig. 1B); this unit
appears in a number of other natural products
(Fig. 1C), although
the biosynthetic steps leading to this reactive moiety (a protected version of
a dicarboxylate) have not been rigorously investigated. Feeding experiments
with 13C-labeled precursors indicated that the anhydride of TTM and
TTN is assembled from a propionate and an as yet undefined C-5 unit
(Fig. 1A), which would
require novel chemistry for polyketide biosynthesis
(22). TTM differentiates
itself from all known PP-1 and PP-2A inhibitors by virtue of its unique
combination of both the dialkymaleic anhydride and spiroketal
functionalities.Multiple total syntheses of TTM and a small number of analogs have been
reported, confirming the predicted structure and absolute stereochemistry and
facilitating structure-activity relationship studies on PP inhibition and
apoptosis induction (19,
23-25).
These studies revealed that: (i) the C22-C26 carbon chain and the
dialkylmaleic anhydride are the minimum requirements for TTM bioactivity; (ii)
the C18-C21 carbon chain and 22-hydroxy group are important for PP inhibition;
(iii) the spiroketal moiety determines the affinity to specific protein
phosphatases; (iv) the active form is most likely the dicarboxylate; and (v)
3′-epi-TTM exhibits 1,000-fold less activity than TTM. However, taken as
a whole, none of the analogs had an improved potency or selectivity for PP-1
inhibition than the natural TTM
(19,
22-25).
As a result, a more specific inhibitor of PP-1 is urgently awaited to
differentiate the physiological roles of PP-1 and PP-2A in vivo and
to explore PPs as therapeutic targets for drug discovery.We have undertaken the cloning and characterization of the TTM biosynthetic
gene cluster from S. spiroverticillatus as the first step toward
engineering TTM biosynthesis for novel analogs
(26). We report here: (i)
cloning and sequencing of the complete ttm gene cluster, (ii)
determination of the ttm gene cluster boundaries, (iii)
bioinformatics analysis of the ttm cluster and a proposal for TTM
biosynthesis, and (iv) genetic characterization of the TTM pathway to support
the proposed pathway. Of particular interest has been the identification of
genes possibly related to dialkylmaleic anhydride biosynthesis, the unveiling
of the ttm polyketide synthase (PKS) genes predicted to select and
incorporate four different starter and extender units for TTM production, and
the apparent lack of candidate genes associated with spiroketal formation.
These findings now set the stage to engineer TTM analogs for novel PP-1- and
PP-2A-specific inhibitors by applying combinatorial biosynthetic methods to
the TTM biosynthetic machinery. 相似文献
10.
11.
C. Martin Lawrence Smita Menon Brian J. Eilers Brian Bothner Reza Khayat Trevor Douglas Mark J. Young 《The Journal of biological chemistry》2009,284(19):12599-12603
Viruses populate virtually every ecosystem on the planet, including the
extreme acidic, thermal, and saline environments where archaeal organisms can
dominate. For example, recent studies have identified crenarchaeal viruses in
the hot springs of Yellowstone National Park and other high temperature
environments worldwide. These viruses are often morphologically and
genetically unique, with genomes that show little similarity to genes of known
function, complicating efforts to understand their viral life cycles. Here, we
review progress in understanding these fascinating viruses at the molecular
level and the evolutionary insights coming from these studies.The last decade has seen resurgent interest in the study of viruses that
lie outside traditional agricultural and medical interests. One reason is the
growing appreciation of the enormous abundance and impact of viruses on the
greater biosphere. For example, the oceans are thought to contain
∼1031 viruses, a truly astronomical number
(1), making viruses the most
abundant biological entities in this ecosystem, where they catalyze turnover
of 20% of the oceanic biomass per day
(1). Remarkably, the virosphere
has now been shown to extend to almost every known environment on earth,
including the extreme acidic, thermal, and saline environments where archaeal
organisms can be dominant. Thus, because of their abundance and variety,
viruses are now thought to represent the greatest reservoir of genetic
diversity on the planet
(2).A second reason to study archaeal viruses is a growing appreciation for the
roles viruses play in evolution. Remarkably with >500 cellular genomes
sequenced to date, most show a significant amount of viral or virus-like
sequence within their genome, further evidence that viruses play a central
role in horizontal gene transfer and help drive the evolution of their hosts.
Roles for viruses in cellular evolution are also being considered. Current
hypotheses contend that viruses have catalyzed several major evolutionary
transitions, including the invention of DNA and DNA replication mechanisms
(3), the origin of the
eukaryotic nucleus (4), and
thus a role in the formation of the three domains of life. In addition, there
is also considerable interest in viral genesis and evolution in and of itself.
To evaluate these hypotheses and to analyze evolutionary relationships among
viruses, knowledge of viruses infecting the archaea is essential, yet these
viruses are vastly understudied. Finally, interest in archaeal viruses stems
also from the exceptional molecular insight viruses have traditionally
provided into host processes; archaeal viruses are certain to provide new
insights into the molecular biology of this poorly understood domain of
life.Pioneering studies by Wolfram Zillig et al.
(5) identified the first
archaeal viruses. Although initial studies suggested that viruses infecting
the euryarchaea (principally halophiles and methanogens) were similar to
head-tail bacteriophage, studies of viruses infecting the hyperthermophilic
crenarchaea revealed morphologies suggesting new viral families. Indeed, work
by several laboratories has led to the identification of seven new viral
families infecting the crenarchaea, the Globuloviridae, Guttaviridae,
Fuselloviridae, Bicaudaviridae, Ampullaviridae, Rudiviridae, and
Lipothrixviridae (Fig. 1)
(6,
7), with
STIV3
(8) and STSV1
(9) awaiting assignment. All of
these viruses contain double-stranded DNA genomes ranging in size from 13.7 to
75.3 kilobase pairs, encoding 31–74 ORFs. Although many package a
circular genome, the filamentous Lipothrixviridae and rod-shaped Rudiviridae
are notable exceptions and are the only viruses in any domain known to
encapsidate linear double-stranded DNA. Although most crenarchaeal viruses are
enveloped, the Rudiviridae are devoid of lipid, and with the
exception of the Fuselloviridae, they employ a lytic life cycle,
although only STIV and ATV (Bicaudaviridae) are known to cause cell
lysis
(11).4Open in a separate windowFIGURE 1.Morphological diversity in crenarchaeal viruses. A,
clockwise, beginning at upper left: STIV
(8), a PSV-like virus,
Sulfolobus neozealandicus droplet-shaped virus (SNDV)
(47), SSV1
(48), STSV1
(9), an ATV-like virus, an SIRV
virus, and S. icelandicus filamentous virus (SIFV)
(10). Micrographs of SIRV,
PSV-like, and ATV-like viruses from Yellowstone National Park are the courtesy
of M. J. Y. Other panels are reproduced, with permission, from Refs.
8–10,
47, and
48. B, cryoelectron
microscopy reconstruction of the STIV particle
(8) showing a cutaway view
(20) of the T = 31
icosahedral capsid with turret-like projections that extend from each of the
5-fold vertices. Portions of the protein shell (blue) and inner lipid
layer (yellow) have been removed to reveal the interior.The exceptional morphology of these viruses has been reviewed
(6,
7) and thus is only summarized
here (Fig. 1). For the
rod-shaped Rudiviridae, plugs are seen at both ends, from which three
short tail fibers emanate, whereas the Lipothrixviridae show mop- or claw-like
structures at both ends (6).
Similarly, the non-tailed icosahedral viruses, STIV and euryarchaeal SH1, have
large turrets or spikes that project from the surface
(8,
12). In each case, these
structures are thought to facilitate virus-host interactions. In contrast,
other crenarchaeal viruses utilize a fusiform or lemon-shaped virion, a
morphology unique to archaeal viruses. These fusiform viruses generally
contain tail fibers or an extended tail on one end that is also involved in
host recognition. For ATV, however, nascent particles are devoid of tails when
released from the host (13).
Remarkably, extended tails develop at both ends of the virion in an
extracellular maturation process. Finally, Acidianus bottle-shaped
virus (Ampullaviridae) shows an exceptional morphology that differs in its
basic architecture from any known virus. 相似文献
12.
13.
ATP-binding cassette (ABC) transporters transduce the free energy of ATP
hydrolysis to power the mechanical work of substrate translocation across cell
membranes. MsbA is an ABC transporter implicated in trafficking lipid A across
the inner membrane of Escherichia coli. It has sequence similarity
and overlapping substrate specificity with multidrug ABC transporters that
export cytotoxic molecules in humans and prokaryotes. Despite rapid advances
in structure determination of ABC efflux transporters, little is known
regarding the location of substrate-binding sites in the transmembrane segment
and the translocation pathway across the membrane. In this study, we have
mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using
site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin
labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster
at the cytoplasmic end of helices 3 and 6 at a site accessible from the
membrane/water interface and extending into an aqueous chamber formed at the
interface between the two transmembrane domains. Binding of a nonhydrolyzable
ATP analog inverts the transporter to an outward-facing conformation and
relieves DNR quenching by spin labels suggesting DNR exclusion from proximity
to the spin labels. The simplest model consistent with our data has DNR
entering near an elbow helix parallel to the water/membrane interface,
partitioning into the open chamber, and then translocating toward the
periplasm upon ATP binding.ATP-binding cassette
(ABC)2 transporters
transduce the energy of ATP hydrolysis to power the movement of a wide range
of substrates across the cell membranes
(1,
2). They constitute the largest
family of prokaryotic transporters, import essential cell nutrients, flip
lipids, and export toxic molecules
(3). Forty eight human ABC
transporters have been identified, including ABCB1, or P-glycoprotein, which
is implicated in cross-resistance to drugs and cytotoxic molecules
(4,
5). Inherited mutations in
these proteins are linked to diseases such as cystic fibrosis, persistent
hypoglycemia of infancy, and immune deficiency
(6).The functional unit of an ABC transporter consists of four modules. Two
highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze
ATP to supply the active energy for transport
(7). ABCs drive the mechanical
work of proteins with diverse functions ranging from membrane transport to DNA
repair (3,
5). Substrate specificity is
determined by two transmembrane domains (TMDs) that also provide the
translocation pathway across the bilayer
(7). Bacterial ABC exporters
are expressed as monomers, each consisting of one NBD and one TMD, that
dimerize to form the active transporter
(3). The number of
transmembrane helices and their organization differ significantly between ABC
importers and exporters reflecting the divergent structural and chemical
nature of their substrates (1,
8,
9). Furthermore, ABC exporters
bind substrates directly from the cytoplasm or bilayer inner leaflet and
release them to the periplasm or bilayer outer leaflet
(10,
11). In contrast, bacterial
importers have their substrates delivered to the TMD by a dedicated high
affinity substrate-binding protein
(12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on
the inner membrane to its final destination in the outer membrane requires the
ABC transporter MsbA (13).
Although MsbA has not been directly shown to transport lipid A, suppression of
MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits
bacterial growth strongly suggesting a role in translocation
(14-16).
In addition to this role in lipid A transport, MsbA shares sequence similarity
with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus
lactis, and Sav1866 of Staphylococcus aureus
(16-19).
ABCB1, a prototype of the ABC family, is a plasma membrane protein whose
overexpression provides resistance to chemotherapeutic agents in cancer cells
(1). LmrA and MsbA have
overlapping substrate specificity with ABCB1 suggesting that both proteins can
function as drug exporters
(18,
20). Indeed, cells expressing
MsbA confer resistance to erythromycin and ethidium bromide
(21). MsbA can be photolabeled
with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342
() across membrane vesicles in an energy-dependent manner
( H3334221).The structural mechanics of ABC exporters was revealed from comparison of
the MsbA crystal structures in the apo- and nucleotide-bound states as well as
from analysis by spin labeling EPR spectroscopy in liposomes
(17,
19,
22,
23). The energy harnessed from
ATP binding and hydrolysis drives a cycle of NBD association and dissociation
that is transmitted to induce reorientation of the TMD from an inward- to
outward-facing conformation
(17,
19,
22). Large amplitude motion
closes the cytoplasmic end of a chamber found at the interface between the two
TMDs and opens it to the periplasm
(23). These rearrangements
lead to significant changes in chamber hydration, which may drive substrate
translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile,
wasting the energy of ATP hydrolysis without substrate extrusion
(7). Consistent with this
model, ATP binding reduces ABCB1 substrate affinity, potentially through
binding site occlusion
(24-26).
Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP
hydrolysis increasing transport efficiency
(1,
27,
28). However, there is a
paucity of information regarding the location of substrate binding, the
transport pathway, and the structural basis of substrate recognition by ABC
exporters. In vitro studies of MsbA substrate specificity identify a
broad range of substrates that stimulate ATPase activity
(29). In addition to the
putative physiological substrates lipid A and lipopolysaccharide (LPS), the
ABCB1 substrates Ilmofosine, , and verapamil differentially enhance ATP
hydrolysis of MsbA ( H3334229,
30). Intrinsic MsbA tryptophan
(Trp) fluorescence quenching by these putative substrate molecules provides
further support of interaction
(29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model
of substrate binding to ABC efflux exporters. This so-called
“hydrophobic cleaner model” describes substrates binding from the
inner leaflet of the bilayer and then translocating through the TMD
(10,
31,
32). These studies also
identified a large number of residues involved in substrate binding and
selectivity (33). When these
crucial residues are mapped onto the crystal structures of MsbA, a subset of
homologous residues clusters to helices 3 and 6 lining the putative substrate
pathway (34). Consistent with
a role in substrate binding and specificity, simultaneous replacement of two
serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport
of ethidium and taxol, although and erythromycin interactions remain
unaffected ( H3334234).The tendency of lipophilic substrates to partition into membranes confounds
direct analysis of substrate interactions with ABC exporters
(35,
36). Such partitioning may
promote dynamic collisions with exposed Trp residues and nonspecific
cross-linking in photo-affinity labeling experiments. In this study, we
utilize a site-specific quenching approach to identify residues in the
vicinity of the daunorubicin (DNR)-binding site
(37). Although the data on DNR
stimulation of ATP hydrolysis is inconclusive
(20,
29,
30), the quenching of MsbA Trp
fluorescence suggests a specific interaction. Spin labels were introduced
along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent
quenching of DNR fluorescence. Residues that quench DNR cluster along the
cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR.
Furthermore, many of these residues are not lipid-exposed but face the
putative substrate chamber formed between the two TMDs. These residues are
proximal to two Trps, which likely explains the previously reported quenching
(29). Our results suggest DNR
partitions to the membrane and then binds MsbA in a manner consistent with the
hydrophobic cleaner model. Interpretation in the context of the crystal
structures of MsbA identifies a putative translocation pathway through the
transmembrane segment. 相似文献
14.
Formin-homology (FH) 2 domains from formin proteins associate processively
with the barbed ends of actin filaments through many rounds of actin subunit
addition before dissociating completely. Interaction of the actin
monomer-binding protein profilin with the FH1 domain speeds processive barbed
end elongation by FH2 domains. In this study, we examined the energetic
requirements for fast processive elongation. In contrast to previous
proposals, direct microscopic observations of single molecules of the formin
Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed
that profilin is not required for formin-mediated processive elongation of
growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin
release the γ-phosphate of ATP on average >2.5 min after becoming
incorporated into filaments. Therefore, the release of γ-phosphate from
actin does not drive processive elongation. We compared experimentally
observed rates of processive elongation by a number of different FH2 domains
to kinetic computer simulations and found that actin subunit addition alone
likely provides the energy for fast processive elongation of filaments
mediated by FH1FH2-formin and profilin. We also studied the role of FH2
structure in processive elongation. We found that the flexible linker joining
the two halves of the FH2 dimer has a strong influence on dissociation of
formins from barbed ends but only a weak effect on elongation rates. Because
formins are most vulnerable to dissociation during translocation along the
growing barbed end, we propose that the flexible linker influences the
lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament
structures for diverse processes in eukaryotic cells (reviewed in Ref.
1). Formins stimulate
nucleation of actin filaments and, in the presence of the actin
monomer-binding protein profilin, speed elongation of the barbed ends of
filaments
(2-6).
The ability of formins to influence elongation depends on the ability of
single formin molecules to remain bound to a growing barbed end through
multiple rounds of actin subunit addition
(7,
8). To stay associated during
subunit addition, a formin molecule must translocate processively on the
barbed end as each actin subunit is added
(1,
9-12).
This processive elongation of a barbed end by a formin is terminated when the
formin dissociates stochastically from the growing end during translocation
(4,
10).The formin-homology
(FH)2 1 and
2 domains are the best conserved domains of formin proteins
(2,
13,
14). The FH2 domain is the
signature domain of formins, and in many cases, is sufficient for both
nucleation and processive elongation of barbed ends
(2-4,
7,
15). Head-to-tail homodimers
of FH2 domains (12,
16) encircle the barbed ends
of actin filaments (9). In
vitro, association of barbed ends with FH2 domains slows elongation by
limiting addition of free actin monomers. This “gating” behavior
is usually explained by a rapid equilibrium of the FH2-associated end between
an open state competent for actin monomer association and a closed state that
blocks monomer binding (4,
9,
17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for
profilin to stimulate formin-mediated elongation. Individual tracks of
polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer
the actin directly to the FH2-associated barbed end to increase processive
elongation rates
(4-6,
8,
10,
17).Rates of elongation and dissociation from growing barbed ends differ widely
for FH1FH2 fragments from different formin homologs
(4). We understand few aspects
of FH1FH2 domains that influence gating, elongation or dissociation. In this
study, we examined the source of energy for formin-mediated processive
elongation, and the influence of FH2 structure on elongation and dissociation
from growing ends. In contrast to previous proposals
(6,
18), we found that fast
processive elongation mediated by FH1FH2-formins is not driven by energy from
the release of the γ-phosphate from ATP-actin filaments. Instead, the
data show that the binding of an actin subunit to the barbed end provides the
energy for processive elongation. We found that in similar polymerizing
conditions, different natural FH2 domains dissociate from growing barbed ends
at substantially different rates. We further observed that the length of the
flexible linker between the subunits of a FH2 dimer influences dissociation
much more than elongation. 相似文献
15.
Tatsuhiro Sato Akio Nakashima Lea Guo Fuyuhiko Tamanoi 《The Journal of biological chemistry》2009,284(19):12783-12791
Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway
by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully
reproduced in vitro by using mTORC1 immunoprecipitated by the use of
anti-raptor antibody from mammalian cells starved for nutrients. The low
in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is
dramatically increased by the addition of recombinant Rheb. On the other hand,
the addition of Rheb does not activate mTORC2 immunoprecipitated from
mammalian cells by the use of anti-rictor antibody. The activation of mTORC1
is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42
did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition,
the activation is dependent on the presence of bound GTP. We also find that
the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a
recently proposed mediator of Rheb action, appears not to be involved in the
Rheb-dependent activation of mTORC1 in vitro, because the preparation
of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of
Rheb results in a significant increase of binding of the substrate protein
4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that
competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation
of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated
by Rheb. Rheb does not induce autophosphorylation of mTOR. These results
suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to
regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins
(1). We have shown that Rheb
proteins are conserved and are found from yeast to human
(2). Although yeast and fruit
fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or
simply Rheb) and Rheb2 (RhebL1)
(2). Structurally, these
proteins contain G1-G5 boxes, short stretches of amino acids that define the
function of the Ras superfamily G-proteins including guanine nucleotide
binding (1,
3,
4). Rheb proteins have a
conserved arginine at residue 15 that corresponds to residue 12 of Ras
(1). The effector domain
required for the binding with downstream effectors encompasses the G2 box and
its adjacent sequences (1,
5). Structural analysis by
x-ray crystallography further shows that the effector domain is exposed to
solvent, is located close to the phosphates of GTP especially at residues
35–38, and undergoes conformational change during GTP/GDP exchange
(6). In addition, all Rheb
proteins end with the CAAX (C is cysteine, A is an aliphatic amino
acid, and X is the C-terminal amino acid) motif that signals
farnesylation. In fact, we as well as others have shown that these proteins
are farnesylated
(7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling
pathway that plays central roles in regulating protein synthesis and growth in
response to nutrient, energy, and growth conditions
(10–14).
Rheb is down-regulated by a TSC1·TSC2 complex that acts as a
GTPase-activating protein for Rheb
(15–19).
Recent studies established that the GAP domain of TSC2 defines the functional
domain for the down-regulation of Rheb
(20). Mutations in the
Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms
include the appearance of benign tumors called hamartomas at different parts
of the body as well as neurological symptoms
(21,
22). Overexpression of Rheb
results in constitutive activation of mTOR even in the absence of nutrients
(15,
16). Two mTOR complexes,
mTORC1 and mTORC2, have been identified
(23,
24). Whereas mTORC1 is
involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is
involved in the phosphorylation of Akt in response to insulin. It has been
suggested that Rheb is involved in the activation of mTORC1 but not mTORC2
(25).Although Rheb is clearly involved in the activation of mTOR, the mechanism
of activation has not been established. We as well as others have suggested a
model that involves the interaction of Rheb with the TOR complex
(26–28).
Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was
reported (29). Rheb has been
shown to interact with mTOR
(27,
30), and this may involve
direct interaction of Rheb with the kinase domain of mTOR
(27). However, this Rheb/mTOR
interaction is a weak interaction and is not dependent on the presence of GTP
bound to Rheb (27,
28). Recently, a different
model proposing that FKBP38 (FK506-binding protein
38) mediates the activation of
mTORC1 by Rheb was proposed
(31,
32). In this model, FKBP38
binds mTOR and negatively regulates mTOR activity, and this negative
regulation is blocked by the binding of Rheb to FKBP38. However, recent
reports dispute this idea
(33).To further characterize Rheb activation of mTOR, we have utilized an in
vitro system that reproduces activation of mTORC1 by the addition of
recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved
cells using anti-raptor antibody and have shown that its kinase activity
against 4E-BP1 is dramatically increased by the addition of recombinant Rheb.
Importantly, the activation of mTORC1 is specific to Rheb and is dependent on
the presence of bound GTP as well as an intact effector domain. FKBP38 is not
detected in our preparation and further investigation suggests that FKBP38 is
not an essential component for the activation of mTORC1 by Rheb. Our study
revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1
rather than increasing the kinase activity of mTOR. 相似文献
16.
Zhemin Zhou Yoshiteru Hashimoto Michihiko Kobayashi 《The Journal of biological chemistry》2009,284(22):14930-14938
17.
Takafumi Tasaki Adriana Zakrzewska Drew D. Dudgeon Yonghua Jiang John S. Lazo Yong Tae Kwon 《The Journal of biological chemistry》2009,284(3):1884-1895
The N-end rule pathway is a ubiquitin-dependent system where E3 ligases
called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and
type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have
recently reported an E3 family (termed UBR1 through UBR7) characterized by the
70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured
during affinity-based proteomics with synthetic degrons. Here we characterized
substrate binding specificity and recognition domains of UBR proteins.
Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and
300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding
assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations
narrow down the degron-binding activity to a 72-residue UBR box-only fragment
that recognizes type-1 but not type-2 residues. A surface plasmon resonance
assay shows that the UBR box binds to the type-1 substrate Arg-peptide with
Kd of ∼3.4 μm. Downstream from the UBR
box, we identify a second substrate recognition domain, termed the N-domain,
required for type-2 substrate recognition. The ∼80-residue N-domain shows
structural and functional similarity to 106-residue Escherichia coli
ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box
functions as a common structural element essential for binding to all known
destabilizing N-terminal residues, whereas specific residues localized in the
UBR box (for type 1) or the N-domain (for type 2) provide substrate
selectivity through interaction with the side group of an N-terminal amino
acid. Our work provides new insights into substrate recognition in the N-end
rule pathway.The N-end rule pathway is a ubiquitin
(Ub)2-dependent
proteolytic system in which N-terminal residues of short-lived proteins
function as an essential component of degradation signals (degrons) called
N-degrons (Fig. 1A)
(1-15).
An N-degron can be created from a pre-N-degron through specific N-terminal
modifications (12).
Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing
residues that function through their deamidation by N-terminal amidohydrolases
into the secondary destabilizing N-terminal residues Asp and Glu, respectively
(6,
16)
(Fig. 1A). N-terminal
Asp and Glu are secondary destabilizing residues that function through their
arginylation by ATE1 R-transferase, which creates the primary
destabilizing residue Arg at the N terminus
(4,
8)
(Fig. 1A). N-terminal
Cys can also function as a tertiary destabilizing residue through its
oxidation in a manner depending on nitric oxide and oxygen (O2);
the oxidized Cys residue is subsequently arginylated by ATE1
(8,
13,
17).Open in a separate windowFIGURE 1.A, the mammalian N-end rule pathway. N-terminal residues are
indicated by single-letter abbreviations for amino acids. Yellow
ovals denote the rest of a protein substrate. C*
denotes oxidized N-terminal Cys, either Cys-sulfinic acid
[CysO2(H)] or Cys-sulfonic acid [CysO3(H)]. The Cys
oxidation requires nitric oxide and oxygen (O2) or its derivatives.
The oxidized Cys is arginylated by ATE1 Arg-tRNA-protein transferase
(R-transferase). N-recognins also recognize internal (non-N-terminal)
degrons in other substrates of the N-end rule pathway. B, the
X-peptide pull-down assay. Left, a 12-mer peptide bearing N-terminal
Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residue
was cross-linked through its C-terminal Cys residue to Ultralink Iodoacetyl
beads. Right, the otherwise identical 12-mer peptide, bearing
C-terminal biotinylated Lys instead of Cys, was conjugated, via biotin, to the
streptavidin-Sepharose beads. C, the X-peptide pull-down assay of
endogenous UBR proteins using testes extracts. Extracts from mouse testes were
mixed with bead-conjugated X-peptides bearing N-terminal Phe (F), Gly
(G), or Arg (R). After centrifugation, captured proteins
were separated and subjected to anti-UBR immunoblotting. Mo, a
pull-down reaction with mock beads. D, the X-peptide pull-down assays
using rat testis extracts were performed in the presence of varying
concentrations of NaCl. After incubation and washing, bound proteins were
eluted by 10 mm Tyr-Ala for Phe-peptide, 10 mm Arg-Ala
for Arg-peptide, and 5 mm Tyr-Ala and 5 mm Arg-Ala for
Val-peptide. Eluted proteins were subjected to immunoblotting for UBR1 and
UBR5. E, cytoplasmic fractions of wild-type (+/+),
Ubr1-/-, Ubr2-/-,
Ubr1-/-Ubr2-/-, and
Ubr1-/-Ubr2-/-Ubr4RNAi
MEFs were subjected to X-peptide pull-down assay. Precipitated proteins were
separated and analyzed by immunoblotting for UBR1 and UBR4.N-terminal Arg together with other primary destabilizing N-terminal
residues are directly bound by specific E3 Ub ligases called N-recognins
(3,
7,
9). Destabilizing N-terminal
residues can be created through the removal of N-terminal Met or the
endoproteolytic cleavage of a protein, which exposes a new amino acid at the N
terminus (12,
13). N-terminal degradation
signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2
(bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues
(2,
12). In addition to a
destabilizing N-terminal residue, a functional N-degron requires at least one
internal Lys residue (the site of a poly-Ub chain formation) and a
conformational feature required for optimal ubiquitylation
(1,
2,
18). UBR1 and UBR2 are
functionally overlapping N-recognins
(3,
7,
9). Our proteomic approach
using synthetic peptides bearing destabilizing N-terminal residues captured a
set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa
UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR
box
(10-12).
UBR5 is a HECT E3 ligase known as EDD (E3 identified by
differential display)
(19) and a homolog of
Drosophila hyperplastic discs
(20). The mammalian genome
encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7
(10). UBR box proteins are
generally heterogeneous in size and sequence but contain, with the exception
of UBR4, specific signatures unique to E3s or a substrate recognition subunit
of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in
UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7
(Fig. 2B). The
biochemical properties of more recently identified UBR box proteins, such as
UBR3 through UBR7, are largely unknown.Open in a separate windowFIGURE 2.The binding properties of the UBR box family members to type-1 and
type-2 destabilizing N-terminal residues. A, the X-peptide
pull-down assay with overexpressed, full-length UBR proteins: UBR2, UBR3 (in
S. cerevisiae cells), UBR4, UBR5 (in COS7 cells), and UBR6 and UBR7
(in the wheat germ lysates). Precipitates were analyzed by immunoblotting (for
UBR2, UBR3, UBR4, and UBR5) with tag-specific antibodies as indicated in
B or autoradiography (for UBR6 and UBR7). B, the structures
of UBR box proteins. Shown are locations of the UBR box, the N-domain, and
other E3-related domains. UBR, UBR box; RING, RING finger;
UAIN, UBR-specific autoinhibitory domain; CRD, cysteine-rich
domain; PHD, plant homeodomain; HECT, HECT domain.Studies using knock-out mice implicated the N-end rule pathway in cardiac
development and signaling, angiogenesis
(8,
15), meiosis
(9), DNA repair
(21), neurogenesis
(15), pancreatic functions
(22), learning and memory
(23,
24), female development
(9), muscle atrophy
(25), and olfaction
(11). Mutations in human
UBR1 is a cause of Johanson-Blizzard syndrome
(22), an autosomal recessive
disorder with multiple developmental abnormalities
(26). Other functions of the
pathway include: (i) a nitric oxide and oxygen (O2) sensor
controlling the proteolysis of RGS4, RGS5, and RGS16
(8,
13,
17), (ii) a heme sensor
through hemin-dependent inhibition of ATE1 function
(27), (iii) the regulation of
short peptide import through the peptide-modulated degradation of the
repressor of the import (28,
29), (iv) the control of
chromosome segregation through the degradation of a separate produced cohesin
fragment (30), (v) the
regulation of apoptosis through the degradation of a caspase-processed
inhibitor of apoptosis (31,
32), (vi) the control of the
human immunodeficiency virus replication cycle through the degradation of
human immunodeficiency virus integrase
(10,
33), and (vii) the regulation
of leaf senescence in plants
(34).In the present study we characterized substrate binding specificities and
recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4,
and UBR5 were captured by N-terminal degradation determinants, whereas UBR3,
UBR6, and UBR7 were not. We also report that in contrast to other E3 systems
that usually recognize substrates through protein-protein interface, UBR1 and
UBR2 have a general substrate recognition domain termed the UBR box.
Remarkably, a 72-residue UBR box-only fragment fully retains its structural
integrity and thereby the ability to recognize type-1 N-end rule substrates.
We also report that the N-domain, structurally and functionally related with
bacterial N-recognins, is required for recognizing type-2 N-end rule
substrates. We discuss the evolutionary relationship between eukaryotic and
prokaryotic N-recognins. 相似文献
18.
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
19.
Eun-Yeong Bergsdorf Anselm A. Zdebik Thomas J. Jentsch 《The Journal of biological chemistry》2009,284(17):11184-11193
Members of the CLC gene family either function as chloride channels or as
anion/proton exchangers. The plant AtClC-a uses the pH gradient across the
vacuolar membrane to accumulate the nutrient
in this organelle. When AtClC-a was
expressed in Xenopus oocytes, it mediated
exchange
and less efficiently mediated Cl–/H+ exchange.
Mutating the “gating glutamate” Glu-203 to alanine resulted in an
uncoupled anion conductance that was larger for Cl– than
. Replacing the “proton
glutamate” Glu-270 by alanine abolished currents. These could be
restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4
and ClC-5 mediate stoichiometrically coupled
2Cl–/H+ exchange, their
transport is largely uncoupled from
protons. By contrast, the AtClC-a-mediated
accumulation in plant vacuoles
requires tight
coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in
AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this
proline was mutated to serine (P160S), Cl–/H+
exchange of AtClC-a proceeded as efficiently as
exchange, suggesting a role of this residue in
exchange. Indeed, when the corresponding serine of ClC-5 was replaced by
proline, this Cl–/H+ exchanger gained efficient
coupling. When inserted into the model Torpedo chloride channel
ClC-0, the equivalent mutation increased nitrate relative to chloride
conductance. Hence, proline in the CLC pore signature sequence is important
for
exchange and conductance both in
plants and mammals. Gating and proton glutamates play similar roles in
bacterial, plant, and mammalian CLC anion/proton exchangers.CLC proteins are found in all phyla from bacteria to humans and either
mediate electrogenic anion/proton exchange or function as chloride channels
(1). In mammals, the roles of
plasma membrane CLC Cl– channels include transepithelial
transport
(2–5)
and control of muscle excitability
(6), whereas vesicular CLC
exchangers may facilitate endocytosis
(7) and lysosomal function
(8–10)
by electrically shunting vesicular proton pump currents
(11). In the plant
Arabidopsis thaliana, there are seven CLC isoforms
(AtClC-a–AtClC-g)2
(12–15),
which may mostly reside in intracellular membranes. AtClC-a uses the pH
gradient across the vacuolar membrane to transport the nutrient nitrate into
that organelle (16). This
secondary active transport requires a tightly coupled
exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1
(one of the two CLC isoforms in Escherichia coli) display tightly
coupled Cl–/H+ exchange, but anion flux is largely
uncoupled from H+ when
is transported
(17–21).
The lack of appropriate expression systems for plant CLC transporters
(12) has so far impeded
structure-function analysis that may shed light on the ability of AtClC-a to
perform efficient
exchange. This dearth of data contrasts with the extensive mutagenesis work
performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues
(22,
23) and the investigation of
mutants (17,
19–21,
24–29)
have yielded important insights into their structure and function. CLC
proteins form dimers with two largely independent permeation pathways
(22,
25,
30,
31). Each of the monomers
displays two anion binding sites
(22). A third binding site is
observed when a certain key glutamate residue, which is located halfway in the
permeation pathway of almost all CLC proteins, is mutated to alanine
(23). Mutating this gating
glutamate in CLC Cl– channels strongly affects or even
completely suppresses single pore gating
(23), whereas CLC exchangers
are transformed by such mutations into pure anion conductances that are not
coupled to proton transport
(17,
19,
20). Another key glutamate,
located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark
of CLC anion/proton exchangers. Mutating this proton glutamate to
nontitratable amino acids uncouples anion transport from protons in the
bacterial EcClC-1 protein (27)
but seems to abolish transport altogether in mammalian ClC-4 and -5
(21). In those latter
proteins, anion transport could be restored by additionally introducing an
uncoupling mutation at the gating glutamate
(21).The functional complementation by AtClC-c and -d
(12,
32) of growth phenotypes of a
yeast strain deleted for the single yeast CLC Gef1
(33) suggested that these
plant CLC proteins function in anion transport but could not reveal details of
their biophysical properties. We report here the first functional expression
of a plant CLC in animal cells. Expression of wild-type (WT) and mutant
AtClC-a in Xenopus oocytes indicate a general role of gating and
proton glutamate residues in anion/proton coupling across different isoforms
and species. We identified a proline in the CLC signature sequence of AtClC-a
that plays a crucial role in
exchange. Mutating it to serine, the residue present in mammalian CLC proteins
at this position, rendered AtClC-a Cl–/H+ exchange
as efficient as
exchange. Conversely, changing the corresponding serine of ClC-5 to proline
converted it into an efficient
exchanger. When proline replaced the critical serine in Torpedo
ClC-0, the relative conductance of
this model Cl– channel was drastically increased, and
“fast” protopore gating was slowed. 相似文献
20.
Maika Deffieu Ingrid Bhatia-Ki??ová Bénédicte Salin Anne Galinier Stéphen Manon Nadine Camougrand 《The Journal of biological chemistry》2009,284(22):14828-14837
The antioxidant N-acetyl-l-cysteine prevented the
autophagy-dependent delivery of mitochondria to the vacuoles, as examined by
fluorescence microscopy of mitochondria-targeted green fluorescent protein,
transmission electron microscopy, and Western blot analysis of mitochondrial
proteins. The effect of N-acetyl-l-cysteine was specific
to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation
of alkaline phosphatase and the presence of hallmarks of non-selective
microautophagy were not altered by N-acetyl-l-cysteine.
The effect of N-acetyl-l-cysteine was not related to its
scavenging properties, but rather to its fueling effect of the glutathione
pool. As a matter of fact, the decrease of the glutathione pool induced by
chemical or genetical manipulation did stimulate mitophagy but not general
autophagy. Conversely, the addition of a cell-permeable form of glutathione
inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the
strain Δuth1, which is deficient in selective mitochondrial
degradation. These data show that mitophagy can be regulated independently of
general autophagy, and that its implementation may depend on the cellular
redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of
long-lived proteins and organelles, where they are degraded and recycled.
Autophagy plays a crucial role in differentiation and cellular response to
stress and is conserved in eukaryotic cells from yeast to mammals
(1,
2). The main form of autophagy,
macroautophagy, involves the non-selective sequestration of large portions of
the cytoplasm into double-membrane structures termed autophagosomes, and their
delivery to the vacuole/lysosome for degradation. Another process,
microautophagy, involves the direct sequestration of parts of the cytoplasm by
vacuole/lysosomes. The two processes coexist in yeast cells but their extent
may depend on different factors including metabolic state: for example, we
have observed that nitrogen-starved lactate-grown yeast cells develop
microautophagy, whereas nitrogen-starved glucose-grown cells preferentially
develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in
the way that autophagosomes and vacuole invaginations do not appear to
discriminate the sequestered material. However, selective forms of autophagy
have been observed (4) that
target namely peroxisomes (5,
6), chromatin
(7,
8), endoplasmic reticulum
(9), ribosomes
(10), and mitochondria
(3,
11–13).
Although non-selective autophagy plays an essential role in survival by
nitrogen starvation, by providing amino acids to the cell, selective autophagy
is more likely to have a function in the maintenance of cellular structures,
both under normal conditions as a “housecleaning” process, and
under stress conditions by eliminating altered organelles and macromolecular
structures
(14–16).
Selective autophagy targeting mitochondria, termed mitophagy, may be
particularly relevant to stress conditions. The mitochondrial respiratory
chain is both the main site and target of
ROS4 production
(17). Consequently, the
maintenance of a pool of healthy mitochondria is a crucial challenge for the
cells. The progressive accumulation of altered mitochondria
(18) caused by the loss of
efficiency of the maintenance process (degradation/biogenesis de
novo) is often considered as a major cause of cellular aging
(19–23).
In mammalian cells, autophagic removal of mitochondria has been shown to be
triggered following induction/blockade of apoptosis
(23), suggesting that
autophagy of mitochondria was required for cell survival following
mitochondria injury (14).
Consistent with this idea, a direct alteration of mitochondrial permeability
properties has been shown to induce mitochondrial autophagy
(13,
24,
25). Furthermore, inactivation
of catalase induced the autophagic elimination of altered mitochondria
(26). In the yeast
Saccharomyces cerevisiae, the alteration of
F0F1-ATPase biogenesis in a conditional mutant has been
shown to trigger autophagy
(27). Alterations of
mitochondrial ion homeostasis caused by the inactivation of the
K+/H+ exchanger was shown to cause both autophagy and
mitophagy (28). We have
reported that treatment of cells with rapamycin induced early ROS production
and mitochondrial lipid oxidation that could be inhibited by the hydrophobic
antioxidant resveratrol (29).
Furthermore, resveratrol treatment impaired autophagic degradation of both
cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death,
suggesting that mitochondrial oxidation events may play a crucial role in the
regulation of autophagy. This existence of regulation of autophagy by ROS has
received molecular support in HeLa cells
(30): these authors showed
that starvation stimulated ROS production, namely H2O2,
which was essential for autophagy. Furthermore, they identified the cysteine
protease hsAtg4 as a direct target for oxidation by
H2O2. This provided a possible connection between the
mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown
yeast cells have established the existence of two distinct processes: the
first one occurring very early, is selective for mitochondria and is dependent
on the presence of the mitochondrial protein Uth1p; the second one occurring
later, is not selective for mitochondria, is not dependent on Uth1p, and is a
form of bulk microautophagy
(3). The absence of the
selective process in the Δuth1 mutant strongly delays and
decreases mitochondrial protein degradation
(3,
12). The putative protein
phosphatase Aup1p has been also shown to be essential in inducing mitophagy
(31). Additionally several Atg
proteins were shown to be involved in vacuolar sequestration of mitochondrial
GFP (3,
12,
32,
33). Recently, the protein
Atg11p, which had been already identified as an essential protein for
selective autophagy has also been reported as being essential for mitophagy
(33).The question remains as to identify of the signals that trigger selective
mitophagy. It is particularly intriguing that selective mitophagy is activated
very early after the shift to a nitrogen-deprived medium
(3). Furthermore, selective
mitophagy is very active on lactate-grown cells (with fully differentiated
mitochondria) but is nearly absent in glucose-grown cells
(3). In the present paper, we
investigated the relationships between the redox status of the cells and
selective mitophagy, namely by manipulating glutathione. Our results support
the view that redox imbalance is a trigger for the selective elimination of
mitochondria. 相似文献