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1.
Nodar Makharashvili Tian Mi Olga Koroleva Sergey Korolev 《The Journal of biological chemistry》2009,284(3):1425-1434
RecF pathway proteins play an important role in the restart of stalled
replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR,
and RecO initiate homologous recombination (HR) by loading of the RecA
recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein.
The specific role of RecF in this process is not well understood. Previous
studies have proposed that RecF directs the RecOR complex to boundaries of
damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA
junctions. RecF belongs to ABC-type ATPases, which function through an
ATP-dependent dimerization. Here, we demonstrate that the RecF of
Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer,
and that the DNA binding and ATPase activity of RecF depend on both the
structure of DNA substrate, and the presence of RecR. We found that RecR
interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity
to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to
ssDNA and dimerization, likely due to increasing the ATPase rate. The
DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific
protein-protein interactions without significant contributions from RecR-DNA
interactions. Finally, RecF neither alone nor in complex with RecR
preferentially binds to the ss/dsDNA junction. Our data suggest that the
specificity of the RecFOR complex toward the boundaries of DNA damaged regions
may result from a network of protein-protein and DNA-protein interactions,
rather than a simple recognition of the ss/dsDNA junction by RecF.Homologous recombination
(HR)2 is one of the
primary mechanisms by which cells repair dsDNA breaks (DSBs) and ssDNA gaps
(SSGs), and is important for restart of stalled DNA replication
(1). HR is initiated when
RecA-like recombinases bind to ssDNA forming an extended nucleoprotein
filament, referred to as a presynaptic complex
(2). The potential for genetic
rearrangements dictates that HR initiation is tightly regulated at multiple
levels (1). During replication,
the ssDNA-binding protein (SSB) protects transiently unwound DNA chains,
preventing interactions with recombinases. Following DNA damage, recombination
mediator proteins (RMPs) initiate HR by facilitating the formation of the
recombinase filaments with ssDNA, while removing SSB
(3,
4). Mutations in human proteins
involved in HR initiation are linked to cancer predisposition, chromosome
instability, UV sensitivity, and premature aging diseases
(4–8).
To date, little is known about the mechanism by which RMPs regulate the
formation of the recombinase filaments on the SSB-protected ssDNA.In Escherichia coli, there are two major recombination pathways,
RecBCD and RecF (9,
10). A helicase/nuclease
RecBCD complex processes DSBs and recruits RecA on ssDNA in a
sequence-specific manner
(11–13).
The principle players in the RecF pathway are the RecF, RecO, and RecR
proteins, which form an epistatic group that is important for SSG repair, for
restart of stalled DNA replication, and under specific conditions, can also
process DSBs
(14–20).
Homologs of RecF, -O, and -R are present in the majority of known bacteria
(21), including
Deinococcus radiodurans, extremely radiation-resistant bacteria that
lacks the RecBCD pathway, yet is capable of repairing thousands of DSBs
(22,
23). In addition, the sequence
or functional homologs of RecF pathway proteins are involved in similar
pathways in eukaryotes that include among others WRN, BLM, RAD52, and BRCA2
proteins
(4–8).The involvement of all three RecF, -O, and -R proteins in HR initiation is
well documented by genetic and cellular approaches
(18,
24–30),
yet their biochemical functions in the initiation process remain unclear,
particularly with respect to RecF. RecO and RecR proteins are sufficient to
promote formation of the RecA filament on SSB-bound ssDNA in vitro
(27). The UV-sensitive
phenotype of recF mutants can be suppressed by RecOR overexpression,
suggesting that RecF may direct the RMP complex to DNA-damaged regions where
HR initiation is required
(31). In agreement with this
hypothesis, RecF dramatically increases the efficiency of the RecA loading at
ds/ssDNA junctions with a 3′ ssDNA extension under specific conditions
(32). RecF and RecR proteins
also prevent the RecA filaments from extending into dsDNA regions adjacent to
SSGs (33). These data suggest
that RecF may directly recognize an ss/dsDNA junction structure
(34). However, DNA binding
experiments have not provided clear evidence to support such a hypothesis
(11).The targeting promoted by RecF may also occur through more complex
processes. RecF shares a high structural similarity with the head domain of
Rad50, an ABC-type ATPase that recognizes DSBs and initiates repair in archaea
and eukaryotes (35). All known
ABC-type ATPases function as oligomeric complexes in which a sequence of
inter- and intra-molecular interactions is triggered by the ATP-dependent
dimerization and the dimer-dependent ATP hydrolysis
(36–39).
RecF is also an ATP-dependent DNA-binding protein and a weak DNA-dependent
ATPase (11,
40). RecF forms an
ATP-dependent dimer and all three conserved motifs (Walker A, Walker B, and
“signature”) of RecF are important for ATP-dependent dimerization,
ATP hydrolysis, and functional resistance to DNA damage
(35). Thus, RecF may function
in recombination initiation through a complex pathway of protein-protein and
DNA-protein interactions regulated by ATP-dependent RecF dimerization.In this report, we present a detailed characterization of the RecF
dimerization, and its role in the RecF interaction with various DNA
substrates, with RecR, and in ATP hydrolysis. Our data outline the following
key findings. First, RecF interacts with DNA as a dimer. Second, neither RecF
alone nor the RecFR complex preferentially binds the ss/dsDNA junction.
Finally, RecR changes the ATPase activity and the DNA binding of RecF by
destabilizing the interaction with ssDNA, and greatly enhancing the
interaction with dsDNA. Our results suggest that the specificity of RecF for
the boundaries of SSGs is likely to result from a sequence of protein-protein
interaction events rather than a simple RecF ss/dsDNA binding, underlining a
highly regulated mechanism of the HR initiation by the RecFOR proteins. 相似文献
2.
3.
Martin J. Sergeant Jian-Jun Li Christine Fox Nicola Brookbank Dean Rea Timothy D. H. Bugg Andrew J. Thompson 《The Journal of biological chemistry》2009,284(8):5257-5264
Members of the carotenoid cleavage dioxygenase family catalyze the
oxidative cleavage of carotenoids at various chain positions, leading to the
formation of a wide range of apocarotenoid signaling molecules. To explore the
functions of this diverse enzyme family, we have used a chemical genetic
approach to design selective inhibitors for different classes of carotenoid
cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized
in which the distance between an iron-chelating hydroxamic acid and an
aromatic ring was varied; these compounds were screened as inhibitors of four
different enzyme classes, either in vitro or in vivo. Potent
inhibitors were found that selectively inhibited enzymes that cleave
carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar
concentrations. Application of certain inhibitors at 100 μm to
Arabidopsis node explants or whole plants led to increased shoot
branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as
photoprotective molecules and are key components in animal diets, an example
being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids
occurs in plants, animals, and micro-organisms and leads to the release of a
range of apocarotenoids that function as signaling molecules with a diverse
range of functions (1). The
first gene identified as encoding a carotenoid cleavage dioxygenase
(CCD)2 was the maize
Vp14 gene that is required for the formation of abscisic acid (ABA),
an important hormone that mediates responses to drought stress and aspects of
plant development such as seed and bud dormancy
(2). The VP14 enzyme cleaves at
the 11,12 position (Fig. 1) of
the epoxycarotenoids 9′-cis-neoxanthin and/or
9-cis-violaxanthin and is now classified as a
9-cis-epoxycarotenoid dioxygenase (NCED)
(3), a subclass of the larger
CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases.
a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by
NCED; b, oxidative cleavage reactions on β-carotene and
zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be
involved in the production of a variety of apocarotenoids
(Fig. 1). In insects, the
visual pigment retinal is formed by oxidative cleavage of β-carotene by
β-carotene-15,15′-dioxygenase
(4). Retinal is produced by an
orthologous enzyme in vertebrates, where it is also converted to retinoic
acid, a regulator of differentiation during embryogenesis
(5). A distinct mammalian CCD
is believed to cleave carotenoids asymmetrically at the 9,10 position
(6) and, although its function
is unclear, recent evidence suggests a role in the metabolism of dietary
lycopene (7). The plant
volatiles β-ionone and geranylacetone are produced from an enzyme that
cleaves at the 9,10 position
(8) and the pigment
α-crocin found in the spice saffron results from an 7,8-cleavage enzyme
(9).Other CCDs have been identified where biological function is unknown, for
example, in cyanobacteria where a variety of cleavage specificities have been
described
(10-12).
In other cases, there are apocarotenoids with known functions, but the
identity or involvement of CCDs have not yet been described: grasshopper
ketone is a defensive secretion of the flightless grasshopper Romalea
microptera (13),
mycorradicin is produced by plant roots during symbiosis with arbuscular
mycorrhyza (14), and
strigolactones (15) are plant
metabolites that act as germination signals to parasitic weeds such as
Striga and Orobanche
(16).Recently it was discovered that strigolactones also function as a branching
hormone in plants (17,
18). The existence of such a
branching hormone has been known for some time, but its identity proved
elusive. However, it was known that the hormone was derived from the action of
at least two CCDs, max3 and max4 (more axillary growth)
(19), because deletion of
either of these genes in Arabidopsis thaliana, leads to a bushy
phenotype (20,
21). In Escherichia
coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position
and the apocarotenoid product (10-apo-β-carotene) is reported to be
further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene
(22). Also recent evidence
suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene
(23). How the production of
13-apo-β-carotene leads to the synthesis of the complex strigolactone is
unknown. The possibility remains that the enzymes may have different
specificities and cleavage activities in planta. In addition, a
cytochrome P450 enzyme (24) is
believed to be involved in strigolactone synthesis and acts in the pathway
downstream of the CCD genes. Strigolactone is thought to effect branching by
regulating auxin transport
(25). Because of the
involvement of CCDs in strigolactone synthesis, the possibility arises that
plant architecture and interaction with parasitic weeds and mycorrhyza could
be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to
probe function and substrate specificity of CCDs in their native biological
contexts, particularly in plant species with simple genetic systems or that
are amenable to transgenesis, there are many systems where genetic approaches
are difficult or impossible. Also, when recombinant CCDs are studied either
in vitro or in heterologous in vivo assays, such as in
E. coli strains engineered to accumulate carotenoids
(26), they are often active
against a broad range of substrates
(5,
21,
27), and in many cases the
true in vivo substrate of a particular CCD remains unknown. Therefore
additional experimental tools are needed to investigate both apocarotenoid and
CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified
that are active against known target proteins; they are then applied to a
biological system to investigate protein function in vivo
(28,
29). This approach is
complementary to conventional genetics since the small molecules can be
applied easily to a broad range of species, their application can be
controlled in dose, time, and space to provide detailed studies of biological
functions, and individual proteins or whole protein classes may be targeted by
varying the specificity of the small molecules. Notably, functions of the
plant hormones gibberellin, brassinosteroid, and abscisic acid have been
successfully probed using this approach by adapting triazoles to inhibit
specific cytochrome P450 monooxygenases involved in the metabolism of these
hormones (30).In the case of the CCD family, the tertiary amines abamine
(31) and the more active
abamineSG (32) were reported
as specific inhibitors of NCED, and abamine was used to show new functions of
abscisic acid in legume nodulation
(33). However, no selective
inhibitors for other types of CCD are known. Here we have designed a novel
class of CCD inhibitor based on hydroxamic acids, where variable chain length
was used to direct inhibition of CCD enzymes that cleave carotenoids at
specific positions. We demonstrate the use of such novel inhibitors to control
shoot branching in a model plant. 相似文献
4.
5.
Susan R. Ferrari Jennifer Grubb Douglas K. Bishop 《The Journal of biological chemistry》2009,284(18):11766-11770
During homologous recombination, a number of proteins cooperate to catalyze
the loading of recombinases onto single-stranded DNA. Single-stranded
DNA-binding proteins stimulate recombination by coating single-stranded DNA
and keeping it free of secondary structure; however, in order for recombinases
to load on single-stranded-DNA-binding protein-coated DNA, the activity of a
class of proteins known as recombination mediators is required. Mediator
proteins coordinate the handoff of single-stranded DNA from single-stranded
DNA-binding protein to recombinase. Here we show that a complex of Mei5 and
Sae3 from Saccharomyces cerevisiae preferentially binds
single-stranded DNA and relieves the inhibition of the strand assimilation and
DNA binding abilities of the meiotic recombinase Dmc1 imposed by the
single-stranded DNA-binding protein replication protein A. Additionally, we
demonstrate the physical interaction of Mei5-Sae3 with replication protein A.
Our results, together with previous in vivo studies, indicate that
Mei5-Sae3 is a mediator of Dmc1 assembly during meiotic recombination in
S. cerevisiae.During meiosis, recombination between homologous chromosomes ensures proper
segregation into haploid products. Recombination events are initiated by the
formation of double strand breaks
(DSBs)2 in DNA
(1). This is followed by
resection of free DNA ends to yield 3′ single-stranded tails, upon which
recombinase assembles to form nucleoprotein filaments. Following recombinase
assembly, the nucleoprotein filament engages a donor chromatid, searches for
homologous DNA sequences on that chromatid, and promotes strand exchange to
yield a heteroduplex DNA intermediate often referred to as a joint molecule.
Although recombinase alone is capable of promoting homology search and strand
exchange in vitro, genetic and biochemical studies have demonstrated
that normal recombinase function in vivo requires the activity of a
number of accessory factors
(2). These factors enhance the
assembly of nucleoprotein filaments, target capture, homology search, and
dissociation of recombinase from duplex DNA.Most eukaryotes possess two recombinases, both homologues of the
Escherichia coli recombinase RecA: Rad51, which is the major
recombinase in mitotic cells and is also important during meiotic
recombination, and Dmc1, which functions only in meiosis. Dmc1 and Rad51 have
been shown to assemble at DSBs by immunofluorescence and chromatin
immunoprecipitation
(3–6),
and both proteins oligomerize on single-stranded DNA (ssDNA) to form
nucleofilaments that catalyze strand invasion
(7–9).A number of biochemical studies have defined the role of accessory factors
in stimulating the activity of Rad51
(10–12).
Replication protein A (RPA), the yeast ssDNA-binding protein (SSB), removes
secondary structure in ssDNA that otherwise prevents formation of fully
functional nucleoprotein filaments
(13). Both Rad52 protein
(11,
12) and the heterodimeric
protein Rad55/Rad57 (14) can
overcome the inhibitory effect of RPA on Rad51 nucleoprotein filament
formation in purified systems, mediating a handoff between RPA and Rad51. It
is thought that the mechanism for the mediator activity of Rad52 involves
Rad52 recognizing and binding to RPA-coated ssDNA, where it provides
nucleation sites for the recruitment of free molecules of Rad51
(15). The tumor suppressor
protein BRCA2 also serves as an assembly factor for Rad51 during mitosis in a
variety of species that encode orthologues of this protein, including mice
(16), corn smut
(17), and humans
(18).The meiosis-specific recombinase Dmc1 is stimulated by a distinct set of
accessory factors. Immunostaining studies suggest that the Rad51 mediators
Rad52 and Rad55/Rad57 are not required for assembly of Dmc1 foci in
vivo, although Rad51 itself promotes Dmc1 foci
(19–21).
More recently, immunostaining and chromatin immunoprecipitation experiments
demonstrated a role for the Mei5 and Sae3 proteins of Saccharomyces
cerevisiae in assembly of Dmc1 at sites of DSBs in vivo
(22,
23). Consistent with these
observations, mei5 and sae3 mutants display markedly similar
meiotic defects as compared with dmc1 mutants, including defects in
sporulation, spore viability, crossing over, DSB repair, progression through
meiosis, and synaptonemal complex formation
(19,
22–24).
Finally, the three proteins have been shown to physically interact; Mei5 and
Sae3 have been co-purified and co-immunoprecipitated, and an N-terminal
portion of Mei5 has been shown to interact with Dmc1 in a two-hybrid assay
(22).The fission yeast Schizosaccharomyces pombe encodes two proteins,
Swi5 and Sfr1, which share sequence homology with Sae3 and Mei5, respectively
(22). Swi5 and Sfr1 have been
shown to stimulate the strand exchange activity of Rhp51 (the S.
pombe Rad51 homologue) and Dmc1
(25). Although some results
indicate functional similarity of Swi5-Sfr1 and Mei5-Sae3, there are also
clear differences. The Mei5-Sae3 complex of budding yeast is expressed solely
during meiosis, and no mitotic phenotypes have been reported for mei5
or sae3 mutants (22,
24,
26). In contrast, the
Swi5-Sfr1 complex of fission yeast is expressed in mitotic and meiotic cells,
and mutations in SWI5 have been shown to cause defects in mitotic
recombination (27).
Furthermore, although mei5 and sae3 mutants are
phenotypically similar to dmc1 mutants, swi5 and
sfr1 mutants display more severe meiotic defects during fission yeast
meiosis than do dmc1 mutants
(27–29).
These data suggest that although Swi5-Sfr1 clearly contributes to Rad51
activity in fission yeast, it is possible that the activity of Mei5-Sae3 is
restricted to stimulating Dmc1 in budding yeast.In this study, a biochemical approach is used to test the budding yeast
Mei5-Sae3 complex for properties expected of a recombinase assembly mediator.
We show that Mei5-Sae3 binds both ssDNA and double-stranded DNA (dsDNA) but
binds ssDNA preferentially. We also show that Mei5-Sae3 can overcome the
inhibitory effects of RPA on the ssDNA binding and strand assimilation
activities of Dmc1. Finally, we show that Mei5-Sae3 and RPA bind one another
directly. These results indicate that Mei5-Sae3 acts directly as a mediator
protein for assembly of Dmc1. 相似文献
6.
7.
8.
Denise A. Berti Cain Morano Lilian C. Russo Leandro M. Castro Fernanda M. Cunha Xin Zhang Juan Sironi Cl��cio F. Klitzke Emer S. Ferro Lloyd D. Fricker 《The Journal of biological chemistry》2009,284(21):14105-14116
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme
that has been proposed to metabolize peptides within cells, thereby affecting
antigen presentation and G protein-coupled receptor signal transduction.
However, only a small number of intracellular substrates of EP24.15 have been
reported previously. Here we have identified over 100 peptides in human
embryonic kidney 293 (HEK293) cells that are derived from intracellular
proteins; many but not all of these peptides are substrates or products of
EP24.15. First, cellular peptides were extracted from HEK293 cells and
incubated in vitro with purified EP24.15. Then the peptides were
labeled with isotopic tags and analyzed by mass spectrometry to obtain
quantitative data on the extent of cleavage. A related series of experiments
tested the effect of overexpression of EP24.15 on the cellular levels of
peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10
of the cellular peptides were incubated with purified EP24.15 in
vitro, and the cleavage was monitored by high pressure liquid
chromatography and mass spectrometry. Many of the EP24.15 substrates
identified by these approaches are 9–11 amino acids in length,
supporting the proposal that EP24.15 can function in the degradation of
peptides that could be used for antigen presentation. However, EP24.15 also
converts some peptides into products that are 8–10 amino acids, thus
contributing to the formation of peptides for antigen presentation. In
addition, the intracellular peptides described here are potential candidates
to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and
if this process is impaired, the elevated levels of aged proteins usually lead
to the formation of intracellular insoluble aggregates that can cause severe
pathologies (1). In mammalian
cells, most proteins destined for degradation are initially tagged with a
polyubiquitin chain in an energy-dependent process and then digested to small
peptides by the 26 S proteasome, a large proteolytic complex involved in the
regulation of cell division, gene expression, and other key processes
(2,
3). In eukaryotes, 30–90%
of newly synthesized proteins may be degraded by proteasomes within minutes of
synthesis (3,
4). In addition to proteasomes,
other extralysosomal proteolytic systems have been reported
(5,
6). The proteasome cleaves
proteins into peptides that are typically 2–20 amino acids in length
(7). In most cases, these
peptides are thought to be rapidly hydrolyzed into amino acids by
aminopeptidases
(8–10).
However, some intracellular peptides escape complete degradation and are
imported into the endoplasmic reticulum where they associate with major
histocompatibility complex class I
(MHC-I)3 molecules and
traffic to the cell surface for presentation to the immune system
(10–12).
Additionally, based on the fact that free peptides added to the intracellular
milieu can regulate cellular functions mediated by protein interactions such
as gene regulation, metabolism, cell signaling, and protein targeting
(13,
14), intracellular peptides
generated by proteasomes that escape degradation have been suggested to play a
role in regulating protein interactions
(15). Indeed, oligopeptides
isolated from rat brain tissue using the catalytically inactive EP24.15 (EC
3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and
were found capable of altering G protein-coupled receptor signal transduction
(16). Moreover, EP24.15
overexpression itself changed both angiotensin II and isoproterenol signal
transduction, suggesting a physiological function for its intracellular
substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family
that contains the HEXXH motif
(17). This enzyme was first
described as a neuropeptide-degrading enzyme present in the soluble fraction
of brain homogenates (18).
Whereas EP24.15 can be secreted
(19,
20), its predominant location
in the cytosol and nucleus suggests that the primary function of this enzyme
is not the extracellular degradation of neuropeptides and hormones
(21,
22). EP24.15 was shown in
vivo to participate in antigen presentation through MHC-I
(23–25)
and in vitro to bind
(26) or degrade
(27) some MHC-I associated
peptides. EP24.15 has also been shown in vitro to degrade peptides
containing 5–17 amino acids produced after proteasome digestion of
β-casein (28). EP24.15
shows substrate size restriction to peptides containing from 5 to 17 amino
acids because of its catalytic center that is located in a deep channel
(29). Despite the size
restriction, EP24.15 has a broad substrate specificity
(30), probably because a
significant portion of the enzyme-binding site is lined with potentially
flexible loops that allow reorganization of the active site following
substrate binding (29).
Recently, it has also been suggested that certain substrates may be cleaved by
an open form of EP24.15 (31).
This characteristic is supported by the ability of EP24.15 to accommodate
different amino acid residues at subsites S4 to S3′, which even includes
the uncommon post-proline cleavage
(30). Such biochemical and
structural features make EP24.15 a versatile enzyme to degrade structurally
unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15
were isolated and identified using mass spectrometry
(22). The majority of peptides
captured by the inactive enzyme were intracellular protein fragments that
efficiently interacted with EP24.15; the smallest peptide isolated in these
assays contained 5 and the largest 17 amino acids
(15,
16,
22,
32), which is within the size
range previously reported for natural and synthetic substrates of EP24.15
(18,
30,
33,
34). Interestingly, the
peptides released by the proteasome are in the same size range of EP24.15
competitive inhibitors/substrates
(7,
35,
36). Taken altogether, these
data suggest that in the intracellular environment EP24.15 could further
cleave proteasome-generated peptides unrelated to MHC-I antigen presentation
(15).Although the mutated inactive enzyme “capture” assay was
successful in identifying several cellular protein fragments that were
substrates for EP24.15, it also found some interacting peptides that were not
substrates. In this study, we used several approaches to directly screen for
cellular peptides that were cleaved by EP24.15. The first approach involved
the extraction of cellular peptides from the HEK293 cell line, incubation
in vitro with purified EP24.15, labeling with isotopic tags, and
analysis by mass spectrometry to obtain quantitative data on the extent of
cleavage. The second approach examined the effect of EP24.15 overexpression on
the cellular levels of peptides in the HEK293 cell line. The third set of
experiments tested synthetic peptides with purified EP24.15 in vitro,
and examined cleavage by high pressure liquid chromatography and mass
spectrometry. Collectively, these studies have identified a large number of
intracellular peptides, including those that likely represent the endogenous
substrates and products of EP24.15, and this original information contributes
to a better understanding of the function of this enzyme in vivo. 相似文献
9.
10.
11.
12.
Gaetan Pascreau Frank Eckerdt Andrea L. Lewellyn Claude Prigent James L. Maller 《The Journal of biological chemistry》2009,284(9):5497-5505
p53 is an important tumor suppressor regulating the cell cycle at multiple
stages in higher vertebrates. The p53 gene is frequently deleted or mutated in
human cancers, resulting in loss of p53 activity. This leads to centrosome
amplification, aneuploidy, and tumorigenesis, three phenotypes also observed
after overexpression of the oncogenic kinase Aurora A. Accordingly, recent
studies have focused on the relationship between these two proteins. p53 and
Aurora A have been reported to interact in mammalian cells, but the function
of this interaction remains unclear. We recently reported that
Xenopus p53 can inhibit Aurora A activity in vitro but only
in the absence of TPX2. Here we investigate the interplay between
Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2
is required for nearly all Aurora A activation and for full p53 synthesis and
phosphorylation in vivo during oocyte maturation. In vitro,
phosphorylation mediated by Aurora A targets serines 129 and 190 within the
DNA binding domain of p53. Glutathione S-transferase pull-down
studies indicate that the interaction occurs via the p53 transactivation
domain and the Aurora A catalytic domain around the T-loop. Our studies
suggest that targeting of TPX2 might be an effective strategy for specifically
inhibiting the phosphorylation of Aurora A substrates, including p53.Aurora A is an oncogenic protein kinase that is active in mitosis and plays
important roles in spindle assembly and centrosome function
(1). Overexpression of either
human or Xenopus Aurora A transforms mammalian cells, but only when
the p53 pathway is altered
(2–4).
Aurora A is localized on centrosomes during mitosis, and overexpression of the
protein leads to centrosome amplification and aneuploidy
(2,
3,
5,
6), two likely contributors to
genomic instability (7,
8). Because of its oncogenic
potential and amplification in human tumors, considerable attention has been
focused on the mechanism of Aurora A activation in mitosis. Evidence from
several laboratories indicates that activation occurs as a result of
phosphorylation of a threonine residue in the T-loop of the kinase
(4,
9,
10). Purification of Aurora
A-activating activity from M phase Xenopus egg extracts led to an
apparent activation mechanism in which autophosphorylation at the T-loop is
stimulated by binding of the targeting protein for Xklp2 (TPX2)
(11–14).
On the other hand, it has been shown that Aurora A activity can be inhibited
by interaction with several proteins, including PP1 (protein phosphatase 1),
AIP (Aurora A kinase-interacting protein), and, more recently, p53
(9,
15–17).p53 is a well known tumor suppressor able to drive cell cycle arrest,
apoptosis, or senescence when DNA is damaged or cell integrity is threatened
(18,
19). In human cancers, the p53
gene is frequently deleted or mutated, leading to inactivation of p53
functions (20). p53 protein is
almost undetectable in “normal cells,” mainly due to its
instability. Indeed, during a normal cell cycle, p53 associates with Mdm2 in
the nucleus and thereafter undergoes nuclear exclusion, allowing its
ubiquitination and subsequent degradation
(21). In cells under stress,
p53 is stabilized through the disruption of its interaction with Mdm2
(21), leading to p53
accumulation in the nucleus and triggering different responses, as described
above.Although p53 has mostly been characterized as a nuclear protein, it has
also been shown to localize on centrosomes
(22–24)
and regulate centrosome duplication
(23,
24). Centrosomes are believed
to act as scaffolds that concentrate many regulatory molecules involved in
signal transduction, including multiple protein kinases
(25). Thus, centrosomal
localization of p53 might be important for its own regulation by
phosphorylation/dephosphorylation, and one of its regulators could be the
mitotic kinase Aurora A. Indeed, phenotypes associated with the misexpression
of these two proteins are very similar. For example, overexpression of Aurora
A kinase leads to centrosome amplification, aneuploidy, and tumorigenesis, and
the same effects are often observed after down-regulation of p53
transactivation activity or deletion/mutation of its gene
(26,
27).Several recent studies performed in mammalian models show interplay between
p53 and Aurora A, with each protein having the ability to inhibit the other,
depending on the stage of the cell cycle and the stress level of the cell
(17,
28,
29). These studies reported
that p53 is a substrate of Aurora A, and serines 215 and 315 were demonstrated
to be the two major Aurora A phosphorylation sites in human p53 in
vitro and in vivo. Phosphorylation of Ser-215 within the DNA
binding domain of human p53 inhibited both p53 DNA binding and transactivation
activities (29). Recently, our
group showed that Xenopus p53 is able to inhibit Aurora A kinase
activity in vitro, but this inhibitory effect can be suppressed by
prior binding of Aurora A to TPX2
(9). Contrary to somatic cells,
where p53 is nuclear, unstable, and expressed at a very low level, p53 is
highly expressed in the cytoplasm of Xenopus oocytes and stable until
later stages of development
(30,
31). The high concentration of
both p53 and Aurora A in the oocyte provided a suitable basis for
investigating p53-Aurora A interaction and also evaluating Xenopus
p53 as a substrate of Aurora A. 相似文献
13.
Motoki Takaku Shinichi Machida Noriko Hosoya Shugo Nakayama Yoshimasa Takizawa Isao Sakane Takehiko Shibata Kiyoshi Miyagawa Hitoshi Kurumizaka 《The Journal of biological chemistry》2009,284(21):14326-14336
The RAD51 protein is a central player in homologous recombinational repair.
The RAD51B protein is one of five RAD51 paralogs that function in the
homologous recombinational repair pathway in higher eukaryotes. In the present
study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested
to be involved in actin-remodeling processes, unexpectedly binds to the RAD51
and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and
strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged
DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone
promotes single-stranded DNA annealing, and the recombination activities of
the EVL protein are further enhanced by the RAD51B protein. The expression of
the EVL protein is not ubiquitous, but it is significantly expressed in breast
cancer-derived MCF7 cells. These results suggest that the EVL protein is a
novel recombination factor that may be required for repairing specific DNA
lesions, and that may cause tumor malignancy by its inappropriate
expression.Chromosomal DNA double strand breaks
(DSBs)2 are potential
inducers of chromosomal aberrations and tumorigenesis, and they are accurately
repaired by the homologous recombinational repair (HRR) pathway, without base
substitutions, deletions, and insertions
(1–3).
In the HRR pathway (4,
5), single-stranded DNA (ssDNA)
tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue
of the bacterial RecA protein, binds to the ssDNA tail and forms a helical
nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact
double-stranded DNA (dsDNA) to form a three-component complex, containing
ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the
RAD51 protein promotes recombination reactions, such as homologous pairing and
strand exchange
(6–9).The RAD51 protein requires auxiliary proteins to promote the homologous
pairing and strand exchange reactions efficiently in cells
(10–12).
In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the
RAD51 protein
(13–17)
and stimulate the RAD51-mediated homologous pairing and/or strand exchange
reactions in vitro
(18–21).
The human RAD51AP1 protein, which directly binds to the RAD51 protein
(22), was also found to
stimulate RAD51-mediated homologous pairing in vitro
(23,
24). The BRCA2 protein
contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs
(25–33),
and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated
RAD51-mediated strand exchange
(34,
35). Most of these
RAD51-interacting factors are known to be required for efficient RAD51
assembly onto DSB sites in cells treated with ionizing radiation
(10–12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which
share about 20–30% amino acid sequence similarity with the RAD51 protein
(36–38).
RAD51B-deficient cells are hypersensitive to DSB-inducing agents,
such as cisplatin, mitomycin C (MMC), and γ-rays, indicating that the
RAD51B protein is involved in the HRR pathway
(39–44).
Genetic experiments revealed that RAD51B-deficient cells exhibited
impaired RAD51 assembly onto DSB sites
(39,
44), suggesting that the
RAD51B protein functions in the early stage of the HRR pathway. Biochemical
experiments also suggested that the RAD51B protein participates in the early
to late stages of the HRR pathway
(45–47).In the present study, we found that the human EVL (Ena/Vasp-like) protein
binds to the RAD51 and RAD51B proteins in a HeLa cell extract. The EVL protein
is known to be involved in cytoplasmic actin remodeling
(48) and is also overexpressed
in breast cancer (49). Like
the RAD51B knockdown cells, the EVL knockdown cells partially impaired RAD51
foci formation after DSB induction, suggesting that the EVL protein enhances
RAD51 assembly onto DSB sites. The purified EVL protein preferentially bound
to ssDNA and stimulated RAD51-mediated homologous pairing and strand exchange.
The EVL protein also promoted the annealing of complementary strands. These
recombination reactions that were stimulated or promoted by the EVL protein
were further enhanced by the RAD51B protein. These results strongly suggested
that the EVL protein is a novel factor that activates RAD51-mediated
recombination reactions, probably with the RAD51B protein. We anticipate that,
in addition to its involvement in cytoplasmic actin dynamics, the EVL protein
may be required in homologous recombination for repairing specific DNA
lesions, and it may cause tumor malignancy by inappropriate recombination
enhanced by EVL overexpression in certain types of tumor cells. 相似文献
14.
The procofactor, factor VIII, is activated by thrombin or factor
Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689.
The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at
either Arg-1689 or Arg-372 and influences reaction rates at these sites.
Because cleavage at Arg-372 appears rate-limiting and dependent upon initial
cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences
catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H
and R1689Q were prepared and stably expressed to slow and eliminate cleavage,
respectively. Specific activity values for the His and Gln mutations were
∼50 and ∼10%, respectively, that of wild type. Thrombin activation of
the R1689H variant showed an ∼340-fold reduction in the rate of Arg-1689
cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at
this site. Examination of heavy chain cleavages showed ∼4- and 11-fold
reductions in A2 subunit generation and ∼3- and 7-fold reductions in A1
subunit generation for the R1689H and R1689Q mutants, respectively. These
results suggest a linkage between light chain cleavage and cleavages in heavy
chain. Results obtained evaluating proteolysis of the factor VIII mutants by
factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1
subunits and in cleaving light chain at Arg-1721 from either variant,
suggesting little dependence upon prior cleavage at residue 1689 as compared
with thrombin. Overall, these results are consistent with a competition
between heavy and light chains for thrombin exosite binding and subsequent
proteolysis with binding of the former chain preferred.Factor VIII, a plasma protein missing or defective in individuals with
hemophilia A, is synthesized as an ∼300-kDa single chain polypeptide
corresponding to 2332 amino acids. Within the protein are six domains based on
internal homologies and ordered as NH2-A1-A2-B-A3-C1-C2-COOH
(1,
2). Bordering the A domains are
short segments containing high concentrations of acidic residues that follow
the A1 and A2 domains and precede the A3 domain and are designated a1
(residues 337–372), a2 (residues 711–740), and a3
(1649–1689). Factor VIII is processed by cleavage at the B-A3 junction
to generate a divalent metal ion-dependent heterodimeric protein composed of a
heavy chain (A1-a1-A2-a2-B domains) and a light chain (a3-A3-C1-C2 domains)
(3).The activated form of factor VIII, factor VIIIa, functions as a cofactor
for factor IXa, increasing its catalytic efficiency by several orders of
magnitude in the phospholipid- and Ca2+-dependent conversion of
factor X to factor Xa (4). The
factor VIII procofactor is converted to factor VIIIa through limited
proteolysis catalyzed by thrombin or factor Xa
(5,
6). Thrombin is believed to act
as the physiological activator of factor VIII, as association of factor VIII
with von Willebrand factor impairs the capacity for the membrane-dependent
factor Xa to efficiently activate the procofactor
(5,
7). Activation of factor VIII
occurs through proteolysis by either protease via cleavage of three P1
residues at Arg-740 (A2-B domain junction), Arg-372 (A1-A2 domain junction),
and Arg-1689 (a3-A3 junction)
(5). After factor VIII
activation, there is a weak electrostatic interaction between the A1 and A2
domains of factor VIIIa (8,
9) and spontaneous inactivation
of the cofactor occurs through A2 subunit dissociation from the A1/A3-C1-C2
dimer, consequently dampening factor Xase
(3).Thrombin cleavage of factor VIII appears to be an ordered pathway, with
relative rates at Arg-740 > Arg-1689 > Arg-372 and the initial
proteolysis at Arg-740 facilitating proteolysis at Arg-372 as well as Arg-1689
(10). This latter observation
was based upon results showing that mutations at Arg-740, impairing this
cleavage, significantly reduced cleavage rates at the two other P1 sites.
Thrombin-catalyzed activation of factor VIII is dependent upon interactions
involving the anion binding exosites of the proteinase
(11,
12). Exosite binding is
believed to determine substrate affinity, whereas subsequent active site
docking primarily affects Vmax
(13). Furthermore, the complex
interactions involving multiple cleavages within a single substrate may
utilize a ratcheting mechanism
(14) where presentation of the
scissile bond is facilitated by a prior cleavage event.Cleavage at Arg-372 is a critical step in thrombin activation of factor
VIII as it exposes a cryptic functional factor IXa-interactive site in the A2
domain (15), whereas cleavage
at Arg-1689 liberates factor VIII from von Willebrand factor
(16) and contributes to factor
VIIIa specific activity (17,
18). Although cleavage at
Arg-740 represents a fast step relative to cleavages at other P1 residues in
the activation of factor VIII
(19), the influence of
Arg-1689 cleavage on cleavages in the heavy chain remains unknown. In the
present study cleavage at Arg-1689 is examined using recombinant factor VIII
variants possessing single point mutations of R1689Q and R1689H. Results
indicating reduced rates of A1 and A2 subunit generation, which are dependent
upon the residue at position 1689, suggest that cleavage at Arg-1689 affects
rates of proteolysis at Arg-740 and Arg-372. These observations are consistent
with a mechanism whereby heavy chain and light chain compete for a binding
thrombin exosite(s), with heavy chain preferred over light chain. In this
competition mechanism, cleavage at Arg-740 is favored over Arg-1689.
Subsequent cleavage at Arg-372 in heavy chain may involve a ratcheting
mechanism after initial cleavage at Arg-740. On the other hand, the mechanism
for factor Xa-catalyzed activation of factor VIII appears to be less dependent
on cleavage at the Arg-1689 site as compared with thrombin. 相似文献
15.
Tzung-Ju Wu Yi-Hsuan Chiang Yi-Chien Lin Chang-Ru Tsai Tai-Yuan Yu Ming-Ta Sung Yan-Hwa Wu Lee Jing-Jer Lin 《The Journal of biological chemistry》2009,284(19):12801-12808
Ku is a heterodimeric protein involved in nonhomologous end-joining of the
DNA double-stranded break repair pathway. It binds to the double-stranded DNA
ends and then activates a series of repair enzymes that join the broken DNA.
In addition to its function in DNA repair, the yeast Saccharomyces
cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes
that affect telomere function. The yeast telomeres are composed of duplex
C1–3(A/T)G1–3 telomeric DNA repeats plus
single-stranded TG1–3 telomeric DNA tails. Here we show that
Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that
mimics the structure of telomeres. Addition of Cdc13p, a single-stranded
telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of
a ternary complex with Cdc13p binding to the single-stranded tail of the DNA
substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail
inhibits the binding of Yku, the results suggested that loading of Yku and
Cdc13p to telomeres is sequential. Through generating a double-stranded break
near telomeric DNA sequences, we found that Ku protein appears to bind to the
de novo synthesized telomeres earlier than that of Cdc13p in
vivo. Thus, our results indicated that Yku interacts directly with
telomeres and that sequential loading of Yku followed by Cdc13p to telomeres
is required for both proteins to form a ternary complex on telomeres. Our
results also offer a mechanism that the binding of Cdc13p to telomeres might
prevent Yku from initiating DNA double-stranded break repair pathway on
telomeres.DNA damages in the form of double-stranded breaks
(DSBs)4 compromise the
integrity of genomes. Failure in repairing or mis-repairing double-stranded
breaks can lead to chromosome instability and eventually cell death or cancer
(1). Double-stranded breaks are
repaired by two main pathways, the homologous recombination and nonhomologous
DNA end-joining. In nonhomologous DNA end-joining, Ku is the first protein to
bind to the DNA ends to initiate the repair pathway
(2). Upon binding, Ku then
recruits a series of repair enzymes to join the broken ends
(2). Ku is a heterodimeric
protein composed of 70- and ∼80-kDa subunits. In Saccharomyces
cerevisiae, Ku includes Yku70 and Yku80 subunits. Because the biochemical
configuration of the broken ends could be very diverse on DSBs, Ku binds to
double-stranded ends in a sequence- and energy-independent manner. It is
capable of binding to DNA ends with blunt 3′-overhangs or
5′-overhangs as well as double-stranded DNA with nicks, gaps, or
internal loops
(3–7).
However, Ku does not have high affinity to single-stranded DNA. The crystal
structure of human Ku heterodimer indicates that it forms a ring structure
that encircles duplex DNA (7).
This unique structure feature enables Ku to recognize DNA ends and achieves
its high affinity binding.In additional to the role in double-stranded break repair, Ku was shown to
be a component of telomeric protein-DNA complex in yeast and mammals
(8–10).
Telomeres are terminal structures of chromosomes composed of short tandem
repeated sequences (11,
12). Mutation of
YKU70 or YKU80 causes defects in telomere structure
(13–15),
telomere silencing
(16–19),
and replication timing of telomeres
(20). The function of yeast Ku
(Yku) on telomeres could mediate through protein-protein interaction with
Sir4p or protein-RNA interaction with Tlc1 RNA
(21,
22). For example, through the
interaction with Sir4p, Yku selectively affects telomeres silencing but not
the silent mating type loci
(17). Yku could also bind to
telomerase Tlc1 RNA for telomere length maintenance
(22). Judged by the DNA
binding activity of Yku, it is reasonable to suggest that it may bind directly
to telomeric DNA. Indeed, it was shown that human Ku is capable of binding
directly to telomeric DNA in vitro
(15). Moreover, because the
deletion of SIR4 in budding yeast
(23) or Taz1 in
fission yeast (24) does not
abolish the association of Ku with chromosomal ends, this suggests that Ku
might bind directly to telomeric DNA in cells. However, because yeast
telomeres have a short 12–14-mer single-stranded tail
(25), it is uncertain whether
Yku could pass the single-stranded region to reach its binding site. The
direct binding of Yku to telomeric DNA has not been experimentally
determined.In contrast to double-stranded breaks, the ends of linear chromosomes are
not recognized by repair enzymes as DNA damage. In S. cerevisiae,
Cdc13p is the single-stranded TG1–3 DNA-binding protein that
enables cells to differentiate whether the ends of a linear DNA are telomeres
or broken ends
(26–29).
Thus, although the mechanism of how cells prevent the activation of DSB repair
pathway in telomere is unclear, it is likely that binding of Cdc13p to
telomeres might inhibit the initiation of DNA damage response by the Ku
protein. Here, using a tailed-duplex DNA synthesized by telomeric DNA
sequences to mimic telomere structure, we showed that Yku binds directly to
this tailed-duplex DNA substrate and forms a ternary complex with Cdc13p. Our
results also showed that Yku loaded to a de novo synthesized telomere
earlier than Cdc13p in vivo. These results support the direct binding
of Yku to telomeric DNA and that the spatial orientation of Cdc13p might block
the activation of DSB repair pathway on telomeres. 相似文献
16.
17.
18.
19.
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