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Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
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Shary N. Shelton Mary E. Shawgo John D. Robertson 《The Journal of biological chemistry》2009,284(17):11247-11255
The extent to which the BH3-only protein Bid is important for intrinsic
(mitochondria-mediated) apoptotic cell death induced by genotoxic stress
remains controversial. In the present study, we examine this issue using a
panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably
depleted of Bid were far less sensitive than control-transfected cells to
etoposide-induced apoptosis. In particular, drug-induced Bak activation,
cytochrome c release, loss of mitochondrial membrane potential, and
caspase activation were all decreased in cells lacking Bid. Reconstitution
experiments using recombinant proteins and permeabilized Bid-deficient cells
demonstrated that truncated Bid (tBid), but not full-length Bid, potently
induced Bak activation and the release of cytochrome c. Further,
caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to
drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as
cells overexpressing full-length X-linked inhibitor of apoptosis protein
(XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to
genotoxic stress. These data suggest that tBid plays an important regulatory
role in the execution of DNA damage-induced cytochrome c release and
apoptosis. However, the fact that cleavage of Bid to tBid is mediated by
executioner caspases suggests that a self-amplifying feed forward loop
involving caspases, Bid, and mitochondria may help determine irreversible
commitment to apoptosis.Apoptosis is an active form of cell death that plays an essential role
during normal embryonic development and in the maintenance of tissue
homeostasis in the adult organism
(1). Consequently,
dysregulation of apoptosis has been implicated as a contributing factor to the
onset of different pathological conditions, including cancer. In addition, it
is now generally accepted that many genotoxic anticancer drugs are effective
against tumor cells for their ability to induce mitochondria-mediated
apoptosis (2). Similarly,
mutations or the altered expression of pro- and anti-apoptotic proteins can
contribute to the development of drug resistance.Execution of apoptosis is mediated by a family of cysteine-dependent
aspartate-specific proteases (caspases). During true mitochondria-mediated
apoptosis, members of the Bcl-2 family of proteins are the primary regulators
of caspase activation for their role in controlling mitochondrial outer
membrane permeabilization
(MOMP)2
(3). The process of MOMP
results in the release of cytochrome c, second mitochondria-derived
activator of caspase (Smac, also known as DIABLO), and Omi (also known as
HtrA2) into the cytosol where they converge to promote the activation of
caspase-9 within the apoptotic protease-activating factor-1 (Apaf-1)
apoptosome complex. The Bcl-2 family contains proteins with opposing
functions, and it is generally thought that the induction of MOMP requires the
activation of either Bak or Bax triggered by a Bcl-2 homology 3 (BH3)-only
protein
(4–6).
Indeed, evidence in the literature indicates that cells lacking either Bak or
Bax exhibit only subtle defects in MOMP, whereas doubly deficient cells are
often found to be highly resistant to mitochondria-mediated apoptosis
(7,
8).At present, there are two models for the activation of Bax or Bak by
BH3-only proteins. One model argues that BH3-only proteins function as either
“sensitizer” (e.g. Bad and Noxa) or
“activator” proteins (e.g. truncated Bid (tBid), Bim, and
perhaps Puma) (9). In this
scenario, a sensitizer protein is needed to displace an activator protein from
a prosurvival protein (e.g. Bcl-2, Bcl-xL, or Mcl-1) to
activate Bak or Bax. The second model argues that BH3-only proteins bind and
inhibit the function of prosurvival Bcl-2 proteins, which normally bind to and
inhibit Bak and Bax (10,
11). Of the seven or so known
BH3-only proteins (6), Bid is
unique in that it requires post-translational modification for activation,
most notably involving caspase-8-mediated cleavage to tBid
(12–14).
Bid normally resides in the cytosol and possibly the nucleus
(15). Upon being cleaved, the
C-terminal fragment (tBid) is myristoylated at its newly exposed N terminus,
translocates to the outer mitochondrial membrane (OMM), and/or activates Bak
or Bax protein (16). Recently,
it was shown that the N-terminal cleavage fragment of Bid is quickly
ubiquitinated for degradation and that this degradation is necessary for the
pro-apoptotic function of tBid
(17). The same study also
concluded that, although full-length Bid is capable of translocating to the
OMM, it is not able to induce MOMP on its own
(17). A well characterized
example of tBid involvement during apoptosis is in the engagement of the
mitochondrial apoptotic pathway in so-called type II cells upon activation of
the extrinsic pathway
(18).Here, we have investigated whether Bid plays a functional role in the
induction of MOMP during apoptosis in response to the genotoxic anticancer
drug etoposide. To that end, we used Bax-deficient Jurkat cells that are
stably depleted of Bid and evaluated the extent to which these cells underwent
drug-induced MOMP. In addition, Jurkat clones in which the intrinsic pathway
had been inhibited due to the stable knockdown of Apaf-1 or the overexpression
of full-length XIAP or the baculoviral IAP repeats 1 and 2 (BIR1/BIR2) of XIAP
were used to gain insight into the molecular requirements necessary for
cleavage of Bid to tBid during drug-induced apoptosis. Strikingly, the data
showed that etoposide-induced apoptosis was decreased in Bid-deficient Jurkat
cells. In particular, cells lacking Bid expression exhibited decreased Bak
activation, cytochrome c release, loss of mitochondrial membrane
potential (ΔΨ), and caspase activation. Further, incubation of
permeabilized Bid-deficient cells with recombinant tBid, but not full-length
Bid, induced Bak dimerization and cytochrome c release.
Significantly, we also found that cleavage of Bid to tBid occurred strictly
downstream of Apaf-1 by a mechanism that required active executioner
caspases. 相似文献
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Jenny Erales Sabrina Lignon Brigitte Gontero 《The Journal of biological chemistry》2009,284(19):12735-12744
A new role is reported for CP12, a highly unfolded and flexible protein,
mainly known for its redox function with A4
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized
CP12 can prevent the in vitro thermal inactivation and aggregation of
GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not
redox-dependent. The protection is specific to CP12, because other proteins,
such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do
not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific
chaperone, since it does not protect other proteins, such as catalase, alcohol
dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is
necessary to prevent the aggregation and inactivation, since the mutant C66S
that does not form any complex with GAPDH cannot accomplish this protection.
Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is
partially able to protect and to slow down the inactivation and aggregation.
Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of
GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox
function but also behaves as a specific “chaperone-like protein”
for GAPDH, although a stable and not transitory interaction is observed. This
new function of CP12 may explain why it is also present in complexes involving
A2B2 GAPDHs that possess a regulatory C-terminal
extension (GapB subunit) and therefore do not require CP12 to be
redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most
photosynthetic organisms, including cyanobacteria
(1,
2), higher plants
(3), the diatom
Asterionella formosa
(4,
5), and green
(1) and red algae
(6). It allows the formation of
a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH),3 two key
enzymes of the Calvin cycle pathway, and was recently shown to interact with
fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway
(7). The
phosphoribulokinase·GAPDH·CP12 complex has been extensively
studied in Chlamydomonas reinhardtii
(8,
9) and in Arabidopsis
thaliana (10,
11). In the green alga C.
reinhardtii, the interaction between CP12 and GAPDH is strong
(8). GAPDH may exist as a
homotetramer composed of four GapA subunits (A4) in higher plants,
cyanobacteria, and green and red algae
(6,
12), but in higher plants, it
can also exist as a heterotetramer (A2B2), composed of
two subunits, GapA and GapB
(13,
14). GapB, up to now, has
exclusively been found in Streptophyta, but recently two
prasinophycean green algae, Ostreococcus tauri and Ostreococcus
lucimarinus, were also shown to possess a GapB gene, whereas
CP12 is missing (15).
The GapB subunit is similar to the GapA subunit but has a C-terminal extension
containing two redox-regulated cysteine residues
(16). Thus, although the
A4 GAPDHs lack these regulatory cysteine residues
(13,
14,
17–20),
they are also redox-regulated through its interaction with CP12, since the C
terminus of this small protein resembles the C-terminal extension of the GapB
subunit. The regulatory cysteine residues for GapA are thus supplied by CP12,
as is well documented in the literature
(1,
8,
11,
16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs)
(21–26).
The amino acid composition of these proteins causes them to have no or few
secondary structures. Their total or partial lack of structure and their high
flexibility allow them to be molecular adaptors
(27,
28). They are often able to
bind to several partners and are involved in most cellular functions
(29,
30). Recently, some IUPs have
been described in photosynthetic organisms
(31,
32).There are many functional categories of IUPs
(22,
33). They can be, for
instance, involved in permanent binding and have (i) a scavenger role,
neutralizing or storing small ligands; (ii) an assembler role by forming
complexes; and (iii) an effector role by modulating the activity of a partner
molecule (33). These functions
are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating
its redox properties (8,
34,
35), and can also bind a metal
ion (36,
37). IUPs can also bind
transiently to partners, and some of them have been found to possess a
chaperone activity (31,
38). This chaperone function
was first shown for α-synuclein
(39) and for α-casein
(40), which are fully
disordered. The amino acid composition of IUPs is less hydrophobic than those
of soluble proteins; hence, they lack hydrophobic cores and do not become
insoluble when heated. Since CP12 belongs to this family, we tested if it was
resistant to heat treatment and finally, since it is tightly bound to GAPDH,
if it could prevent aggregation of its partner, GAPDH, an enzyme well known
for its tendency to aggregate
(41–44)
and consequently a substrate commonly used in chaperone studies
(45,
46).Unlike chaperones, which form transient, dynamic complexes with their
protein substrates through hydrophobic interactions
(47,
48), CP12 forms a stable
complex with GAPDH. The interaction involves the C-terminal part of the
protein and the presence of negatively charged residues on CP12
(35). However, only a
site-directed mutagenesis has been performed to characterize the interaction
site on GAPDH. Although the mutation could have an indirect effect, the
residue Arg-197 was shown to be a good candidate for the interaction site
(49).In this report, we accordingly used proteolysis experiments coupled with
mass spectrometry to detect which regions of GAPDH are protected by its
association with CP12. To conclude, the aim of this report was to characterize
a chaperone function of CP12 that had never been described before and to map
the interaction site on GAPDH using an approach that does not involve
site-directed mutagenesis. 相似文献
8.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
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10.
11.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
12.
Tushar K. Beuria Srinivas Mullapudi Eugenia Mileykovskaya Mahalakshmi Sadasivam William Dowhan William Margolin 《The Journal of biological chemistry》2009,284(21):14079-14086
Cytokinesis in bacteria depends upon the contractile Z ring, which is
composed of dynamic polymers of the tubulin homolog FtsZ as well as other
membrane-associated proteins such as FtsA, a homolog of actin that is required
for membrane attachment of the Z ring and its subsequent constriction. Here we
show that a previously characterized hypermorphic mutant FtsA (FtsA*)
partially disassembled FtsZ polymers in vitro. This effect was
strictly dependent on ATP or ADP binding to FtsA* and occurred at
substoichiometric levels relative to FtsZ, similar to cellular levels.
Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical
concentration for FtsZ assembly but was able to disassemble preformed FtsZ
polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination
of the inhibited FtsZ polymers revealed a transition from long, straight
polymers and polymer bundles to mainly short, curved protofilaments. These
results indicate that a bacterial actin, when activated by adenine
nucleotides, can modify the length distribution of bacterial tubulin polymers,
analogous to the effects of actin-depolymerizing factor/cofilin on
F-actin.Bacterial cell division requires a large number of proteins that colocalize
to form a putative protein machine at the cell membrane
(1). This machine, sometimes
called the divisome, recruits enzymes to synthesize the septum cell wall and
to initiate and coordinate the invagination of the cytoplasmic membrane (and
in Gram-negative bacteria, the outer membrane). The most widely conserved and
key protein for this process is FtsZ, a homolog of tubulin that forms a ring
structure called the Z ring, which marks the site of septum formation
(2,
3). Like tubulin, FtsZ
assembles into filaments with GTP but does not form microtubules
(4). The precise assembly state
and conformation of these FtsZ filaments at the division ring is not clear,
although recent electron tomography work suggests that the FtsZ ring consists
of multiple short filaments tethered to the membrane at discrete junctures
(5), which may represent points
along the filaments bridged by membrane anchor proteins.In Escherichia coli, two of these anchor proteins are known. One
of these, ZipA, is not well conserved but is an essential protein in E.
coli. ZipA binds to the C-terminal tail of FtsZ
(6–8),
and purified ZipA promotes bundling of FtsZ filaments in vitro
(9,
10). The other, FtsA, is also
essential in E. coli and is more widely conserved among bacterial
species. FtsA is a member of the HSP70/actin superfamily
(11,
12), and like ZipA, it
interacts with the C-terminal tail of FtsZ
(7,
13–15).
FtsA can self-associate (16,
17) and bind ATP
(12,
18), but reports of ATPase
activity vary, with Bacillus subtilis FtsA having high activity
(19) and Streptococcus
pneumoniae FtsA exhibiting no detectable activity
(20). There are no reports of
any other in vitro activities of FtsA, including effects on FtsZ
assembly.Understanding how FtsA affects FtsZ assembly is important because FtsA has
a number of key activities in the cell. It is required for recruitment of a
number of divisome proteins
(21,
22) and helps to tether the Z
ring to the membrane via a C-terminal membrane-targeting sequence
(23). FtsA, like ZipA and
other divisome proteins, is necessary to activate the contraction of the Z
ring (24,
25). In E. coli, the
FtsA:FtsZ ratio is crucial for proper cell division, with either too high or
too low a ratio inhibiting septum formation
(26,
27). This ratio is roughly
1:5, with ∼700 molecules of FtsA and 3200 molecules of FtsZ per cell
(28), which works out to
concentrations of 1–2 and 5–10 μm, respectively.Another interesting property of FtsA is that single residue alterations in
the protein can result in significant enhancement of divisome activity. For
example, the R286W mutation of FtsA, also called FtsA*, can substitute for the
native FtsA and divide the cell. However, this mutant FtsA causes E.
coli cells to divide at less than 80% of their normal length
(29) and allows efficient
division of E. coli cells in the absence of ZipA
(30), indicating that it has
gain-of-function activity. FtsA* and other hypermorphic mutations such as
E124A and I143L can also increase division activity in cells lacking other
essential divisome components
(31–33).
The R286W and E124A mutants of FtsA also bypass the FtsA:FtsZ ratio rule,
allowing cell division to occur at higher ratios than with
WT2 FtsA. This may be
because the altered FtsA proteins self-associate more readily than WT FtsA,
which may cause different changes in FtsZ assembly state as compared with WT
FtsA (17,
34).In this study, we use an in vitro system with purified FtsZ and a
purified tagged version of FtsA* to elucidate the role of FtsA in activating
constriction of the Z ring in vivo. We show that FtsA*, at
physiological concentrations in the presence of ATP or ADP, has significant
effects on the assembly of FtsZ filaments. 相似文献
13.
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
14.
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
15.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
16.
17.
18.
19.
Aggregation of the Ure2 protein is at the origin of the [URE3]
prion trait in the yeast Saccharomyces cerevisiae. The N-terminal
region of Ure2p is necessary and sufficient to induce the [URE3]
phenotype in vivo and to polymerize into amyloid-like fibrils in
vitro. However, as the N-terminal region is poorly ordered in the native
state, making it difficult to detect structural changes in this region by
spectroscopic methods, detailed information about the fibril assembly process
is therefore lacking. Short fibril-forming peptide regions (4–7
residues) have been identified in a number of prion and other amyloid-related
proteins, but such short regions have not yet been identified in Ure2p. In
this study, we identify a unique cysteine mutant (R17C) that can greatly
accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions.
We found that the segment QVNI, corresponding to residues 18–21 in
Ure2p, plays a critical role in the fast assembly properties of R17C,
suggesting that this segment represents a potential amyloid-forming region. A
series of peptides containing the QVNI segment were found to form fibrils
in vitro. Furthermore, the peptide fibrils could seed fibril
formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a
hydrophobic residue, instead of a charged residue, caused the rate of assembly
into fibrils to increase greatly for both peptides and full-length Ure2p. Our
results indicate that the potential amyloid stretch and its preceding residue
can modulate the fibril assembly of Ure2p to control the initiation of prion
formation.The [URE3] phenotype of Saccharomyces cerevisiae arises
because of conversion of the Ure2 protein to an aggregated propagatable prion
state (1,
2). Ure2p contains two regions:
a poorly structured N-terminal region and a compactly folded C-terminal region
(3,
4). The N-terminal region is
rich in Asn and Gln residues, is highly flexible, and is without any
detectable ordered secondary structure
(4–6).
This region is necessary and sufficient for prion behavior in vivo
(2) and amyloid-forming
capacity in vitro (5,
7), so it is referred to as the
prion domain (PrD).2
The C-terminal region has a fold similar to the glutathione
S-transferase superfamily
(8,
9) and possesses
glutathione-dependent peroxidase activity
(10). Upon fibril formation,
the N-terminal region undergoes a significant conformational change from an
unfolded to a thermally resistant conformation
(11), whereas the glutathione
S-transferase-like C-terminal domain retains its enzymatic activity,
suggesting that little conformational change occurs
(10,
12). Ure2p fibrils show
various morphologies, including variations in thickness and the presence or
absence of a periodic twist
(13–16).
The overall structure of the fibrils imaged by cryoelectron microscopy
suggests that the intact fibrils contain a 4-nm amyloid filament backbone
surrounded by C-terminal globular domains
(17).It is widely accepted that disulfide bonds play a critical role in
maintaining protein stability
(18–21)
and also affect the process of protein folding by influencing the folding
pathway
(22–25).
A recent study shows that the presence of a disulfide bond in a protein can
markedly accelerate the folding process
(26). Therefore, a disulfide
bond is a useful tool to study protein folding. In the study of prion and
other amyloid-related proteins, cysteine scanning has been widely used to
study the structure of amyloid fibrils, the driving force of amyloid
formation, and the plasticity of amyloid fibrils
(13,
27–31).Short segments from amyloid-related proteins, including IAPP
(islet amyloid polypeptide),
β2-microglobulin, insulin, and the amyloid-β peptide,
show amyloid-forming capacity
(32–34).
Hence, the amyloid stretch hypothesis has been proposed, which suggests that a
short amino acid stretch bearing a highly amyloidogenic motif might supply
most of the driving force needed to trigger the self-catalytic assembly
process of a protein to form fibrils
(35,
36). In support of this
hypothesis, it was found that the insertion of an amyloidogenic stretch into a
non-amyloid-related protein can trigger the amyloidosis of the protein
(36). At the same time, the
structural information obtained from microcrystals formed by amyloidogenic
stretches and bearing cross-β-structure has contributed significantly to
our understanding of the structure of intact fibrils at the atomic level
(34,
37). However, no amyloidogenic
stretches <10 amino acids have so far been identified in the yeast prion
protein Ure2.In this study, we performed a cysteine scan within the N-terminal PrD of
Ure2p and found a unique cysteine mutant (R17C) that eliminates the lag phase
of the Ure2p fibril assembly reaction upon the addition of oxidizing agents.
Furthermore, we identified a 4-residue region adjacent to Arg17 as
a potential amyloid stretch in Ure2p. 相似文献
20.
Eva Brombacher Simon Urwyler Curdin Ragaz Stefan S. Weber Keiichiro Kami Michael Overduin Hubert Hilbi 《The Journal of biological chemistry》2009,284(8):4846-4856
The causative agent of Legionnaires disease, Legionella
pneumophila, forms a replicative vacuole in phagocytes by means of the
intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV
secretion system and translocated effector proteins, some of which subvert
host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC
anchors to the membrane of Legionella-containing vacuoles (LCVs) by
specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a
nonbiased screen for novel L. pneumophila PI-binding proteins, we
identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the
predominant PtdIns(4)P-binding protein. Purified SidM specifically and
directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate
LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L.
pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding
domain of SidM was mapped to the 12-kDa C-terminal sequence, termed
“P4M” (PtdIns4P binding of
SidM/DrrA). The isolated P4M domain is largely helical and
displayed higher PtdIns(4)P binding activity in the context of the
α-helical, monomeric full-length protein. SidM constructs containing P4M
were translocated by Icm/Dot-proficient L. pneumophila and localized
to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via
its P4M domain. An L. pneumophila ΔsidM mutant strain
displayed significantly higher amounts of SidC on LCVs, suggesting that SidM
and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally,
RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by
host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits
PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC
and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative
agent of Legionnaires disease, but it evolved as a parasite of various species
of environmental predatory protozoa, including the social amoeba
Dictyostelium discoideum
(1,
2). The human disease is linked
to the inhalation of contaminated aerosols, followed by replication in
alveolar macrophages. To accommodate the transfer between host cells, L.
pneumophila alternates between replicative and transmissive phases, the
regulation of which includes an apparent quorum-sensing system
(3–5).In macrophages and amoebae, L. pneumophila forms a replicative
compartment, the Legionella-containing vacuole
(LCV).3 LCVs avoid
fusion with lysosomes (6),
intercept vesicular traffic at endoplasmic reticulum (ER) exit sites
(7), and fuse with the ER
(8–10).
The uptake of L. pneumophila and formation of LCVs in macrophages and
amoebae depends on the Icm/Dot type IV secretion system (T4SS)
(11–14).
Although more than 100 Icm/Dot substrates (“effector” proteins)
have been identified to date, only few are functionally characterized,
including effectors that interfere with host cell signal transduction, vesicle
trafficking, or apoptotic pathways
(15–18).Two Icm/Dot-translocated substrates, SidM/DrrA
(19,
20) and RalF
(21), have been characterized
as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small
GTPases. These bacterial GEFs are recruited to and activate their targets on
LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic
signal transduction pathways and in actin cytoskeleton regulation
(22). Inactive Rho GTPases
bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases
are activated by removal of the GDI and the exchange of GDP with GTP by GEFs,
which promotes the interaction with downstream effector proteins, such as
protein or lipid kinases and various adaptor proteins. The cycle is closed by
hydrolysis of the bound GTP, which is mediated by GTPase-activating
proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle
transport, and additionally, SidM acts as a GDI displacement factor (GDF) to
activate Rab1 (23,
24). The function of SidM is
assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA
preferentially binds to activated Rab1, thus supporting the recruitment of
early secretory vesicles by SidM
(19,
20,
23,
25,
26). Another Icm/Dot
substrate, LepB (27),
contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its
GTPase-activating protein function, thus acting as an antagonist of SidM
(24).The Icm/Dot substrate RalF recruits and activates the small GTPase
ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle
transport from Golgi to ER
(21). Dominant negative Arf1
(7,
28) or knockdown of Arf1 by
RNA interference (29) impairs
the formation of LCVs, as well as the recruitment of the Icm/Dot substrate
SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane
(31), where the proteins
specifically bind to the host cell lipid phosphatidylinositol 4-phosphate
(PtdIns(4)P) (32,
33). Phosphoinositides (PIs)
regulate eukaryotic receptor-mediated signal transduction, actin remodeling,
and membrane dynamics (34,
35). PtdIns(4)P is present on
the cytoplasmic membrane, but localizes preferentially to the
trans-Golgi network (TGN), where this PI is produced by an
Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ)
(36) to promote trafficking
along the secretory pathway. Recently, PtdIns(4)P was found to also mediate
the export of early secretory vesicles from ER exit sites
(37). At present, the L.
pneumophila effector proteins that mediate exploitation of host PI
signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using
different PIs coupled to agarose beads, we identified SidM as a major
PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a
novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs,
including the P4M domain, were found to be translocated and bind the LCV
membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ. 相似文献