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1.
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
2.
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
3.
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
4.
5.
6.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
7.
8.
9.
10.
11.
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
12.
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
13.
Jonathan M. Budzik So-Young Oh Olaf Schneewind 《The Journal of biological chemistry》2009,284(19):12989-12997
Bacillus cereus and other Gram-positive bacteria elaborate pili
via a sortase D-catalyzed transpeptidation mechanism from major and minor
pilin precursor substrates. After cleavage of the LPXTG sorting
signal of the major pilin, BcpA, sortase D forms an amide bond between the
C-terminal threonine and the amino group of lysine within the YPKN motif of
another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the
BcpA sorting signal, resulting in a covalent bond between BcpA and the cell
wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the
minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal
threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby
positioning BcpB at the tip of pili. Thus, unique attributes of the sorting
signals of minor pilins provide Gram-positive bacteria with a universal
mechanism ordering assembly of pili.Sortases catalyze transpeptidation reactions to assemble proteins in the
envelope of Gram-positive bacteria
(1). Secreted proteins require
a C-terminal sorting signal for sortase recognition such that sortase cleaves
the substrate at a short peptide motif and forms a thioester-linked
intermediate to its active site cysteine
(2–4).
Nucleophilic attack by an amino group within the bacterial envelope resolves
the thioester intermediate, generating an amide bond tethering surface
proteins at their C terminus onto Gram-positive bacteria
(5). Four classes of sortases
can be distinguished on the basis of sequence homology and substrate
recognition (6,
7). Sortase A cleaves secreted
protein at LPXTG sorting signals and recognizes the amino group of
lipid II peptidoglycan precursors as a nucleophile
(8,
9). Sortase B cleaves protein
substrates at NPQTN sorting signals
(10). This enzyme immobilizes
proteins within fully assembled cell walls, utilizing the cell wall
cross-bridge as a nucleophile
(11). Sortase C cuts LPNTA
sorting signals and anchors proteins to the peptidoglycan cross-bridges in
sporulating bacteria (12,
13). Finally, sortase D
catalyzes transpeptidation reactions in the assembly of pili
(14,
15). Sortase D recognizes the
amino group of lysine residues within the YPKN motif of pilin subunits as
nucleophiles (16). The
resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow
the pilus fiber (16,
17).Pili of Gram-positive bacteria comprised either two or three different
pilin subunits synthesized as cytoplasmic precursors with N-terminal signal
peptides and C-terminal sorting signals (P1 precursors)
(14,
18). After translocation
across the plasma membrane, P2 precursor species arise from removal of the
signal peptide from P1 precursors by a signal peptidase
(16). Bacillus cereus
pili are composed of two subunits; that is, the major pilin, BcpA, and the
minor pilin, BcpB (15). In
contrast to BcpA, which is deposited throughout the pilus, BcpB is found at
fiber tip (15). Sortase D
cleaves the BcpA LPXTG motif sorting signal between the threonine and
glycine residues to form an amide bond to the ε-amino group of the lysine
within the YPKN motif of adjacent BcpA subunits
(16). However, sortase A also
cleaves BcpA precursors, which are subsequently linked to the side chain amino
group of meso-diaminopimelic acid within lipid II
(19). The latter reaction
serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall
envelope (19).The conservation of sortase D, the YPKN motif, and C-terminal sorting
signal in major pilin subunits suggest a universal pilus assembly mechanism
among Gram-positive bacteria
(14,
20). However, the molecular
mechanism whereby bacilli deposit BcpB, the minor pilin, at the tip of BcpA
pili is not known. Although the BcpB precursor harbors an N-terminal signal
peptide and a C-terminal IPNTG sorting signal, it lacks the YPKN pilin motif
of the major subunit (15).
Furthermore, the substrate properties of the BcpB IPNTG sorting signal for the
four classes of sortases expressed by bacilli has yet to be established. 相似文献
14.
15.
Graham H. Diering John Church Masayuki Numata 《The Journal of biological chemistry》2009,284(20):13892-13903
NHE5 is a brain-enriched Na+/H+ exchanger that
dynamically shuttles between the plasma membrane and recycling endosomes,
serving as a mechanism that acutely controls the local pH environment. In the
current study we show that secretory carrier membrane proteins (SCAMPs), a
group of tetraspanning integral membrane proteins that reside in multiple
secretory and endocytic organelles, bind to NHE5 and co-localize predominantly
in the recycling endosomes. In vitro protein-protein interaction
assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic
extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5
increased cell-surface abundance as well as transporter activity of NHE5
across the plasma membrane. Expression of a deletion mutant lacking the
SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the
N-terminal extension, reduced the transporter activity. Although both Arf6 and
Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across
the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by
dominant-negative Arf6 but not by dominant-negative Rab11. Together, these
results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes
and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are
especially sensitive to perturbations of pH
(1). Many voltage- and
ligand-gated ion channels that control membrane excitability are sensitive to
changes in cellular pH
(1-3).
Neurotransmitter release and uptake are also influenced by cellular and
organellar pH (4,
5). Moreover, the intra- and
extracellular pH of both neurons and glia are modulated in a highly transient
and localized manner by neuronal activity
(6,
7). Thus, neurons and glia
require sophisticated mechanisms to finely tune ion and pH homeostasis to
maintain their normal functions.Na+/H+ exchangers
(NHEs)3 were
originally identified as a class of plasma membrane-bound ion transporters
that exchange extracellular Na+ for intracellular H+,
and thereby regulate cellular pH and volume. Since the discovery of NHE1 as
the first mammalian NHE (8),
eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have
been isolated in mammals (9,
10). NHE1-5 commonly exhibit
transporter activity across the plasma membrane, whereas NHE6-9 are mostly
found in organelle membranes and are believed to regulate organellar pH in
most cell types at steady state
(11). More recently, NHE10 was
identified in human and mouse osteoclasts
(12,
13). However, the cDNA
encoding NHE10 shares only a low degree of sequence similarity with other
known members of the NHE gene family, raising the possibility that
this sodium-proton exchanger may belong to a separate gene family distantly
related to NHE1-9 (see Ref.
9).NHE gene family members contain 12 putative transmembrane domains
at the N terminus followed by a C-terminal cytosolic extension that plays a
role in regulation of the transporter activity by protein-protein interactions
and phosphorylation. NHEs have been shown to regulate the pH environment of
synaptic nerve terminals and to regulate the release of neurotransmitters from
multiple neuronal populations
(14-16).
The importance of NHEs in brain function is further exemplified by the
findings that spontaneous or directed mutations of the ubiquitously expressed
NHE1 gene lead to the progression of epileptic seizures, ataxia, and
increased mortality in mice
(17,
18). The progression of the
disease phenotype is associated with loss of specific neuron populations and
increased neuronal excitability. However, NHE1-null mice appear to
develop normally until 2 weeks after birth when symptoms begin to appear.
Therefore, other mechanisms may compensate for the loss of NHE1
during early development and play a protective role in the surviving neurons
after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene
family whose mRNA is expressed almost exclusively in the brain
(19,
20), although more recent
studies have suggested that NHE5 might be functional in other cell
types such as sperm (21,
22) and osteosarcoma cells
(23). Curiously, mutations
found in several forms of congenital neurological disorders such as
spinocerebellar ataxia type 4
(24-26)
and autosomal dominant cerebellar ataxia
(27-29)
have been mapped to chromosome 16q22.1, a region containing NHE5.
However, much remains unknown as to the molecular regulation of NHE5 and its
role in brain function.Very few if any proteins work in isolation. Therefore identification and
characterization of binding proteins often reveal novel functions and
regulation mechanisms of the protein of interest. To begin to elucidate the
biological role of NHE5, we have started to explore NHE5-binding proteins.
Previously, β-arrestins, multifunctional scaffold proteins that play a
key role in desensitization of G-protein-coupled receptors, were shown to
directly bind to NHE5 and promote its endocytosis
(30). This study demonstrated
that NHE5 trafficking between endosomes and the plasma membrane is regulated
by protein-protein interactions with scaffold proteins. More recently, we
demonstrated that receptor for activated
C-kinase 1 (RACK1), a scaffold protein that links
signaling molecules such as activated protein kinase C, integrins, and Src
kinase (31), directly
interacts with and activates NHE5 via integrin-dependent and independent
pathways (32). These results
further indicate that NHE5 is partly associated with focal adhesions and that
its targeting to the specialized microdomain of the plasma membrane may be
regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily
conserved tetra-spanning integral membrane proteins. SCAMPs are found in
multiple organelles such as the Golgi apparatus, trans-Golgi network,
recycling endosomes, synaptic vesicles, and the plasma membrane
(33,
34) and have been shown to
play a role in exocytosis
(35-38)
and endocytosis (39).
Currently, five isoforms of SCAMP have been identified in mammals. The
extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats,
which may allow these isoforms to participate in clathrin coat assembly and
vesicle budding by binding to Eps15 homology (EH)-domain proteins
(40,
41). Further, SCAMP2 was shown
recently to bind to the small GTPase Arf6
(38), which is believed to
participate in traffic between the recycling endosomes and the cell surface
(42,
43). More recent studies have
suggested that SCAMPs bind to organellar membrane type NHE7
(44) and the serotonin
transporter SERT (45) and
facilitate targeting of these integral membrane proteins to specific
intracellular compartments. We show in the current study that SCAMP2 binds to
NHE5, facilitates the cell-surface targeting of NHE5, and elevates
Na+/H+ exchange activity at the plasma membrane, whereas
expression of a SCAMP2 deletion mutant lacking the N-terminal domain
containing the NPF repeats suppresses the effect. Further we show that this
activity of SCAMP2 requires an active form of a small GTPase Arf6, but not
Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal
compartment and control its cell-surface abundance via an Arf6-dependent
pathway. 相似文献
16.
17.
John M. Harrington Sawyer Howell Stephen L. Hajduk 《The Journal of biological chemistry》2009,284(20):13505-13512
Trypanosome lytic factor (TLF) is a subclass of human high density
lipoprotein (HDL) that mediates an innate immune killing of certain mammalian
trypanosomes, most notably Trypanosoma brucei brucei, the causative
agent of a wasting disease in cattle. Mechanistically, killing is initiated in
the lysosome of the target trypanosome where the acidic pH facilitates a
membrane-disrupting activity by TLF. Here we utilize a model liposome system
to characterize the membrane binding and permeabilizing activity of TLF and
its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein
L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently
binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas
non-lytic human HDL exhibits inefficient permeabilizing activity. Purified,
delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low
pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for
anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic
membranes. Analysis of the relative particle sizes of susceptible liposomes
reveals distinctly different membrane-active behavior for native TLF and the
delipidated protein components. We propose that lysosomal membrane damage in
TLF-susceptible trypanosomes is initiated by the stable association of the TLF
particle with the lysosomal membrane and that this is a property unique to
this subclass of human HDL.High density lipoproteins
(HDL)2 are complex yet
ordered macromolecules consisting of characteristic proteins embedded in a
phospholipid monolayer that surrounds a hydrophobic core of esterified
cholesterol and triglycerides. A subclass of HDL is responsible for an innate
immune killing of the African blood stream parasite Trypanosoma brucei
brucei
(1–3),
and very recently, has been shown to be cytotoxic to intracellular
Leishmania promastigotes
(4). The trypanolytic HDL
particle, termed trypanosome lytic factor (TLF), is characterized by the
presence of two proteins, apolipoprotein L-1 (apoL-1) and haptoglobin-related
protein (Hpr), as well as the HDL ubiquitous apolipoprotein A-1 (apoA-1)
(1,
5–7).
Killing of the susceptible parasite involves high affinity binding to a
cell-surface receptor, endocytosis, and trafficking of the TLF particle to the
lysosome
(8–12).
The acidic lysosomal environment facilitates a membrane-disrupting activity by
the TLF particle and subsequent cell death
(9,
13). It has been shown that
purified, delipidated apoL-1 or Hpr are sufficient for trypanosome killing.
When these proteins are incorporated into the same lipoprotein particle, a
several hundredfold increase in killing activity is exhibited
(5). In addition,
Molina-Portela et al.
(14) show that maximal
protection against T. b. brucei in a transgenic mouse model requires
the expression of human Hpr, apoL-1, and apoA-1, supporting a synergistic mode
of action.Haptoglobin-related protein evolved during primate evolution and is
restricted to apes, old world monkeys, and humans
(15). Haptoglobin-related
protein is highly similar (92%) to the acute phase serum protein haptoglobin
(Hp) (16). All mammals use Hp
as a scavenger of hemoglobin (Hb) released during hemolysis associated with
infection or trauma. Haptoglobin binds cell-free Hb with high affinity and
facilitates its removal from the circulation through a receptor-mediated
process in the liver (17).
Like Hp, Hpr binds free Hb, yet this Hpr·Hb complex is not recognized
by the requisite receptors in mammals and is thus not removed from the
circulation (18). TLF uptake
by susceptible trypanosomes requires specific binding to an Hpr·Hb
complex that facilitates trafficking of the TLF particle to the lysosome
(10). It has been proposed
that once inside the lysosomal compartment, Hpr·Hb contributes directly
to membrane disruption through the generation of oxygen radicals with the
bound Hb providing the iron necessary for Fenton chemistry
(7,
10,
19).Apolipoprotein L-1 is a unique member of the apolipoprotein L protein
family in that, unlike the remaining apoL proteins, it possesses an N-terminal
signal sequence and is thus secreted from cells. As is the case for Hpr,
apoL-1 appeared during primate evolution
(20–22).
Within the circulation of primates, apoL-1 is exclusively associated with HDL,
and the majority of the protein is in the TLF subclass
(5). The apoL family members
are all predicted to adopt amphipathic α-helical conformations,
suggesting that their physiological role involves membrane interaction
(20). Apolipoprotein L-1
shares limited homology with channel-forming colicins and, consistent with
this observation, has been shown to function as an ion channel when
incorporated into lipid bilayers
(23).The ultimate fate of TLF-targeted lysosomal membranes is not firmly
established. Several studies employing both in vivo cellular analysis
and artificial membrane systems address this point with conflicting results.
Electron microscopy studies with gold-conjugated TLF revealed accumulation of
TLF in intracellular vesicles and subsequent vesicle membrane breakdown and
appearance of gold particles in the cytoplasm
(9). Widener et al.
(10) observed efflux of
lysosomally localized large molecular mass dextrans (500 kDa) in TLF-treated
T. b. brucei. These data suggest that the lysosomal membrane
experiences large scale disruption. In contrast, Perez-Morga et al.
(23) and Vanhollebeke et
al. (24) report
uncontrollable lysosomal swelling in susceptible trypanosomes treated with
normal human serum, suggesting stability of the lamellar structure of the
lysosomal membrane after TLF attack. Swelling is attributed to apoL-1-mediated
influx of Cl– ions and concomitant osmotic flow of water into
the lysosome. However, Molina-Portela et al.
(25) observed the formation of
cation-selective pores in TLF-treated planar lipid bilayers composed of
trypanosome lipids. The diversity of activities reported for TLF and normal
human serum may reflect the packaging of multiple toxins within the same
complex that can act synergistically to provide optimal killing activity
(5,
14).Here we utilize model liposomes to monitor the membrane activity of TLF and
its protein constituents. We describe the effects of TLF, delipidated Hpr,
apoL-1, and apoA-1 on the permeability of unilamellar liposomes. Additionally,
we show that TLF, apoL-1, and apoA-1 exhibit lipid specificity and that Hpr,
apoL-1, and apoA-1 induce large scale changes in the geometry of liposomes.
These results provide a molecular basis for the recognition of lysosomal
membranes by this toxic HDL and support a multicomponent mechanism for
trypanosome killing. 相似文献
18.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
19.
20.
Ivana I. Knezevic Sanda A. Predescu Radu F. Neamu Matvey S. Gorovoy Nebojsa M. Knezevic Cordus Easington Asrar B. Malik Dan N. Predescu 《The Journal of biological chemistry》2009,284(8):5381-5394
It is known that platelet-activating factor (PAF) induces severe
endothelial barrier leakiness, but the signaling mechanisms remain unclear.
Here, using a wide range of biochemical and morphological approaches applied
in both mouse models and cultured endothelial cells, we addressed the
mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and
of increased endothelial permeability. The formation of interendothelial gaps
filled with filopodia and lamellipodia is the cellular event responsible for
the disruption of endothelial barrier. We observed that PAF ligation of its
receptor induced the activation of the Rho GTPase Rac1. Following PAF
exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were
found associated with a membrane fraction from which they
co-immunoprecipitated with PAF receptor. In the same time frame with
Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were
relocated from the IEJs, and formation of numerous interendothelial gaps was
recorded. Notably, the response was independent of myosin light chain
phosphorylation and thus distinct from other mediators, such as histamine and
thrombin. The changes in actin status are driven by the PAF-induced localized
actin polymerization as a consequence of Rac1 translocation and activation.
Tiam1 was required for the activation of Rac1, actin polymerization,
relocation of junctional associated proteins, and disruption of IEJs. Thus,
PAF-induced IEJ disruption and increased endothelial permeability requires the
activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic
target against increased vascular permeability associated with inflammatory
diseases.The endothelial barrier is made up of endothelial cells
(ECs)4 connected to
each other by interendothelial junctions (IEJs) consisting of protein
complexes organized as tight junctions (TJs) and adherens junctions (AJs). In
addition, the focal adhesion complex located at the basal plasma membrane
enables firm contact of ECs with the underlying basement membrane and also
contributes to the barrier function
(1-3).
The glycocalyx, the endothelial monolayer, and the basement membrane all
together constitute the vascular barrier.The structural integrity of the ECs along with their proper functionality
are the two most important factors controlling the tightness of the
endothelial barrier. Changes affecting these factors cause loss of barrier
restrictiveness and leakiness. Therefore, defining and understanding the
cellular and molecular mechanisms controlling these processes is of paramount
importance. Increased width of IEJs in response to permeability-increasing
mediators (4) regulates the
magnitude of transendothelial exchange of fluid and solutes. Disruption of
IEJs and the resultant barrier leakiness contribute to the genesis of diverse
pathological conditions, such as inflammation
(5), metastasis
(6,
7), and uncontrolled
angiogenesis (8,
9).Accumulated evidence demonstrated that IEJs changes are responsible for
increased or decreased vascular permeability, and the generally accepted
mechanism responsible for them was the myosin light chain (MLC)-mediated
contraction of ECs (5,
10). However, published
evidence showed that an increase in vascular permeability could be obtained
without a direct involvement of any contractile mechanism
(11-16).The main component of the vascular barrier, the ECs, has more than 10% of
their total protein represented by actin
(17), which under
physiological salt concentrations subsists as monomers (G-actin) and assembled
into filaments (F-actin). A large number of actin-interacting proteins may
modulate the assembly, disassembly, and organization of G-actin and of actin
filaments within a given cell type. Similar to the complexity of
actin-interacting proteins found in other cell types, the ECs utilize their
actin binding proteins to stabilize the endothelial monolayer in order to
efficiently function as a selective barrier
(11). In undisturbed ECs, the
actin microfilaments are organized as different networks with distinctive
functional and morphological characteristics: the peripheral filaments also
known as peripheral dense band (PDB), the cytoplasmic fibers identified as
stress fibers (SF), and the actin from the membrane cytoskeleton
(18). The peripheral web,
localized immediately under the membrane, is associated with (i) the luminal
plasmalemma (on the apical side), (ii) the IEJ complexes on the lateral
surfaces, and (iii) the focal adhesion complexes on the abluminal side (the
basal part) of polarized ECs. The SF reside inside the endothelial cytoplasm
and are believed to be directly connected with the plasmalemma proper on the
luminal as well as on the abluminal side of the cell. As described, the
endothelial actin cytoskeleton (specifically the SF) seems to be a stable
structure helping the cells to remain flat under flow
(19). It is also established
that the actin fibers participate in correct localization of different
junctional complexes while keeping them in place
(20). However, it was
suggested that the dynamic equilibrium between F- and G-actin might modulate
the tightness of endothelial barrier in response to different challenges
(13).Mediators effective at nanomolar concentrations or less that disrupt the
endothelial barrier and increase vascular permeability include C2 toxin of
Clostridium botulinum, vascular permeability factor, better known as
vascular endothelial growth factor, and PAF
(21). C2 toxin increases
endothelial permeability by ribosylating monomeric G-actin at Arg-177
(22). This results in the
impairment of actin polymerization
(23), followed by rounding of
ECs (16) and the disruption of
junctional integrity. Vascular permeability factor was shown to open IEJs by
redistribution of junctional proteins
(24,
25) and by interfering with
the equilibrium of actin pools
(26). PAF
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocoline), a naturally
synthesized phospholipid is active at 10-10 m or less
(27). PAF is synthesized by
and acts on a variety of cell types, including platelets
(28), neutrophils
(29), monocytes
(30), and ECs
(31). PAF-mediated activation
of ECs induced cell migration
(32), angiogenesis
(7), and vascular
hyperpermeability (33)
secondary to disassembly of IEJs
(34). The effects of PAF on
the endothelium are initiated through a G protein-coupled receptor (PAF-R)
localized at the plasmalemma, in a large endosomal compartment inside the cell
(34), and also in the nuclear
membrane (35). In ECs, PAF-R
was shown to signal through Gαq and downstream activation of
phospholipase C isozymes (PLCβ3 and PLCγ1),
and via cSrc (32,
36). Studies have shown that
PAF challenge induced endothelial actin cytoskeletal rearrangement
(37) and marked vascular
leakiness (38); however, the
signaling pathways have not been elucidated.Therefore, in the present study, we carried out a systematic analysis of
PAF-induced morphological and biochemical changes of endothelial barrier
in vivo and in cultured ECs. We found that the opening of endothelial
barrier and the increased vascular leakiness induced by PAF are the result of
a shift in actin pools without involvement of EC contraction, followed by a
redistribution of tight junctional associated protein ZO-1 and adherens
junctional protein VE-cadherin. 相似文献