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1.
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
2.
3.
4.
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
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.
Daniel Lingwood Sebastian Schuck Charles Ferguson Mathias J. Gerl Kai Simons 《The Journal of biological chemistry》2009,284(18):12041-12048
Cell membranes predominantly consist of lamellar lipid bilayers. When
studied in vitro, however, many membrane lipids can exhibit
non-lamellar morphologies, often with cubic symmetries. An open issue is how
lipid polymorphisms influence organelle and cell shape. Here, we used
controlled dimerization of artificial membrane proteins in mammalian tissue
culture cells to induce an expansion of the endoplasmic reticulum (ER) with
cubic symmetry. Although this observation emphasizes ER architectural
plasticity, we found that the changed ER membrane became sequestered into
large autophagic vacuoles, positive for the autophagy protein LC3. Autophagy
may be targeting irregular membrane shapes and/or aggregated protein. We
suggest that membrane morphology can be controlled in cells.The observation that simple mixtures of amphiphilic (polar) lipids and
water yield a rich flora of phase structures has opened a long-standing debate
as to whether such membrane polymorphisms are relevant for living organisms
(1–7).
Lipid bilayers with planar geometry, termed lamellar symmetry, dominate the
membrane structure of cells. However, this architecture comprises only a
fraction of the structures seen with in vitro lipid-water systems
(7–11).
The propensity to form lamellar bilayers (a property exclusive to
cylindrically shaped lipids) is flanked by a continuum of lipid structures
that occur in a number of exotic and probably non-physiological
non-bilayer configurations
(3,
12). However, certain lipids,
particularly those with smaller head groups and more bulky hydrocarbon chains,
can adopt bilayered non-lamellar phases called cubic phases. Here the
bilayer is curved everywhere in the form of saddle shapes corresponding to an
energetically favorable minimal surface of zero mean curvature
(1,
7). Because a substantial
number of the lipids present in biological membranes, when studied as
individual pure lipids, form cubic phases
(13), cubic membranes have
received particular interest in cell biology.Since the application of electron microscopy
(EM)3 to the study of
cell ultrastructure, unusual membrane morphologies have been reported for
virtually every organelle (14,
15). However, interpretation
of three-dimensional structures from two-dimensional electron micrographs is
not easy (16). In seminal
work, Landh (17) developed the
method of direct template correlative matching, a technique that unequivocally
assesses the presence of cubic membranes in biological specimens
(16). Cubic phases adopt
mathematically well defined three-dimensional configurations whose
two-dimensional analogs have been derived
(4,
17). In direct template
correlative matching, electron micrographs are matched to these analogs. Cubic
cell membrane geometries and in vitro cubic phases of purified lipid
mixtures do differ in their lattice parameters; however, such deviations are
thought to relate to differences in water activity and lipid to protein ratios
(10,
14,
18). Direct template
correlative matching has revealed thousands of examples of cellular cubic
membranes in a broad survey of electron micrographs ranging from protozoa to
human cells (14,
17) and, more recently, in the
mitochondria of amoeba (19)
and in subcellular membrane compartments associated with severe acute
respiratory syndrome virus
(20). Analysis of cellular
cubic membranes has also been furthered by the development of EM tomography
that confirmed the presence of cubic bilayers in the mitochondrial membranes
of amoeba (21,
22).Although it is now clear that cubic membranes can exist in living cells,
the generation of such architecture would appear tightly regulated, as
evidenced by the dominance of lamellar bilayers in biology. In this light, we
examined the capability and implications of generating cubic membranes in the
endoplasmic reticulum (ER) of mammalian tissue culture cells. The ER is a
spatially interconnected complex consisting of two domains, the nuclear
envelope and the peripheral ER
(23–26).
The nuclear envelope surrounds the nucleus and is composed of two continuous
sheets of membranes, an inner and outer nuclear membrane connected to each
other at nuclear pores. The peripheral ER constitutes a network of branching
trijunctional tubules that are continuous with membrane sheet regions that
occur in closer proximity to the nucleus. Recently it has been suggested that
the classical morphological definition of rough ER (ribosome-studded) and
smooth ER (ribosome-free) may correspond to sheet-like and tubular ER domains,
respectively (27). The ER has
a strong potential for cubic architectures, as demonstrated by the fact that
the majority of cubic cell membranes in the EM record come from ER-derived
structures (14,
17). Furthermore, ER cubic
symmetries are an inducible class of organized smooth ER (OSER), a definition
collectively referring to ordered smooth ER membranes (=stacked cisternae on
the outer nuclear membrane, also called Karmelle
(28–30),
packed sinusoidal ER (31),
concentric membrane whorls
(30,
32–34),
and arrays of crystalloid ER
(35–37)).
Specifically, weak homotypic interactions between membrane proteins produce
both a whorled and a sinusoidal OSER phenotype
(38), the latter exhibiting a
cubic symmetry (16,
39).We were able to produce OSER with cubic membrane morphology via induction
of homo-dimerization of artificial membrane proteins. Interestingly, the
resultant cubic membrane architecture was removed from the ER system by
incorporation into large autophagic vacuoles. To assess whether these cubic
symmetries were favored in the absence of cellular energy, we depleted ATP. To
our surprise, the cells responded by forming large domains of tubulated
membrane, suggesting that a cubic symmetry was not the preferred conformation
of the system. Our results suggest that whereas the endoplasmic reticulum is
capable of adopting cubic symmetries, both the inherent properties of the ER
system and active cellular mechanisms, such as autophagy, can tightly control
their appearance. 相似文献
8.
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
9.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献
10.
Jaemin Lee Xiaofan Wang Bruno Di Jeso Peter Arvan 《The Journal of biological chemistry》2009,284(19):12752-12761
The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin
(Tg) has been identified as critically important in Tg export from the
endoplasmic reticulum. In a number of human kindreds suffering from congenital
hypothyroidism, and in the cog congenital goiter mouse and
rdw rat dwarf models, thyroid hormone synthesis is inhibited because
of mutations in the ChEL domain that block protein export from the endoplasmic
reticulum. We hypothesize that Tg forms homodimers through noncovalent
interactions involving two predicted α-helices in each ChEL domain that
are homologous to the dimerization helices of acetylcholinesterase. This has
been explored through selective epitope tagging of dimerization partners and
by inserting an extra, unpaired Cys residue to create an opportunity for
intermolecular disulfide pairing. We show that the ChEL domain is necessary
and sufficient for Tg dimerization; specifically, the isolated ChEL domain can
dimerize with full-length Tg or with itself. Insertion of an N-linked
glycan into the putative upstream dimerization helix inhibits homodimerization
of the isolated ChEL domain. However, interestingly, co-expression of upstream
Tg domains, either in cis or in trans, overrides the
dimerization defect of such a mutant. Thus, although the ChEL domain provides
a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL
domain actively stabilizes the Tg dimer complex for intracellular
transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of
thyroglobulin (Tg)2 to
the apical luminal cavity of thyroid follicles
(1). Once secreted, Tg is
iodinated via the activity of thyroid peroxidase
(2). A coupling reaction
involving a quinol-ether linkage especially engages di-iodinated tyrosyl
residues 5 and 130 to form thyroxine within the amino-terminal portion of the
Tg polypeptide (3,
4). Preferential iodination of
Tg hormonogenic sites is dependent not on the specificity of the peroxidase
(5) but upon the native
structure of Tg (6,
7). To date, no other thyroidal
proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746
amino acids) involves three regions called I-II-III comprised of
disulfide-rich repeat domains held together by intradomain disulfide bonds
(8,
9). The final 581 amino acids
of Tg are strongly homologous to acetylcholinesterase
(10–12).
Rate-limiting steps in the overall process of Tg secretion involve its
structural maturation within the endoplasmic reticulum (ER)
(13). Interactions between
regions I-II-III and the cholinesterase-like (ChEL) domain have recently been
suggested to be important in this process, with ChEL functioning as an
intramolecular chaperone and escort for I-II-III
(14). In addition, Tg
conformational maturation culminates in Tg homodimerization
(15,
16) with progression to a
cylindrical, and ultimately, a compact ovoid structure
(17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a
commonly affected site of mutation, including the recently described A2215D
(20,
21), R2223H
(22), G2300D, R2317Q
(23), G2355V, G2356R, and the
skipping of exon 45 (which normally encodes 36 amino acids), as well as the
Q2638stop mutant (24) (in
addition to polymorphisms including P2213L, W2482R, and R2511Q that may be
associated with thyroid overgrowth
(25)). As best as is currently
known, all of the congenital hypothyroidism-inducing Tg mutants are defective
for intracellular transport
(26). A homozygous G2300R
mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is
responsible for congenital hypothyroidism in rdw rats
(27,
28), whereas we identified the
Tg-L2263P point mutation as the cause of hypothyroidism in the cog
mouse (29). Such mutations
perturb intradomain structure
(30), and interestingly, block
homodimerization (31).
Acquisition of quaternary structure has long been thought to be required for
efficient export from the ER
(32) as exemplified by
authentic acetylcholinesterase
(33,
34) in which dimerization
enhances protein stability and export
(35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain)
is blocked within the ER (30),
whereas a secretory version of the isolated ChEL domain of Tg devoid of
I-II-III undergoes rapid and efficient intracellular transport and secretion
(14). A striking homology
positions two predicted α-helices of the ChEL domain to the identical
relative positions of the dimerization helices in acetylcholinesterase. This
raises the possibility that ChEL may serve as a homodimerization domain for
Tg, providing a critical function in maturation for Tg transport to the site
of thyroid hormone synthesis
(1).In this study, we provide unequivocal evidence for homodimerization of the
ChEL domain and “hetero”-dimerization of that domain with
full-length Tg, and we provide significant evidence that the predicted ChEL
dimerization helices provide a nidus for Tg assembly. On the other hand, our
data also suggest that upstream Tg regions known to interact with ChEL
(14) actively stabilize the Tg
dimer complex. Together, I-II-III and ChEL provide unique contributions to the
process of intracellular transport of Tg through the secretory pathway. 相似文献
11.
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.
As obligate intracellular parasites, viruses exploit diverse cellular
signaling machineries, including the mitogen-activated protein-kinase pathway,
during their infections. We have demonstrated previously that the open reading
frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90
ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities
(Kuang, E., Tang, Q., Maul, G. G., and Zhu, F.
(2008) J. Virol. 82
,1838
-1850). Here, we define the
mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45
to RSK increases the association of extracellular signal-regulated kinase
(ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass
protein complexes. We further demonstrated that the complexes shielded active
pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK
and ERK were activated and sustained at high levels. Finally, we provide
evidence that this mechanism contributes to the sustained activation of ERK
and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase
(ERK)2
mitogen-activated protein kinase (MAPK) signaling pathway has been implicated
in diverse cellular physiological processes including proliferation, survival,
growth, differentiation, and motility
(1-4)
and is also exploited by a variety of viruses such as Kaposi
sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human
immunodeficiency virus, respiratory syncytial virus, hepatitis B virus,
coxsackie, vaccinia, coronavirus, and influenza virus
(5-17).
The MAPK kinases relay the extracellular signaling through sequential
phosphorylation to an array of cytoplasmic and nuclear substrates to elicit
specific responses (1,
2,
18). Phosphorylation of MAPK
is reversible. The kinetics of deactivation or duration of signaling dictates
diverse biological outcomes
(19,
20). For example, sustained
but not transient activation of ERK signaling induces the differentiation of
PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells
(20-22).
During viral infection, a unique biphasic ERK activation has been observed for
some viruses (an early transient activation triggered by viral binding or
entry and a late sustained activation correlated with viral gene expression),
but the responsible viral factors and underlying mechanism for the sustained
ERK activation remain largely unknown
(5,
8,
13,
23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine
kinases that lie at the terminus of the ERK pathway
(1,
24-26).
In mammals, four isoforms are known, RSK1 to RSK4. Each one has two
catalytically functional kinase domains, the N-terminal kinase domain (NTKD)
and C-terminal kinase domain (CTKD) as well as a linker region between the
two. The NTKD is responsible for phosphorylation of exogenous substrates, and
the CTKD and linker region regulate RSK activation
(1,
24,
25). In quiescent cells ERK
binds to the docking site in the C terminus of RSK
(27-29).
Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase
(MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker
region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD
activation loop. The activated CTKD then phosphorylates Ser-380 in the linker
region, creating a docking site for 3-phosphoinositide-dependent protein
kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates
Ser-221 of RSK in the activation loop and activates the NTKD. The activated
NTKD autophosphorylates the serine residue near the ERK docking site, causing
a transient dissociation of active ERK from RSK
(25,
26,
28). The stimulation of
quiescent cells by a mitogen such as epidermal growth factor or a phorbol
ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually
results in a transient RSK activation that lasts less than 30 min. RSKs have
been implicated in regulating cell survival, growth, and proliferation.
Mutation or aberrant expression of RSK has been implicated in several human
diseases including Coffin-Lowry syndrome and prostate and breast cancers
(1,
24,
25,
30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma,
primary effusion lymphoma, and a subset of multicentric Castleman disease
(33,
34). Infection and
reactivation of KSHV activate multiple MAPK pathways
(6,
12,
35). Noticeably, the ERK/RSK
activation is sustained late during KSHV primary infection and reactivation
from latency (5,
6,
12,
23), but the mechanism of the
sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45,
an immediate early and also virion tegument protein of KSHV, interacts with
RSK1 and RSK2 and strongly stimulates their kinase activities
(23). We also demonstrated
that the activation of RSK plays an essential role in KSHV lytic replication
(23). In the present study we
determined the mechanism of ORF45-induced sustained ERK/RSK activation. We
found that ORF45 increases the association of RSK with ERK and protects them
from dephosphorylation, causing sustained activation of both ERK and RSK. 相似文献
14.
15.
16.
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
17.
Andrés Norambuena Claudia Metz Lucas Vicu?a Antonia Silva Evelyn Pardo Claudia Oyanadel Loreto Massardo Alfonso González Andrea Soza 《The Journal of biological chemistry》2009,284(19):12670-12679
Galectins have been implicated in T cell homeostasis playing complementary
pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent
pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase
D/phosphatidic acid signaling pathway that has not been reported for any
galectin before. Gal-8 increases phosphatidic signaling, which enhances the
activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a
subsequent decrease in basal protein kinase A activity. Strikingly, rolipram
inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4
activation releases a negative influence of cAMP/protein kinase A on ERK1/2.
The resulting strong ERK1/2 activation leads to expression of the death factor
Fas ligand and caspase-mediated apoptosis. Several conditions that decrease
ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking
antibodies. In addition, experiments with freshly isolated human peripheral
blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28,
show that Gal-8 is pro-apoptotic on activated T cells, most likely on a
subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic
lupus erythematosus block the apoptotic effect of Gal-8. These results
implicate Gal-8 as a novel T cell suppressive factor, which can be
counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly
studied as regulators of the immune response and potential therapeutic agents
for autoimmune disorders (1).
To date, 15 galectins have been identified and classified according with the
structural organization of their distinctive monomeric or dimeric carbohydrate
recognition domain for β-galactosides
(2,
3). Galectins are secreted by
unconventional mechanisms and once outside the cells bind to and cross-link
multiple glycoconjugates both at the cell surface and at the extracellular
matrix, modulating processes as diverse as cell adhesion, migration,
proliferation, differentiation, and apoptosis
(4–10).
Several galectins have been involved in T cell homeostasis because of their
capability to kill thymocytes, activated T cells, and T cell lines
(11–16).
Pro-apoptotic galectins might contribute to shape the T cell repertoire in the
thymus by negative selection, restrict the immune response by eliminating
activated T cells at the periphery
(1), and help cancer cells to
escape the immune system by eliminating cancer-infiltrating T cells
(17). They have also a
promising therapeutic potential to eliminate abnormally activated T cells and
inflammatory cells (1). Studies
on the mostly explored galectins, Gal-1, -3, and -9
(14,
15,
18–20),
as well as in Gal-2 (13),
suggest immunosuppressive complementary roles inducing different pathways to
apoptosis. Galectin-8
(Gal-8)4 is one of the
most widely expressed galectins in human tissues
(21,
22) and cancerous cells
(23,
24). Depending on the cell
context and mode of presentation, either as soluble stimulus or extracellular
matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis
(6,
7,
9,
10,
22,
25). Its role has been mostly
studied in relation to tumor malignancy
(23,
24). However, there is some
evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or
inflammatory disorders. For instance, the intrathymic expression and
pro-apoptotic effect of Gal-8 upon CD4highCD8high
thymocytes suggest a role for Gal-8 in shaping the T cell repertoire
(16). Gal-8 could also
modulate the inflammatory function of neutrophils
(26), Moreover Gal-8-blocking
agents have been detected in chronic autoimmune disorders
(10,
27,
28). In rheumatoid arthritis,
Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid
cells, but can be counteracted by a specific rheumatoid version of CD44
(CD44vRA) (27). In systemic
lupus erythematosus (SLE), a prototypic autoimmune disease, we recently
described function-blocking autoantibodies against Gal-8
(10,
28). Thus it is important to
define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in
immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with
specific integrins, such as α1β1, α3β1, and
α5β1 but not α4β1, and as a matrix protein promotes cell
adhesion and asymmetric spreading through activation of the extracellular
signal-regulated kinases 1 and 2 (ERK1/2)
(10). These early effects
occur within 5–30 min. However, ERK1/2 signaling supports long term
processes such as T cell survival or death, depending on the moment of the
immune response. During T cell activation, ERK1/2 contributes to enhance the
expression of interleukin-2 (IL-2) required for T cell clonal expansion
(29). It also supports T cell
survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by
other previously activated T cells
(30,
31). Later on, ERK1/2 is
required for activation-induced cell death, which controls the extension of
the immune response by eliminating recently activated and restimulated T cells
(32,
33). In activation-induced
cell death, ERK1/2 signaling contributes to enhance the expression of FasL and
its receptor Fas/CD95 (32,
33), which constitute a
preponderant pro-apoptotic system in T cells
(34). Here, we ask whether
Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to
participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling
(35) deserves special
attention. cAMP/PKA signaling plays an immunosuppressive role in T cells
(36) and is altered in SLE
(37). Phosphodiesterases
(PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA
during T cell activation (38,
39). PKA has been described to
control the activity of ERK1/2 either positively or negatively in different
cells and processes (35). A
little explored integration among ERK1/2 and PKA occurs via phosphatidic acid
(PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that
hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA
plays roles in signaling interacting with a variety of targeting proteins that
bear PA-binding domains (40).
In this way PA recruits Raf-1 to the plasma membrane
(41). It is also converted by
phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG),
which among other functions, recruits and activates the GTPase Ras
(42). Both Ras and Raf-1 are
upstream elements of the ERK1/2 activation pathway
(43). In addition, PA binds to
and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP
levels and PKA down-regulation
(44). The regulation and role
of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell
homeostasis, because it is also unknown whether galectins stimulate the PLD/PA
pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering
cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our
results for the first time show that a galectin increases the PA levels,
down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE
activity, and induces an ERK1/2-dependent expression of the pro-apoptotic
factor FasL. The enhanced PDE activity induced by Gal-8 is required for the
activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces
apoptosis in human peripheral blood mononuclear cells (PBMC), especially after
activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other
galectins the property of killing activated T cells contributing to the T cell
homeostasis. The pathway involves a particularly integrated signaling context,
engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for
galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its
susceptibility to inhibition by anti-Gal-8 autoantibodies. 相似文献
18.
19.
Sean R. Stowell Moonjae Cho Christa L. Feasley Connie M. Arthur Xuezheng Song Jennifer K. Colucci Sougata Karmakar Padmaja Mehta Marcelo Dias-Baruffi Rodger P. McEver Richard D. Cummings 《The Journal of biological chemistry》2009,284(8):4989-4999
Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell
surface exposure of phosphatidylserine (PS), a ligand that targets cells for
phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer
equilibrium appears to modulate Gal-1-induced PS exposure, although the
mechanism underlying this regulation remains unclear. Here we show that
monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant
form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired
dimerization and fails to induce cell surface PS exposure while retaining the
ability to recognize carbohydrates and signal Ca2+ flux in
leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas
ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1
dimerization. Continual incubation of leukocytes with Gal-1 resulted in
gradual oxidative inactivation with concomitant loss of cell surface PS,
whereas rapid oxidation prevented mGal-1 from inducing PS exposure.
Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and
mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to
signal sustained PS exposure in leukocytes and mGal-1 to signal both
Ca2+ flux and PS exposure. Taken together, these results
demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to
oxidative inactivation and provides a mechanism whereby ligand partially
protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated
leukocytes following an inflammatory episode. Several factors, including
members of the galectin and tumor necrosis factor families
(1,
2), regulate leukocyte turnover
by inducing apoptotic cell death. In contrast, several galectin family
members, in particular galectin-1
(Gal-1),2 uniquely
regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure,
which normally sensitizes apoptotic cells to phagocytic removal
(3,
4), independent of apoptosis, a
process recently termed preaparesis
(5).Previous studies suggested that dimerization may be required for
Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two
point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays
impaired Gal-1 dimerization and fails to induce PS exposure
(6). However, the manner in
which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear.
Previous studies suggest that dimerization may be required for efficient
cross-linking of functional receptors or the formation of signaling lattices
(7–9).
Consistent with this, monomeric mutants of several other galectins fail to
induce PS exposure or signal leukocytes
(4,
8). Gal-1 signaling of PS
exposure requires initial signaling events, such as mobilization of
intracellular Ca2+ followed by sustained receptor engagement
(10). Although mGal-1 fails to
induce PS exposure (6), whether
mGal-1 can induce these initial signaling events remains unknown
(10).In addition to directly regulating signaling, monomer-dimer equilibrium may
also regulate other aspects of Gal-1 function. Unlike many other proteins
involved in the regulation of immunity, Gal-1 displays unique sensitivity to
oxidative inactivation
(11–15).
Although engagement of ligand partially protects Gal-1 from oxidation
(15), the impact of Gal-1
oxidation on signaling remains enigmatic. During oxidation, Gal-1 forms three
distinct intramolecular disulfide bridges that facilitate profound
conformational changes that preclude ligand binding and Gal-1 dimerization
(12–14),
suggesting that monomerdimer equilibrium may also regulate Gal-1 sensitivity
to oxidative inactivation.Previous studies utilized dithiothreitol (DTT) in treatment conditions to
protect Gal-1 from oxidative inactivation
(16,
17). Indeed, failure to
include DTT precluded Gal-1-induced death in T cells
(3,
18), suggesting that Gal-1
undergoes rapid oxidation in vivo in the absence of reducing
conditions. However, DTT itself can induce apoptosis in leukocytes
(19), leaving questions
regarding the impact of Gal-1 oxidation on these signaling events. In
contrast, recent studies utilizing iodoacetamide-alkylated Gal-1 (iGal-1),
previously shown to protect Gal-1 from oxidative inactivation
(20–29),
demonstrated that DTT actually primes cells to become sensitive to
Gal-1-induced apoptosis regardless of Gal-1 sensitivity to oxidation
(5).As the engagement of leukocyte ligands requires glycan recognition and
oxidation precludes this binding
(11,
15), understanding the impact
of oxidation on Gal-1 signals will facilitate a greater appreciation of the
factors that govern Gal-1 oxidation and therefore function. Our results
demonstrate that Gal-1 monomer-dimer equilibrium provides a key regulatory
point controlling both Gal-1 sensitivity to oxidation and its ability to
signal PS exposure in leukocytes. These results provide novel insights into
Gal-1 function and explain at a biochemical level the mechanisms regulating
Gal-1 oxidative inactivation and signaling. 相似文献
20.
John W. Hardin Francis E. Reyes Robert T. Batey 《The Journal of biological chemistry》2009,284(22):15317-15324
In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are
responsible for 2′-O-methylation of tRNAs and rRNAs. The
archaeal box C/D small RNP complex requires a small RNA component (sRNA)
possessing Watson-Crick complementarity to the target RNA along with three
proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from
S-adenosylmethionine to the target RNA is performed by fibrillarin,
which by itself has no affinity for the sRNA-target duplex. Instead, it is
targeted to the site of methylation through association with Nop5p, which in
turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a
bridge between the targeting and catalytic functions of the box C/D small RNP
complex, we have employed alanine scanning to evaluate the interaction between
the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA
complex. From these data, we were able to construct an isolated RNA-binding
domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography
and binds to the L7Ae box C/D RNA complex with near wild type affinity. These
data demonstrate that the Nop-RBD is an autonomously folding and functional
module important for protein assembly in a number of complexes centered on the
L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full
functionality in the cell (1).
Currently there are over 100 known RNA modification types ranging from small
functional group substitutions to the addition of large multi-cyclic ring
structures (2). Transfer RNA,
one of many functional RNAs targeted for modification
(3-6),
possesses the greatest modification type diversity, many of which are
important for proper biological function
(7). Ribosomal RNA, on the
other hand, contains predominantly two types of modified nucleotides:
pseudouridine and 2′-O-methylribose
(8). The crystal structures of
the ribosome suggest that these modifications are important for proper folding
(9,
10) and structural
stabilization (11) in
vivo as evidenced by their strong tendency to localize to regions
associated with function (8,
12,
13). These roles have been
verified biochemically in a number of cases
(14), whereas newly emerging
functional modifications are continually being investigated.Box C/D ribonucleoprotein
(RNP)3 complexes serve
as RNA-guided site-specific 2′-O-methyltransferases in both
archaea and eukaryotes (15,
16) where they are referred to
as small RNP complexes and small nucleolar RNPs, respectively. Target RNA
pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl
group of the nucleotide five bases upstream of either the D or D′ box
motif of the sRNA (Fig. 1,
star) (17,
18). In archaea, the internal
C′ and D′ motifs generally conform to a box C/D consensus sequence
(19), and each sRNA contains
two guide regions ∼12 nucleotides in length
(20). The bipartite
architecture of the RNP potentially enables the complex to methylate two
distinct RNA targets (21) and
has been shown to be essential for site-specific methylation
(22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components
of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D
sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D,
C′, and D′ boxes are labeled. The target RNA binds the sRNA
through Watson-Crick pairing and is methylated by fibrillarin at the fifth
nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three
proteins for activity (23):
the ribosomal protein L7Ae
(24,
25), fibrillarin, and the
Nop56/Nop58 homolog Nop5p (Fig.
1). L7Ae binds to both box C/D and the C′/D′ motifs
(26), which respectively
comprise kink-turn (27) or
k-loop structures (28), to
initiate the assembly of the RNP
(29,
30). Fibrillarin performs the
methyl group transfer from the cofactor S-adenosylmethionine to the
target RNA
(31-33).
For this to occur, the active site of fibrillarin must be positioned precisely
over the specific 2′-hydroxyl group to be methylated. Although
fibrillarin methylates this functional group in the context of a Watson-Crick
base-paired helix (guide/target), it has little to no binding affinity for
double-stranded RNA or for the L7Ae-sRNA complex
(22,
26,
33,
34). Nop5p serves as an
intermediary protein bringing fibrillarin to the complex through its
association with both the L7Ae-sRNA complex and fibrillarin
(22). Along with its role as
an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses
other functions not yet fully understood. For example, Nop5p self-dimerizes
through a coiled-coil domain
(35) that in most archaea and
eukaryotic homologs includes a small insertion sequence of unknown function
(36,
37). However, dimerization and
fibrillarin binding have been shown to be mutually exclusive in
Methanocaldococcus jannaschii Nop5p, potentially because of the
presence of this insertion sequence
(36). Thus, whether Nop5p is a
monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate
its interaction with a L7Ae box C/D RNA complex because both the
fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal
structures (29,
35,
38). Individual residues on
the surface of a monomeric form of Nop5p (referred to as mNop5p)
(22) were mutated to alanine,
and the effect on binding affinity for a L7Ae box C/D motif RNA complex was
assessed through the use of electrophoretic mobility shift assays. These data
reveal that residues important for binding cluster within the highly conserved
NOP domain (39,
40). To demonstrate that this
domain is solely responsible for the affinity of Nop5p for the preassembled
L7Ae box C/D RNA complex, we expressed and purified it in isolation from the
full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA
complex with nearly wild type affinity, demonstrating that the Nop-RBD is
truly an autonomously folding and functional module. Comparison of our data
with the crystal structure of the homologous spliceosomal hPrp31-15.5K
protein-U4 snRNA complex (41)
suggests the adoption of a similar mode of binding, further supporting a
crucial role for the NOP domain in RNP complex assembly. 相似文献