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1.
Xiaojun Li C. T. Ranjith-Kumar Monica T. Brooks S. Dharmaiah Andrew B. Herr Cheng Kao Pingwei Li 《The Journal of biological chemistry》2009,284(20):13881-13891
The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded
RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate
innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that
lacks signaling capability, regulates the signaling of the RLRs. To establish
the structural basis of dsRNA recognition by the RLRs, we have determined the
2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound
to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of
dsRNA with minimal contacts between the protein molecules. Gel filtration
chromatography and analytical ultracentrifugation demonstrated that LGP2 binds
blunt-ended dsRNA of different lengths, forming complexes with 2:1
stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do
not stimulate the activation of RIG-I efficiently in cells. Surprisingly,
full-length LGP2 containing mutations that abolish dsRNA binding retained the
ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading
pathogens; it is the ubiquitous system of defense against microbial infections
(1). Toll-like receptors
(TLRs)3 and RIG-I
(retinoic acid-inducible gene
1)-like receptors (RLRs) play key roles in innate immune response
toward viral infection
(2-5).
Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the
endosome following phagocytosis of the pathogens
(6). RIG-I-like receptors RIG-I
and MDA5 detect viral RNA from replicating viruses in infected cells
(3,
7,
8). Stimulation of these
receptors leads to the induction of type I interferons (IFNs) and other
proinflammatory cytokines, conferring antiviral activity to the host cells and
activating the acquired immune responses
(4,
9).RIG-I discriminates between viral and host RNA through specific recognition
of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp
ssRNA) generated by viral RNA polymerases
(10,
11). In addition, RIG-I also
recognizes double-stranded RNA generated during RNA virus replication
(7,
12). Transfection of cells
with synthetic double-stranded RNA stimulates the activation of RIG-I
(13,
14). Synthetic dsRNA mimics,
such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5
when introduced into the cytoplasm of cells. Digestion of poly(I·C)
with RNase III transforms poly(I·C) from a ligand for MDA5 into a
ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I
recognizes short dsRNA (15).
Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of
these receptors in antiviral immune responses and demonstrated that they sense
different sets of RNA viruses
(12,
16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N
termini, a DEX(D/H) box RNA helicase domain, and a C-terminal
regulatory or repressor domain (CTD). The helicase domain and the CTD are
responsible for viral RNA binding, whereas the CARDs are required for
signaling (3,
8). The current model of RIG-I
activation suggests that under resting conditions RIG-I is in a suppressed
conformation, and viral RNA binding triggers a conformation change that leads
to the exposure of the CARDs for the recruitment of the downstream protein
IPS-1 (also known as MAVS, Cardif, or VISA)
(14,
17). Limited proteolysis of
the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD
are involved in dsRNA and 5′ ppp ssRNA binding
(14). The CTD of RIG-I
overlaps with the C terminus of the previously identified repressor domain
(18). The structures of RIG-I
and LGP2 (laboratory of genetics and
physiology 2) CTD in isolation have been determined by
x-ray crystallography and NMR spectroscopy
(14,
19,
20). A large, positively
charged surface on RIG-I recognizes the 5′ triphosphate group of viral
ssRNA (14,
19). RNA binding studies by
titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that
overlapping sets of residues on this charged surface are involved in RNA
binding (14). Mutagenesis of
several positively charged residues on this surface either reduces or disrupts
RNA binding by RIG-I, and these mutations also affect the induction of
IFN-β in vivo
(14,
19). However, the exact nature
of how the RLRs recognize viral RNA and how RNA binding activates these
receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no
signaling capability (21,
22). The expression of LGP2 is
inducible by dsRNA or IFN treatment as well as virus infection
(21). Overexpression of LGP2
inhibits Sendai virus and Newcastle disease virus signaling
(21). When coexpressed with
RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD
with the CARD and the helicase domain of RIG-I
(18). LGP2 could suppress
RIG-I signaling by three possible ways
(23): 1) binding RNA with high
affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly
with RIG-I to block the assembly of the signaling complex; and 3) competing
with IKKi (IκB kinase ε) in the NF-κB signaling pathway for a
common binding site on IPS-1. To elucidate the structural basis of dsRNA
recognition by the RLRs, we have crystallized human LGP2 CTD (residues
541-678) bound to an 8-bp double-stranded RNA and determined the structure of
the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD
binds to the termini of dsRNA. Mutagenesis and functional studies showed that
dsRNA binding is likely not required for the inhibition of RIG-I signaling by
LGP2. 相似文献
2.
Jee-Yeon Noh Huikyong Lee Sungmin Song Nam Soon Kim Wooseok Im Manho Kim Hyemyung Seo Chul-Woong Chung Jae-Woong Chang Robert J. Ferrante Young-Jun Yoo Hoon Ryu Yong-Keun Jung 《The Journal of biological chemistry》2009,284(17):11318-11325
Accumulation of expanded polyglutamine proteins is considered to be a major
pathogenic biomarker of Huntington disease. We isolated SCAMP5 as a novel
regulator of cellular accumulation of expanded polyglutamine track protein
using cell-based aggregation assays. Ectopic expression of SCAMP5 augments the
formation of ubiquitin-positive and detergent-resistant aggregates of mutant
huntingtin (mtHTT). Expression of SCAMP5 is markedly increased in the striatum
of Huntington disease patients and is induced in cultured striatal neurons by
endoplasmic reticulum (ER) stress or by mtHTT. The increase of SCAMP5 impairs
endocytosis, which in turn enhances mtHTT aggregation. On the contrary,
down-regulation of SCAMP5 alleviates ER stress-induced mtHTT aggregation and
endocytosis inhibition. Moreover, stereotactic injection into the striatum and
intraperitoneal injection of tunicamycin significantly increase mtHTT
aggregation in the striatum of R6/2 mice and in the cortex of N171-82Q mice,
respectively. Taken together, these results suggest that exposure to ER stress
increases SCAMP5 in the striatum, which positively regulates mtHTT aggregation
via the endocytosis pathway.The expansion of CAG repeats (usually beyond a critical threshold of
∼37 glutamine repeats) encoding polyglutamine
(polyQ)3 causes, to
date, nine late-onset progressive neurodegenerative disorders
(1,
2). Expanded polyQ-containing
huntingtin is the main aggregate component in the affected neurons
(3). Also, molecular
chaperones, such as Hsp70, Hsp40/HDJ1 (dHDJ1), and chaperonin TRiC, perturb
the aggregation of polyQ track protein and reduce polyQ track cytotoxicity in
yeast and cell lines
(4–6)
and in Drosophila and mouse models
(4,
7). Thus, it seems that HD
pathology is closely correlated with the accumulation of insoluble aggregates
of mutant huntingtin (mtHTT) containing expanded polyQ
(2,
3,
8,
9).Endoplasmic reticulum (ER) stress is crucial in many biological responses
and is generated by various signals, such as unfolded protein response,
aberrant calcium regulation, oxidative stress, and inflammation
(10,
11). ER stress response is
generally considered an adaptive reaction of cells to environmental stress,
serving as a survival signal
(10). On the other hand,
increasing evidence also strengthens the importance of ER stress in human
diseases. A malfunction or excess of ER stress response caused by aging,
genetic mutations, and environmental insults is implicated in human diseases,
such as Alzheimer disease, Parkinson disease, diabetes mellitus, and
inflammation
(12–16).
mtHTT also induces ER stress at the early stage of HD, and pathogenic ER
stress from an aging or stressful environment is severe at the late stage of
HD
(17–19).
However, the molecular event linking the aggregation of polyQ track protein to
ER stress response is unknown.The ubiquitin/proteasome pathway, a major protein degradation system, is
altered or impaired in the cell culture model of HD
(20–22).
On the contrary, autophagy employing lysosomal degradation has been recently
considered as a major clearance pathway of insoluble aggregates of polyQ track
protein. Thus, inhibition of autophagy has been suggested to modulate the
aggregate formation of mtHTT and to affect the toxicity of polyglutamine
expansions in fly and mouse models of HD
(23–25).
However, a key molecule controlling the aggregation and clearance of polyQ
track proteins needs to be identified.To further our understanding of the regulation of polyQ track protein
aggregation, we screened human full-length cDNAs and isolated
SCAMP5 (secretory carrier membrane
protein 5) as a modulator of polyQ track protein
aggregation. SCAMP5 is up-regulated by mtHTT and ER stress and functions to
inhibit endocytosis to increase mtHTT aggregation. 相似文献
3.
Parmil K. Bansal Amanda Nourse Rashid Abdulle Katsumi Kitagawa 《The Journal of biological chemistry》2009,284(6):3586-3592
The kinetochore, which consists of DNA sequence elements and structural
proteins, is essential for high-fidelity chromosome transmission during cell
division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore
complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study,
we show that Sgt1 forms homodimers by performing in vitro and in
vivo immunoprecipitation and analytical ultracentrifugation analyses.
Analyses of the dimerization of Sgt1 deletion proteins showed that the
Skp1-binding domain (amino acids 1–211) contains the Sgt1
homodimerization domain. Also, the Sgt1 mutant proteins that were unable to
dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is
important for Sgt1-Skp1 binding. Restoring dimerization activity of a
dimerization-deficient sgt1 mutant (sgt1-L31P) by using the
CENP-B (centromere protein-B) dimerization
domain suppressed the temperature sensitivity, the benomyl sensitivity, and
the chromosome missegregation phenotype of sgt1-L31P. These results
strongly suggest that Sgt1 dimerization is required for kinetochore
assembly.Spindle microtubules are coupled to the centromeric region of the
chromosome by a structural protein complex called the kinetochore
(1,
2). The kinetochore is thought
to generate a signal that arrests cells during mitosis when it is not properly
attached to microtubules, thereby preventing aberrant chromosome transmission
to the daughter cells, which can lead to tumorigenesis
(3,
4). The kinetochore of the
budding yeast Saccharomyces cerevisiae has been characterized
thoroughly, genetically and biochemically; thus, its molecular structure is
the most well detailed to date. More than 70 different proteins comprise the
budding yeast kinetochore, and several of those are conserved in mammals
(2).The budding yeast centromere DNA is a 125-bp region that contains three
conserved regions, CDEI, CDEII, and CDEIII
(5,
6). CDEI is bound by Cbf1
(7–9).
CDEIII (25 bp) is essential for centromere function
(10) and is the site where
CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13
(11–18),
and Skp1 (17,
18), all of which are
essential for viability. Mutations in any of the four CBF3 proteins abolish
the ability of CDEIII to bind to CBF3
(19,
20). All of the described
kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores
dependent on the CBF3 complex
(2). Therefore, the CBF3
complex is the fundamental structure of the kinetochore, and the mechanism of
CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4
kinetochore-defective mutant dosage suppressor
(21). Sgt1 and Skp1 activate
Ctf13; thus, they are required for assembly of the CBF3 complex
(21). The molecular chaperone
Hsp90 is also required for the formation of the Skp1-Ctf13 complex
(22). Sgt1 has two highly
conserved motifs that are required for protein-protein interaction, the
tetratricopeptide repeat
(TPR)2
(21) and the CS
(CHORD protein- and Sgt1-specific) motif. We and others
(23–26)
have found that both domains are important for the interaction with Hsp90. The
Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore
complex; this interaction is an initial step in kinetochore assembly
(24,
26,
27) that is conserved between
yeast and humans (28,
29).In this study, we further characterized the molecular mechanism of this
assembly process. We found that Sgt1 forms dimers in vivo, and our
results strongly suggest that Sgt1 dimerization is required for kinetochore
assembly in budding yeast. 相似文献
4.
5.
6.
7.
Subbareddy Maddika Shirley M.-H. Sy Junjie Chen 《The Journal of biological chemistry》2009,284(19):12998-13003
Proper activation of checkpoint during mitotic stress is an important
mechanism to prevent genomic instability. Chfr (Check point protein
with FHA (Forkhead-associated domain) and RING domains)
is a ubiquitin-protein isopeptide ligase (E3) that is important for the
control of an early mitotic checkpoint, which delays entry into metaphase in
response to mitotic stress. Because several lines of evidence indicate that
Chfr is a potential tumor suppressor, it is critically important for us to
identify Chfr substrates and understand how Chfr may regulate these
substrates, control mitotic transitions, and thus, act as a tumor suppressor
in vivo. Here, we report the discovery of a new Chfr-associated
protein Kif22, a chromokinesin that binds to both DNA and microtubules. We
demonstrated that Kif22 is a novel substrate of Chfr. We showed that
Chfr-mediated Kif22 down-regulation is critical for the maintenance of
chromosome stability. Collectively, our results reveal a new substrate of Chfr
that plays a role in the maintenance of genome integrity.Chfr (Check point protein with FHA and
RING domains) is an early mitotic checkpoint protein that delays
entry into metaphase in response to mitotic stress
(1,
2). The checkpoint function of
Chfr requires both of its
FHA3 and RING domains.
The exact role of FHA domain in Chfr function is largely unknown. Chfr via its
RING domain transfers both lysine 48-linked and lysine 63-linked polyubiquitin
chains to its target proteins, which either promotes the degradation of target
proteins or alters their function
(3,
4). Recently, a PAR-binding
zinc finger motif, which binds directly to polyADP-ribosylated substrates
catalyzed by PARP1, was identified at the C-terminal region of Chfr
(5). This PAR-binding zinc
finger motif was reported to be required for Chfr function in antephase
checkpoint (2,
5).Chfr delays the cell cycle progression at mitosis by inactivating cyclin
B1-bound Cdc2 and then exporting them from nucleus
(6). Further, mechanistic
studies have suggested that the inactivation of Cdc2 may be due to a negative
regulation of Plk1 by Chfr (3).
Polyubiquitination of Plk1 by Chfr negatively regulates the Plk1 protein
levels, which delay the inactivation of Cdc2 inhibitory Wee1 kinase and the
activation of Cdc25 phosphatase and thus maintain Cdc2 at its inactive
state.Several lines of evidence indicate that Chfr is a potential tumor
suppressor. Loss or down-regulation of Chfr has been reported in several types
of cancers including primary breast, lung, esophagus, colon, and gastric
carcinomas (1,
7,
8). To investigate directly
whether Chfr loss contributes to tumorigenesis, our laboratory has generated
Chfr knock-out mice, which were cancer-prone and developed spontaneous tumors
(9). The increased tumor
incidence in Chfr null mice is likely due to a failure in maintaining
chromosomal stability, which occurs at least partially due to the
overexpression of a key mitotic kinase Aurora A
(9). Chfr physically interacts
with Aurora A and promotes its ubiquitination and degradation; thus, higher
protein levels of Aurora A in Chfr null mice may contribute to chromosomal
instability and eventually tumorigenesis. Therefore, our current hypothesis is
that Chfr may regulate the stability of several of its substrates including
Aurora A, and thus, control mitotic progression and prevent chromosomal
instability. In this study, we reported the identification of another Chfr
substrate as chromokinesin protein Kif22 and revealed that Kif22
overexpression also contributes to chromosomal instability observed in
Chfr-deficient cells. 相似文献
8.
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
9.
10.
Il-Ha Lee Craig R. Campbell Sung-Hee Song Margot L. Day Sharad Kumar David I. Cook Anuwat Dinudom 《The Journal of biological chemistry》2009,284(19):12663-12669
It has recently been shown that the epithelial Na+ channel
(ENaC) is compartmentalized in caveolin-rich lipid rafts and that
pharmacological depletion of membrane cholesterol, which disrupts lipid raft
formation, decreases the activity of ENaC. Here we show, for the first time,
that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the
activity and membrane surface expression of ENaC. Physical interaction between
ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found
that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin
protein ligase of the Nedd4 family, which is known to induce ubiquitination
and internalization of ENaC. The effect of Cav-1 on ENaC requires the
proline-rich motifs at the C termini of the β- and γ-subunits of
ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken
together, our data suggest that Cav-1 inhibits the activity of ENaC by
decreasing expression of ENaC at the cell membrane via a mechanism that
involves the promotion of Nedd4-2-dependent internalization of the
channel.Amiloride-sensitive epithelial Na+ channels
(ENaC)3 are membrane
proteins that are expressed in salt-absorptive epithelia, including the distal
collecting tubules of the kidney, the mucosa of the distal colon, the
respiratory epithelium, and the excretory ducts of sweat and salivary glands
(1–4).
Na+ absorption via ENaC is critical to the normal regulation of
Na+ and fluid homeostasis and is important for maintaining blood
pressure (5) and the volume of
fluid in the respiratory passages
(6). Increased ENaC activity
has been implicated in the salt-sensitive inherited form of hypertension,
Liddle''s syndrome (7), and
dehydration of the surface of the airway epithelium in the pathology
associated with cystic fibrosis lung disease
(8).Expression of ENaC at the cell membrane surface is regulated by the E3
ubiquitin protein ligase, Nedd4-2 (neural precursor cell
expressed developmentally down-regulated
protein 4) (9). Interaction
between the WW domains of Nedd4-2 and the proline-rich PY motifs
(PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect
on the channel (10,
11). This interaction leads to
ubiquitination-dependent internalization of ENaC
(12,
13). Several regulators of
ENaC exert their effects on the channel by modulating the action of Nedd4-2.
For instance, serum and glucocorticoid-dependent protein kinase
(14), protein kinase B
(15), and G protein-coupled
receptor kinase (16)
up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of
cellular mechanisms that underlie internalization of ENaC remain to be
elucidated, the physiological significance of Nedd4-dependent internalization
of the channel has been well established. For instance, heritable mutations
that delete the cytosolic termini of the β-or γ-subunit of ENaC,
which contain the proline-rich motifs, are known to cause hyperactivity of
ENaC in the kidney (17) and
increase cell surface expression of the channel
(7,
18).The plasma membranes of most cell types contain lipid raft microdomains
that are enriched with glycosphingolipid and cholesterol
(19), that have distinctive
biophysical properties, and that selectively include or exclude signaling
molecules (20). These
microdomains promote clustering of an array of integral membrane proteins in
the membrane leaflets (21) and
may be important for organizing cascades of signaling molecules
(22,
23). Processes in which raft
microdomains are involved include the intracellular transport of proteins and
lipids to the cell membrane
(24), the endocytotic
retrieval of membrane proteins
(25,
26), and signal transduction
(27,
28). In addition, segregation
of signaling molecules within lipid rafts may facilitate cross-talk between
signal transduction pathways
(29), a phenomenon that may be
important in ensuring rapid and efficient integration of multiple cellular
signaling events (30,
31). Of particular interest is
the subpopulation of lipid rafts enriched with caveolin proteins. Caveolin-1
(Cav-1), a major caveolin isoform expressed in nonmuscle cells, has been
identified as being involved in diverse cellular functions, such as vesicular
transport, cholesterol homeostasis, and signal transduction
(32). Cav-1 also regulates the
activity and membrane expression of ion channels and transporters
(28).In epithelia, the majority of lipid rafts exist at the apical membrane
surface (22). Pools of ENaC
(33–36)
and several proteins that regulate activity of ENaC, such as Nedd4
(37), protein kinase B
(38), protein kinase C
(39), Go
(40), and the G
protein-coupled receptor kinase
(41), have been identified in
detergent-insoluble and cholesterol-rich membrane fractions from a variety of
cell types, consistent with localization of these proteins in lipid rafts.
Furthermore, detergent-free buoyant density separation of lipid rafts has
revealed the presence of Cav-1 with ENaC in the lipid raft-rich membrane
fraction (35). The
physiological role of lipid rafts in the regulation of ENaC has been the
subject of many recent investigations. Most of these studies used a
pharmacological agent, methyl-β-cyclodextrin (MβCD), to promote
redistribution of proteins away from the cholesterol-enriched membrane
domains. The results were, however, inconclusive. In some studies, MβCD
treatment was found to inhibit open probability
(42) or cell surface
expression of ENaC (35),
whereas others found no direct effect of MβCD on the channel
(33,
43).Despite a number of studies into the role of lipid rafts on the regulation
of ENaC, little is known about the physiological relevance of caveolins to the
function of this ion channel. In the present study, we use gene interference
and gene expression techniques to determine the role of Cav-1 in the
regulation of ENaC activity. We provide evidence of the association of Cav-1
with ENaC and evidence that Cav-1 negatively regulates both activity and
abundance of ENaC at the surface of epithelial cells. Importantly, we
demonstrate, for the first time, that the mechanism by which Cav-1 regulates
activity of ENaC involves the E3 ubiquitin protein ligase, Nedd4-2. 相似文献
11.
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
12.
Aggregation of the Ure2 protein is at the origin of the [URE3]
prion trait in the yeast Saccharomyces cerevisiae. The N-terminal
region of Ure2p is necessary and sufficient to induce the [URE3]
phenotype in vivo and to polymerize into amyloid-like fibrils in
vitro. However, as the N-terminal region is poorly ordered in the native
state, making it difficult to detect structural changes in this region by
spectroscopic methods, detailed information about the fibril assembly process
is therefore lacking. Short fibril-forming peptide regions (4–7
residues) have been identified in a number of prion and other amyloid-related
proteins, but such short regions have not yet been identified in Ure2p. In
this study, we identify a unique cysteine mutant (R17C) that can greatly
accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions.
We found that the segment QVNI, corresponding to residues 18–21 in
Ure2p, plays a critical role in the fast assembly properties of R17C,
suggesting that this segment represents a potential amyloid-forming region. A
series of peptides containing the QVNI segment were found to form fibrils
in vitro. Furthermore, the peptide fibrils could seed fibril
formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a
hydrophobic residue, instead of a charged residue, caused the rate of assembly
into fibrils to increase greatly for both peptides and full-length Ure2p. Our
results indicate that the potential amyloid stretch and its preceding residue
can modulate the fibril assembly of Ure2p to control the initiation of prion
formation.The [URE3] phenotype of Saccharomyces cerevisiae arises
because of conversion of the Ure2 protein to an aggregated propagatable prion
state (1,
2). Ure2p contains two regions:
a poorly structured N-terminal region and a compactly folded C-terminal region
(3,
4). The N-terminal region is
rich in Asn and Gln residues, is highly flexible, and is without any
detectable ordered secondary structure
(4–6).
This region is necessary and sufficient for prion behavior in vivo
(2) and amyloid-forming
capacity in vitro (5,
7), so it is referred to as the
prion domain (PrD).2
The C-terminal region has a fold similar to the glutathione
S-transferase superfamily
(8,
9) and possesses
glutathione-dependent peroxidase activity
(10). Upon fibril formation,
the N-terminal region undergoes a significant conformational change from an
unfolded to a thermally resistant conformation
(11), whereas the glutathione
S-transferase-like C-terminal domain retains its enzymatic activity,
suggesting that little conformational change occurs
(10,
12). Ure2p fibrils show
various morphologies, including variations in thickness and the presence or
absence of a periodic twist
(13–16).
The overall structure of the fibrils imaged by cryoelectron microscopy
suggests that the intact fibrils contain a 4-nm amyloid filament backbone
surrounded by C-terminal globular domains
(17).It is widely accepted that disulfide bonds play a critical role in
maintaining protein stability
(18–21)
and also affect the process of protein folding by influencing the folding
pathway
(22–25).
A recent study shows that the presence of a disulfide bond in a protein can
markedly accelerate the folding process
(26). Therefore, a disulfide
bond is a useful tool to study protein folding. In the study of prion and
other amyloid-related proteins, cysteine scanning has been widely used to
study the structure of amyloid fibrils, the driving force of amyloid
formation, and the plasticity of amyloid fibrils
(13,
27–31).Short segments from amyloid-related proteins, including IAPP
(islet amyloid polypeptide),
β2-microglobulin, insulin, and the amyloid-β peptide,
show amyloid-forming capacity
(32–34).
Hence, the amyloid stretch hypothesis has been proposed, which suggests that a
short amino acid stretch bearing a highly amyloidogenic motif might supply
most of the driving force needed to trigger the self-catalytic assembly
process of a protein to form fibrils
(35,
36). In support of this
hypothesis, it was found that the insertion of an amyloidogenic stretch into a
non-amyloid-related protein can trigger the amyloidosis of the protein
(36). At the same time, the
structural information obtained from microcrystals formed by amyloidogenic
stretches and bearing cross-β-structure has contributed significantly to
our understanding of the structure of intact fibrils at the atomic level
(34,
37). However, no amyloidogenic
stretches <10 amino acids have so far been identified in the yeast prion
protein Ure2.In this study, we performed a cysteine scan within the N-terminal PrD of
Ure2p and found a unique cysteine mutant (R17C) that eliminates the lag phase
of the Ure2p fibril assembly reaction upon the addition of oxidizing agents.
Furthermore, we identified a 4-residue region adjacent to Arg17 as
a potential amyloid stretch in Ure2p. 相似文献
13.
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
14.
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
15.
16.
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
17.
18.
Eva Brombacher Simon Urwyler Curdin Ragaz Stefan S. Weber Keiichiro Kami Michael Overduin Hubert Hilbi 《The Journal of biological chemistry》2009,284(8):4846-4856
The causative agent of Legionnaires disease, Legionella
pneumophila, forms a replicative vacuole in phagocytes by means of the
intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV
secretion system and translocated effector proteins, some of which subvert
host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC
anchors to the membrane of Legionella-containing vacuoles (LCVs) by
specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a
nonbiased screen for novel L. pneumophila PI-binding proteins, we
identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the
predominant PtdIns(4)P-binding protein. Purified SidM specifically and
directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate
LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L.
pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding
domain of SidM was mapped to the 12-kDa C-terminal sequence, termed
“P4M” (PtdIns4P binding of
SidM/DrrA). The isolated P4M domain is largely helical and
displayed higher PtdIns(4)P binding activity in the context of the
α-helical, monomeric full-length protein. SidM constructs containing P4M
were translocated by Icm/Dot-proficient L. pneumophila and localized
to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via
its P4M domain. An L. pneumophila ΔsidM mutant strain
displayed significantly higher amounts of SidC on LCVs, suggesting that SidM
and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally,
RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by
host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits
PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC
and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative
agent of Legionnaires disease, but it evolved as a parasite of various species
of environmental predatory protozoa, including the social amoeba
Dictyostelium discoideum
(1,
2). The human disease is linked
to the inhalation of contaminated aerosols, followed by replication in
alveolar macrophages. To accommodate the transfer between host cells, L.
pneumophila alternates between replicative and transmissive phases, the
regulation of which includes an apparent quorum-sensing system
(3–5).In macrophages and amoebae, L. pneumophila forms a replicative
compartment, the Legionella-containing vacuole
(LCV).3 LCVs avoid
fusion with lysosomes (6),
intercept vesicular traffic at endoplasmic reticulum (ER) exit sites
(7), and fuse with the ER
(8–10).
The uptake of L. pneumophila and formation of LCVs in macrophages and
amoebae depends on the Icm/Dot type IV secretion system (T4SS)
(11–14).
Although more than 100 Icm/Dot substrates (“effector” proteins)
have been identified to date, only few are functionally characterized,
including effectors that interfere with host cell signal transduction, vesicle
trafficking, or apoptotic pathways
(15–18).Two Icm/Dot-translocated substrates, SidM/DrrA
(19,
20) and RalF
(21), have been characterized
as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small
GTPases. These bacterial GEFs are recruited to and activate their targets on
LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic
signal transduction pathways and in actin cytoskeleton regulation
(22). Inactive Rho GTPases
bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases
are activated by removal of the GDI and the exchange of GDP with GTP by GEFs,
which promotes the interaction with downstream effector proteins, such as
protein or lipid kinases and various adaptor proteins. The cycle is closed by
hydrolysis of the bound GTP, which is mediated by GTPase-activating
proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle
transport, and additionally, SidM acts as a GDI displacement factor (GDF) to
activate Rab1 (23,
24). The function of SidM is
assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA
preferentially binds to activated Rab1, thus supporting the recruitment of
early secretory vesicles by SidM
(19,
20,
23,
25,
26). Another Icm/Dot
substrate, LepB (27),
contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its
GTPase-activating protein function, thus acting as an antagonist of SidM
(24).The Icm/Dot substrate RalF recruits and activates the small GTPase
ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle
transport from Golgi to ER
(21). Dominant negative Arf1
(7,
28) or knockdown of Arf1 by
RNA interference (29) impairs
the formation of LCVs, as well as the recruitment of the Icm/Dot substrate
SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane
(31), where the proteins
specifically bind to the host cell lipid phosphatidylinositol 4-phosphate
(PtdIns(4)P) (32,
33). Phosphoinositides (PIs)
regulate eukaryotic receptor-mediated signal transduction, actin remodeling,
and membrane dynamics (34,
35). PtdIns(4)P is present on
the cytoplasmic membrane, but localizes preferentially to the
trans-Golgi network (TGN), where this PI is produced by an
Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ)
(36) to promote trafficking
along the secretory pathway. Recently, PtdIns(4)P was found to also mediate
the export of early secretory vesicles from ER exit sites
(37). At present, the L.
pneumophila effector proteins that mediate exploitation of host PI
signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using
different PIs coupled to agarose beads, we identified SidM as a major
PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a
novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs,
including the P4M domain, were found to be translocated and bind the LCV
membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ. 相似文献
19.
20.