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1.
Lisa E. S. Crose Thomas L. Hilder Noah Sciaky Gary L. Johnson 《The Journal of biological chemistry》2009,284(20):13301-13305
Mutation of CCM2 predisposes individuals to cerebral cavernous
malformations, vascular abnormalities that cause seizures and hemorrhagic
stroke. CCM2 has been proposed to regulate the activity of RhoA for
maintenance of vascular integrity. Herein, we define a novel mechanism where
the CCM2 phosphotyrosine binding (PTB) domain binds the ubiquitin ligase (E3)
Smurf1, controlling RhoA degradation. Brain endothelial cells with knockdown
of CCM2 have increased RhoA protein and display impaired directed cell
migration. CCM2 binding of Smurf1 increases Smurf1-mediated degradation of
RhoA. CCM2 does not significantly alter the catalytic activity of Smurf1, nor
is CCM2 a Smurf1 substrate. Rather the CCM2-Smurf1 interaction functions to
localize Smurf1 for RhoA degradation. These findings provide a molecular
mechanism for the pathogenesis of cerebral cavernous malformations (CCM)
resulting from loss of CCM2-mediated localization of Smurf1, which controls
RhoA degradation required for maintenance of normal endothelial cell
physiology.We previously characterized a scaffold-like protein named
osmosensing scaffold for MEKK3 (OSM) for its
ability to bind actin and localize to Rac-containing membrane ruffles and its
obligate requirement for p38 activation in response to hyperosmotic stress
(1). Subsequently, the gene
encoding OSM, CCM2, was found to be mutated in the human disease
cerebral cavernous malformations
(CCM)2
(2). Cerebral cavernous
malformations are vascular lesions of the central nervous system characterized
as clusters of dilated, thin walled blood vessels. CCM lesions are fragile and
prone to vascular leakiness and rupture, leading to hemorrhages that cause
seizure and stroke (3,
4).Recently, CCM2 knockdown endothelial cells were shown to have increased
activation of RhoA (5),
although the mechanism was not defined. Herein, we demonstrate a molecular
mechanism for activation of this pathway. Through a novel CCM2 PTB domain
interaction with the Smurf1 homologous to the E6-AP C terminus (HECT) domain,
we now show that CCM2 binds the E3 ligase Smurf1 for the control of RhoA
degradation. 相似文献
2.
3.
Laura Stanbery Nisha J D'Silva Julia S Lee Carol R Bradford Thomas E Carey Mark E Prince Gregory T Wolf Francis P Worden Kitrina G Cordell Elizabeth M Petty 《Translational oncology》2010,3(4):239-245
The purpose of this work was to determine SEPT9_v1 expression levels in head and neck squamous cell carcinoma (HNSCC) and to analyze whether SEPT9_v1 expression is relevant to clinical outcomes. Recently, the SEPT9 isoform SEPT9_v1 has been implicated in oncogenesis, and methylation of the SEPT9 promoter region was reported in HNSCC. These findings led us to hypothesize that SEPT9_v1 could be differently expressed in HNSCC. To determine whether SEPT9_v1 is expressed in HNSCC, tissue microarray immunohistochemical analysis was performed using a SEPT9_v1-specific antibody. Tissue microarrays stained with a polyclonal SEPT9_v1-specific antibody was used to determine protein expression levels in HNSCC tissue samples, some with known clinical outcomes. This analysis showed that SEPT9_v1 is in fact highly expressed in HNSCC compared with normal epithelium, and high expression levels directly correlated with poor clinical outcomes. Specifically, a high SEPT9_v1 expression was associated with decreased disease-specific survival (P = .012), time to indication of surgery at primary site (P = .008), response to induction chemotherapy (P = .0002), and response to chemotherapy (P = .02), as well as advanced tumor stage (P = .012) and N stage (P = .0014). The expression of SEPT9_v1 was also strongly correlated with smoking status (P = .00094). SEPT9_v1 is highly expressed in HNSCC, and a high expression of SEPT9_v1 is associated with poor clinical outcomes. These data indicate that SEPT9_v1 warrants additional investigation as a potential biomarker for HNSCC. 相似文献
4.
5.
Valentina Gandin Gustavo J. Gutierrez Laurence M. Brill Tal Varsano Yongmei Feng Pedro Aza-Blanc Qingyan Au Shannon McLaughlan Tiago A. Ferreira Tommy Alain Nahum Sonenberg Ivan Topisirovic Ze'ev A. Ronai 《Molecular and cellular biology》2013,33(13):2510-2526
Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold during or shortly after completion of translation. Nonetheless, signaling pathways that govern the degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and an adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress. 相似文献
6.
7.
8.
Evgeny V. Berdyshev Irina Gorshkova Anastasia Skobeleva Robert Bittman Xuequan Lu Steven M. Dudek Tamara Mirzapoiazova Joe G. N. Garcia Viswanathan Natarajan 《The Journal of biological chemistry》2009,284(9):5467-5477
Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin,
but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase.
Although many of the effects of FTY720 are ascribed to its phosphorylation and
subsequent sphingosine 1-phosphate (S1P)-like action through
S1P1,3–5 receptors, studies on modulation of intracellular
balance of signaling sphingolipids by FTY720 are limited. In this study, we
used stable isotope pulse labeling of human pulmonary artery endothelial cells
with l-[U-13C, 15N]serine as well as in
vitro enzymatic assays and liquid chromatography-tandem mass spectrometry
methodology to characterize FTY720 interference with sphingolipid de
novo biosynthesis. In human pulmonary artery endothelial cells, FTY720
inhibited ceramide synthases, resulting in decreased cellular levels of
dihydroceramides, ceramides, sphingosine, and S1P but increased levels of
dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The
FTY720-induced modulation of sphingolipid de novo biosynthesis was
similar to that of fumonisin B1, a classical inhibitor of ceramide synthases,
but differed in the efficiency to inhibit biosynthesis of short-chain
versus long-chain ceramides. In vitro kinetic studies
revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward
dihydrosphingosine with an apparent Ki of 2.15
μm. FTY720-induced up-regulation of DHS1P level was mediated by
sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using
SphK1/2 silencing with small interfering RNA. Our data demonstrate for the
first time the ability of FTY720 to inhibit ceramide synthases and modulate
the intracellular balance of signaling sphingolipids. These findings open a
novel direction for therapeutic applications of FTY720 that focuses on
inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the
up-regulation of DHS1P generation in cells.FTY7202 is a
synthetic analog of sphingosine and is currently being studied as a potent
immunosuppressive and immunomodulatory agent
(1–3).
FTY720-induced immunosuppression is ascribed, in part, to its protective
effect on endothelial cell barrier function that results in inhibition of
lymphocyte egress from lymph nodes and down-regulation of innate and adaptive
immune responses (4). As
endothelial cells predominantly express the sphingosine 1-phosphate 1
(S1P1) receptor and its activation initiates signaling that results
in the assembly of VE-cadherin-based adherens junctions
(5), it is thought that the
phosphorylation of FTY720 and the binding of FTY720-P to the S1P1
receptor determine its effect on vasculature
(1). Recently it became evident
that the action of FTY720 is more complex as several other direct protein
targets were identified. Thus, FTY720 was found to bind to and inhibit the
cannabinoid CB1 receptor (6),
to inhibit cytosolic phospholipase A2 (cPLA2), and to
counteract ceramide 1-phosphate-induced cPLA2 activation
(7). Additionally FTY720 but
not FTY720-P was shown to inhibit S1P lyase
(8), which degrades S1P to
ethanolamine phosphate and (E)-2-hexadecenal and regulates the
removal of sphingoid bases from the cumulative pool of sphingolipids. These
findings characterize FTY720 as a molecule with a multitargeted mode of action
whose cellular effects are complicated by its metabolic transformation to
FTY720-P, a structural and functional analog of S1P.Phosphorylation of FTY720 to FTY720-P by sphingosine kinases (SphKs) is the
only reported metabolic transformation of FTY720 and has been actively
explored because of its link to S1P-mediated signaling
(1,
2,
9,
10). Recent studies suggest
that the endogenous balance between S1P and ceramide molecules regulates
prosurvival and proapoptotic signaling cascades, which determine the outcome
of cellular response to different stress conditions
(11,
12) or the efficiency of
anticancer therapy
(12–14).
However, despite the fact that FTY720 resembles sphingosine (Sph) and is a
substrate of SphK2
(15–17),
there are no reported studies on the effect of FTY720 on the intrinsic balance
of signaling sphingolipids. Metabolic interconnections between proapoptotic
(ceramides) and prosurvival (dihydrosphingosine 1-phosphate (DHS1P)) molecules
are expected because it is known that fumonisin B1 (FB1), an inhibitor of
(dihydro)ceramide synthases, not only blocks the formation of ceramides and
up-regulates the intracellular content of dihydrosphingosine (DHSph) but also
increases the cellular level of DHS1P
(19,
20).In view of these considerations, it is important to know how compounds with
a potential ability to interfere with the sphingolipidome turnover affect the
DHS1P-S1P/ceramide balance in cells. To address this question we have
investigated the effect of FTY720 on metabolic pathways leading to ceramide
and sphingoid base 1-phosphate generation in human pulmonary artery
endothelial cells (HPAECs) by using a stable isotope pulse labeling approach
and quantitative liquid chromatography-tandem mass spectrometry of signaling
sphingolipids. We demonstrate that treatment of HPAECs with FTY720 results in
the inhibition of de novo ceramide formation with a concomitant
increase in DHSph and DHS1P content in cells. Moreover FTY720 showed a direct
inhibition of ceramide synthases in an in vitro assay, albeit it was
less efficient compared with the classical inhibitor of ceramide synthases,
FB1. Our present findings have identified ceramide synthase isozymes as a
novel molecular target for FTY720 action, opening a new direction for its
potential therapeutic application through the inhibition of ceramide
biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P
generation in cells. 相似文献
9.
B��la Z. Schmidt Rebecca J. Watts Meir Aridor Raymond A. Frizzell 《The Journal of biological chemistry》2009,284(7):4168-4178
Cysteine string protein (Csp) is a J-domain-containing protein whose
overexpression blocks the exit of cystic fibrosis transmembrane conductance
regulator (CFTR) from the endoplasmic reticulum (ER). Another method of
blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as
much immature CFTR compared with Csp overexpression. This finding suggested
that Csp not only inhibits CFTR ER exit but also facilitates the degradation
of immature CFTR. This was confirmed by treatment with a proteasome inhibitor,
which returned the level of immature CFTR to that found in cells expressing
Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently,
did not promote CFTR degradation, suggesting that the pro-degradative effect
of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp
overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with
CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding
partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for
proteasome-mediated degradation. Csp overexpression also increased the amount
of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly
with Csp, which was confirmed by in vitro binding experiments. Csp
overexpression also increased CFTR ubiquitylation and reduced the half-life of
immature CFTR. These findings indicate that Csp not only regulates the exit of
CFTR from the ER, but that this action is accompanied by Hsc70/Hsp70 and
CHIP-mediated CFTR degradation.Cysteine string protein (Csp,
DnaJC5)2 is a member
of the DnaJ/Hsp40 protein chaperone family
(1,
2). Csp contains a short
N-terminal sequence, followed by the J-domain, a linker region, and the
central cysteine-rich region, which gives these proteins their name. The
cysteine residues in this region are post-translationally modified by the
palmitate groups that serve for the membrane attachment of Csp
(3). This structure is followed
by a C-terminal domain, which is the most variable region among the three Csp
paralogs in the human genome.Csp was originally discovered as an abundant protein in presynaptic
junctions of Drosophila neurons
(4). Csps are expressed at high
levels in neurons and in cell types involved in regulated exocytosis
(5–7),
where they have been shown to govern exocytic secretory functions. For
example, depolarization-induced synaptic vesicle exocytosis is impaired in
neurons from Csp-deficient Drosophila
(8), and Csp overexpression
produced decreases in stimulated insulin release from β-cells and
stimulated catecholamine release from chromaffin cells
(9–11).
Deletion of the Csp gene proved to be lethal both in Drosophila and
in mice causing a progressive, fatal sensorimotor disorder characterized by
developing neurodegenerative changes
(12,
13).As for other Hsp40 homologs, the J-domain is responsible for the ability of
Csp to bind Hsc70/Hsp70 and stimulate its ATPase activity
(14). Similar to other
chaperone proteins, Csp can be found in many different protein complexes in
the cell, and depending on the composition of these complexes, Csp has been
linked to many different cellular processes, ranging from membrane fusion to
protein folding and G-protein-mediated signaling.Based on its direct interaction with the SNARE proteins syntaxin 1A and
vesicle-associated membrane protein (synaptobrevin) Csp has been proposed to
be a direct regulator of SNARE function
(15,
16), a role further supported
by Csp''s phosphorylation-dependent, direct interaction with synaptotagmin, a
Ca2+-sensing, SNARE-binding protein
(17). As part of a chaperone
complex with Hsp90, Hsp70, and α-guanine nucleotide dissociation
inhibitor, Csp coordinates Ca2+-induced neurotransmitter release by
regulating the retrieval of Rab3b from presynaptic membranes
(18).Csp forms a ternary complex with Hsc70/Hsp70 and small glutamine-rich
tetratricopeptide repeat-containing protein. This complex is present on
synaptic vesicles, and it can re-fold luciferase, leading to the proposal that
Csp assists with the reactivation of unfolded proteins at the synapse
(19). This same
Csp-Hsc70-small glutamine-rich tetratricopeptide repeat-containing protein
complex also interacts with heterotrimeric G-proteins. In this context Csp
functions as a guanine-nucleotide exchange factor for GαS,
which leads to stimulation of G-protein-dependent signaling and to
G-protein-mediated inhibition of N-type Ca2+ channels
(20,
21).Our laboratory previously established a novel role for Csp at endoplasmic
reticulum (ER) membranes in modulating the trafficking of the cystic fibrosis
transmembrane conductance regulator (CFTR). The overexpression of Csp blocked
the maturation of CFTR (22,
23), producing a
dose-dependent reduction in mature (band C) CFTR and an increase in immature
(band B) CFTR, which was localized to the ER. The Csp-induced block of CFTR
maturation required Hsc70/Hsp70, because expression of a J-domain mutant of
Csp (CspH43Q) that cannot stimulate Hsc70/Hsp70 ATPase activity allowed CFTR
maturation. Conversely, the knock down of Csp promoted increased formation of
mature CFTR. Together, these findings indicated that Csp negatively regulates
CFTR progression to post-ER compartments.In the present study, we provide evidence for another ER-based function of
Csp: its involvement in proteasome-mediated CFTR degradation. Overexpression
of Csp increased the association of CFTR with Hsp70/Hsc70 and with the E3
ubiquitin ligase, CHIP (C terminus of Hsp70-interacting protein). Using
co-immunoprecipitation and in vitro binding assays, we demonstrated a
direct interaction between Csp and CHIP. The overexpression of Csp also
increased the ubiquitylation of CFTR, in agreement with its ability to reduce
CFTR steadystate levels and its increased association with CHIP. Pairwise
interactions between Csp, Hsp70/Hsc70, and CHIP suggest a model in which Csp
coordinates the formation of a complex that facilitates the degradation of
CFTR as it blocks CFTR exit from the ER. 相似文献
10.
11.
12.
13.
Renee M. Garza Brian K. Sato Randolph Y. Hampton 《The Journal of biological chemistry》2009,284(22):14710-14722
Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for
the ubiquitin-mediated destruction of both misfolded and normal ER-resident
proteins. ERAD substrates must be moved from the ER to the cytoplasm for
ubiquitination and proteasomal destruction by a process called
retrotranslocation. Many aspects of retrotranslocation are poorly understood,
including its generality, the cellular components required, the energetics,
and the mechanism of transfer through the ER membrane. To address these
questions, we have developed an in vitro assay, using the
8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to
GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We
have now directly demonstrated in vitro retrotranslocation of
full-length, ubiquitinated Hmg2p-GFP to the aqueous phase. Hrd1p was
rate-limiting for Hmg2p-GFP retrotranslocation, which required ATP, the
AAA-ATPase Cdc48p, and its receptor Ubx2p. In addition, the adaptors Dsk2p and
Rad23p, normally implicated in later parts of the pathway, were required.
Hmg2p-GFP retrotranslocation did not depend on any of the proposed ER channel
candidates. To examine the role of the Hrd1p transmembrane domain as a
retrotranslocon, we devised a self-ubiquitinating polytopic substrate
(Hmg1-Hrd1p) that undergoes ERAD in the absence of Hrd1p. In vitro
retrotranslocation of full-length Hmg1-Hrd1p occurred in the absence of the
Hrd1p transmembrane domain, indicating that it did not serve a required
channel function. These studies directly demonstrate polytopic membrane
protein retrotranslocation during ERAD and delineate avenues for mechanistic
understanding of this general process.The endoplasmic reticulum
(ER)2-associated
degradation (ERAD) pathway mediates the destruction of numerous integral
membrane or lumenal ER-localized proteins
(1,
2). ERAD functions mainly in
the disposal of misfolded or unassembled proteins but also participates in the
physiological regulation of some normal residents of the organelle. This
ER-localized degradation pathway has been implicated in a wide variety of
normal and pathophysiological processes, including sterol synthesis
(3,
4), rheumatoid arthritis
(5), fungal differentiation
(6), cystic fibrosis
(7,
8), and several
neurodegenerative diseases (9).
Accordingly, there is great impetus to understand the molecular mechanisms
that mediate this broadly important route of protein degradation.ERAD proceeds by the ubiquitin-proteasome pathway, by which an ER-localized
substrate is covalently modified by the addition of multiple copies of 7.6-kDa
ubiquitin to form a multiubiquitin chain that is recognized by the cytosolic
26S proteasome (10,
11). Ubiquitin is added to the
substrate by the successive action of three enzymes. The E1
ubiquitin-activating enzyme uses ATP to covalently add ubiquitin to an E2
ubiquitin-conjugating (UBC) enzyme. Ubiquitin is then transferred from the
charged E2 to the substrate or the growing ubiquitin chain by the action of an
E3 ubiquitin ligase, resulting in a substrate-attached multiubiquitin chain
that is recognized by the proteasome, leading to degradation of the
ubiquitinated substrate. This is a skeletal picture; in most cases, ancillary
factors participate in substrate recognition and transfer of the ubiquitinated
substrate to the proteasome
(12–14).ERAD substrates are either sequestered in the lumen or embedded in the ER
membrane with lumenal portions. Thus, a critical step in the ERAD pathway
involves transfer of the ERAD substrate to the cytosol for proteasomal
degradation by a process referred to as retrotranslocation or dislocation
(15). Retrotranslocation
requires the hexameric AAA-ATPase called Cdc48p in yeast and p97 in mammals,
and it is thought that a protein channel mediates the movement of substrates
across the ER membrane. Channel candidates include the derlins
(16,
17), the Sec61p anterograde
channel (18,
19), or the multispanning
domains of the ER ligases themselves
(18–20).The yeast HRD pathway mediates ERAD of numerous misfolded ER proteins and
the physiologically regulated degradation of the Hmg2p isozyme of HMG-CoA
reductase, an 8-transmembrane span (8-spanning) integral membrane protein
critical for sterol synthesis
(3). The integral membrane ER
ligase Hrd1p, in conjunction with Hrd3p, is responsible for ubiquitination of
Hmg2p. Efficient delivery of ubiquitinated Hmg2p to the proteasome requires
the Cdc48p-Ufd1p-Npl4p complex presumably by promoting retrotranslocation of
ER-embedded Hmg2p.Due to the requirement for retrotranslocation in all ERAD pathways we have
adapted our in vitro assay of Hrd1p-mediated ubiquitination of the
normally degraded fusion Hmg2p-GFP to study this ER removal step in ERAD. We
have reconstituted Hrd1p-mediated ubiquitination and retrotranslocation of
Hmg2p-GFP in vitro
(21,
22). We have now directly
demonstrated that the entire 8-spanning Hmg2p-GFP protein is removed from the
membrane by this process, remaining intact yet soluble after
retrotranslocation. The dislocation of intact Hmg2p-GFP required both Cdc48p
and hydrolysis of the β–γ bond of ATP. The Ubx2p adaptor
protein functioned in a manner consistent with its proposed role in Cdc48p
anchoring to the ER. Surprisingly, the Dsk2p/Rad23p proteasomal coupling
factors were also required for retrotranslocation. Neither derlins nor Sec61p
were implicated in Hmg2p-GFP retrotranslocation by our assay. Furthermore, an
engineered substrate based on HMG-CoA reductase underwent ERAD in the complete
absence of Hrd1p or Doa10p and in vitro, full-length
retrotranslocation, both indicating that the large transmembrane domains of
either of these ERAD E3 ligases were not required for membrane extraction.
Taken together, these studies define a core set of proteins that can mediate
recognition and retrotranslocation of the HRD substrate Hmg2p-GFP and will
allow mechanistic analysis along all points of the ERAD pathway. 相似文献
14.
Degradation of DNA by 1,-10-phenanthroline 总被引:1,自引:0,他引:1
15.
Bertha C. Elias Takuya Suzuki Ankur Seth Francesco Giorgianni Gautam Kale Le Shen Jerrold R. Turner Anjaparavanda Naren Dominic M. Desiderio Radhakrishna Rao 《The Journal of biological chemistry》2009,284(3):1559-1569
Occludin is phosphorylated on tyrosine residues during the oxidative
stress-induced disruption of tight junction, and in vitro
phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the
present study mass spectrometric analyses of C-terminal domain of occludin
identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation
sites, which are located in a highly conserved sequence of occludin, YETDYTT;
Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion
of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and
the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin
also abolished the c-Src-mediated phosphorylation and regulation of ZO-1
binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding
even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A
mutant was localized at the plasma membrane and cell-cell contact sites in
Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at
the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant
occludin in Madin-Darby canine kidney cells was significantly delayed compared
with that of wild type occludin or its T398A/T402A mutant. Furthermore,
expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for
hydrogen peroxide-induced barrier disruption. This study reveals a unique
motif in the occludin sequence that is involved in the regulation of ZO-1
binding by reversible phosphorylation of specific Tyr residues.Epithelial tight junctions
(TJs)2 form a
selective barrier to the diffusion of toxins, allergens, and pathogens from
the external environment into the tissues in the gastrointestinal tract, lung,
liver, and kidney (1).
Disruption of TJs is associated with the gastrointestinal diseases such as
inflammatory bowel disease, celiac disease, infectious enterocolitis, and
colon cancer
(2–4)
as well as in diseases of lung and kidney
(5,
6). Numerous inflammatory
mediators such as tumor necrosis factor α, interferon γ, and
oxidative stress
(7–12)
are known to disrupt the epithelial TJs and the barrier function. Several
studies have indicated that hydrogen peroxide disrupts the TJs in intestinal
epithelium by a tyrosine kinase-dependent mechanism
(11,
12).Four types of integral proteins, occludin, claudins, junctional adhesion
molecules, and tricellulin are associated with TJs. Occludin, claudins, and
tricellulin are tetraspan proteins, and their extracellular domains interact
with homotypic domains of the adjacent cells
(1,
2,
13). The intracellular domains
of these proteins interact with a variety of soluble proteins such as ZO-1,
ZO-2, ZO-3, 7H6, cingulin, and symplekin
(14–23);
this protein complex interacts with the perijunctional actomyosin ring. The
interactions among TJ proteins are essential for the assembly and the
maintenance of TJs. Therefore, regulation of the interactions among TJ
proteins may regulate the TJ integrity. A significant body of evidence
indicates that numerous signaling molecules are associated with the TJs.
Protein kinases and protein phosphatases such as protein kinase Cζ
(PKCζ), PKCι/λ
(24), c-Src
(25), c-Yes
(26,
27), mitogen-activated protein
kinase (28), PP2A, and PP1
(29) interact with TJs,
indicating that TJs are dynamically regulated by intracellular signal
transduction involving protein phosphorylation. Additionally, other signaling
molecules such as calcium
(30), phosphatidylinositol
3-kinase (31), Rho
(32), and Rac
(33) are involved in the
regulation of TJs.Occludin, a ∼65-kDa protein, has been well characterized to be
assembled into the TJs. Although occludin knock-out mice showed the formation
of intact TJs in different epithelia
(34), numerous studies have
emphasized that it plays an important role in the regulation of TJ integrity.
Occludin spans the membrane four times to form two extracellular loops and one
intracellular loop, and the N-terminal and C-terminal domains hang into the
intracellular compartment
(35–37).
In epithelium with intact TJs, occludin is highly phosphorylated on Ser and
Thr residues (38), whereas Tyr
phosphorylation is undetectable. However, the disruption of TJs in Caco-2 cell
monolayers by oxidative stress and acetaldehyde leads to Tyr phosphorylation
of occludin; the tyrosine kinase inhibitors attenuate the disruption of TJs
(39,
40). Furthermore, a previous
in vitro study demonstrated that Tyr phosphorylation of the
C-terminal domain of occludin leads to the loss of its interaction with ZO-1
and ZO-3 (25).In the present study we identified the Tyr residues in occludin that are
phosphorylated by c-Src and determined their role in regulated interaction
between occludin and ZO-1 and its assembly into the TJs. Results show that 1)
Tyr-379 and Tyr-383 in chicken occludin and Tyr-398 and Tyr-402 in human
occludin are the exclusive sites of phosphorylation by c-Src, and these Tyr
residues are located in a highly conserved sequence of occludin, YET-DYTT, 2)
deletion of YEDTYTT or point mutation of Tyr-398 and Tyr-402 in human occludin
attenuates the phosphorylation-dependent regulation of ZO-1 binding, 3)
Y398D/Y402D mutation of human occludin leads to loss of ZO-1 binding and
prevents its translocation to the plasma membrane and cell-cell contact sites
in Rat-1 cells, 4) Y398D/Y402D mutation of occludin delays its assembly into
the intercellular junctions during the calcium-induced assembly of TJs, and 5)
expression of Y398D/Y402D mutant occludin sensitizes cell monolayers for
hydrogen peroxide-induced disruption of barrier function. 相似文献
16.
Rationale
Cardiomyocytes express neurotrophin receptor TrkA that promotes survival following nerve growth factor (NGF) ligation. Whether TrkA also resides in cardiac fibroblasts (CFs) and underlies cardioprotection is unknown.Objective
To test whether CFs express TrkA that conveys paracrine signals to neighbor cardiomyocytes using, as probe, the Chagas disease parasite Trypanosoma cruzi, which expresses a TrkA-binding neurotrophin mimetic, named PDNF. T cruzi targets the heart, causing chronic debilitating cardiomyopathy in ∼30% patients.Methods and Results
Basal levels of TrkA and TrkC in primary CFs are comparable to those in cardiomyocytes. However, in the myocardium, TrkA expression is significantly lower in fibroblasts than myocytes, and vice versa for TrkC. Yet T cruzi recognition of TrkA on fibroblasts, preferentially over cardiomyocytes, triggers a sharp and sustained increase in NGF, including in the heart of infected mice or of mice administered PDNF intravenously, as early as 3-h post-administration. Further, NGF-containing T cruzi- or PDNF-induced fibroblast-conditioned medium averts cardiomyocyte damage by H2O2, in agreement with the previously recognized cardioprotective role of NGF.Conclusions
TrkA residing in CFs induces an exuberant NGF production in response to T cruzi infection, enabling, in a paracrine fashion, myocytes to resist oxidative stress, a leading Chagas cardiomyopathy trigger. Thus, PDNF-TrkA interaction on CFs may be a mechanism orchestrated by T cruzi to protect its heart habitat, in concert with the long-term (decades) asymptomatic heart parasitism that characterizes Chagas disease. Moreover, as a potent booster of cardioprotective NGF in vivo, PDNF may offer a novel therapeutic opportunity against cardiomyopathies. 相似文献17.
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José Maria Rodrigues da Luz Sirlaine Albino Paes Karla Veloso Gon?alves Ribeiro Igor Rodrigues Mendes Maria Catarina Megumi Kasuya 《PloS one》2015,10(6)
We studied the biodegradation of green polyethylene (GP) by Pleurotus ostreatus. The GP was developed from renewable raw materials to help to reduce the emissions of greenhouse gases. However, little information regarding the biodegradation of GP discarded in the environment is available. P. ostreatus is a lignocellulolytic fungus that has been used in bioremediation processes for agroindustrial residues, pollutants, and recalcitrant compounds. Recently, we showed the potential of this fungus to degrade oxo-biodegradable polyethylene. GP plastic bags were exposed to sunlight for up to 120 days to induce the initial photodegradation of the polymers. After this period, no cracks, pits, or new functional groups in the structure of GP were observed. Fragments of these bags were used as the substrate for the growth of P. ostreatus. After 30 d of incubation, physical and chemical alterations in the structure of GP were observed. We conclude that the exposure of GP to sunlight and its subsequent incubation in the presence of P. ostreatus can decrease the half-life of GP and facilitate the mineralization of these polymers. 相似文献