共查询到20条相似文献,搜索用时 15 毫秒
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Ronit Har-el Esther Marva Mordechai Chevion Jacob Golenser 《Free radical research》1993,18(5):279-290
Based on the unusually high and stage-dependant susceptibility of Plasmodia to oxidant stress it has been proposed that during parasite development, increasing levels of redox-active forms of iron are gradually released. The purpose of this study was to examine this proposal by using an assay monitoring the levels of available forms of iron for redox reactions. Ascorbate-driven and iron-mediated degradation of adventitious DNA served as the basis for this functional assay.
Incubation of DNA with lysate from infected RBC caused massive degradation, which was dose, time-and parasite-stage dependent. In contrast, lysate from non-infected RBC did not induce DNA degradation. Likewise, lysate only from infected RBC enhanced the aerobic oxidation of ascorbate. These effects on both reactions, DNA degradation and ascorbate oxidation, could be reconstructed with hemin, instead of lysate. Also, chelators exerted similar effects on both reactions.
The results suggest that increased levels of redox-active forms of iron are liberated during parasite development. We propose that hemin or hemin-like structures are the appropriate candidates which could catalyze oxidative stress and deregulate the delicate redox balance of the host-parasite system. 相似文献
Incubation of DNA with lysate from infected RBC caused massive degradation, which was dose, time-and parasite-stage dependent. In contrast, lysate from non-infected RBC did not induce DNA degradation. Likewise, lysate only from infected RBC enhanced the aerobic oxidation of ascorbate. These effects on both reactions, DNA degradation and ascorbate oxidation, could be reconstructed with hemin, instead of lysate. Also, chelators exerted similar effects on both reactions.
The results suggest that increased levels of redox-active forms of iron are liberated during parasite development. We propose that hemin or hemin-like structures are the appropriate candidates which could catalyze oxidative stress and deregulate the delicate redox balance of the host-parasite system. 相似文献
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Omar Ramadan Yongxia Qu Raj Wadgaonkar Ghayath Baroudi Eddy Karnabi Mohamed Chahine Mohamed Boutjdir 《The Journal of biological chemistry》2009,284(8):5042-5049
The novel α1D L-type Ca2+ channel is expressed
in supraventricular tissue and has been implicated in the pacemaker activity
of the heart and in atrial fibrillation. We recently demonstrated that PKA
activation led to increased α1D Ca2+ channel
activity in tsA201 cells by phosphorylation of the channel protein. Here we
sought to identify the phosphorylated PKA consensus sites on the
α1 subunit of the α1D Ca2+
channel by generating GST fusion proteins of the intracellular loops, N
terminus, proximal and distal C termini of the α1 subunit of
α1D Ca2+ channel. An in vitro PKA kinase
assay was performed for the GST fusion proteins, and their phosphorylation was
assessed by Western blotting using either anti-PKA substrate or
anti-phosphoserine antibodies. Western blotting showed that the N terminus and
C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus
sites, were phosphorylated by PKA and identified by mass spectrometry. Site
directed mutagenesis and patch clamp studies revealed that serines 1743 and
1816 were major functional PKA consensus sites. Altogether, biochemical and
functional data revealed that serines 1743 and 1816 are major functional PKA
consensus sites on the α1 subunit of α1D
Ca2+ channel. These novel findings provide new insights into the
autonomic regulation of the α1D Ca2+ channel in
the heart.L-type Ca2+ channels are essential for the generation of normal
cardiac rhythm, for induction of rhythm propagation through the
atrioventricular node and for the contraction of the atrial and ventricular
muscles
(1–5).
L-type Ca2+ channel is a multisubunit complex including
α1, β and α2/δ subunits
(5–7).
The α1 subunit contains the voltage sensor, the selectivity
filter, the ion conduction pore, and the binding sites for all known
Ca2+ channel blockers
(6–9).
While α1C Ca2+ channel is expressed in the atria
and ventricles of the heart
(10–13),
expression of α1D Ca2+ channel is restricted to
the sinoatrial (SA)2
and atrioventricular (AV) nodes, as well as in the atria, but not in the adult
ventricles (2,
3,
10).Only recently it has been realized that α1D along with
α1C Ca2+ channels contribute to L-type
Ca2+ current (ICa-L) and they both play important but
unique roles in the physiology/pathophysiology of the heart
(6–9).
Compared with α1C, α1D L-type
Ca2+ channel activates at a more negative voltage range and shows
slower current inactivation during depolarization
(14,
15). These properties may
allow α1D Ca2+ channel to play critical roles in
SA and AV nodes function. Indeed, α1D Ca2+ channel
knock-out mice exhibit significant SA dysfunction and various degrees of AV
block (12,
16–19).The modulation of α1C Ca2+ channel by
cAMP-dependent PKA phosphorylation has been extensively studied, and the C
terminus of α1 was identified as the site of the modulation
(20–22).
Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a
membrane-permeable cAMP analog, increased α1D Ca2+
channel activity using patch clamp studies
(2). However, very little is
known about potential PKA phosphorylation consensus motifs on the
α1D Ca2+ channel. We therefore hypothesized that
the C terminus of the α1 subunit of the α1D
Ca2+ channel mediates its modulation by cAMP-dependent PKA
pathway. 相似文献
6.
Nicholas J. Anthis Jacob R. Haling Camilla L. Oxley Massimiliano Memo Kate L. Wegener Chinten J. Lim Mark H. Ginsberg Iain D. Campbell 《The Journal of biological chemistry》2009,284(52):36700-36710
Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the β integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated 15N-labeled β3, β1A, and β7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of β3, β1A, and β7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of β integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins. 相似文献
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Canwei Xia Jiayu Liu Per Alström Qiao Wu Yanyun Zhang 《Ethology : formerly Zeitschrift fur Tierpsychologie》2013,119(8):653-661
Soft songs have been detected in many songbirds, but in most species, research on soft songs has lagged behind studies of broadcast songs. In this study, we describe the acoustic features of a soft song in the brownish‐flanked bush warbler Cettia fortipes. Compared with the broadcast song, the warbler's soft song was characterized by a lower minimum frequency and longer duration, and it had a higher proportion of rapid frequency modulation notes. Using playback experiments, some in combination with mounted specimens, we found different responses to soft and broadcast songs, and we found that soft song can predict aggressive escalation (attack). We conclude that the soft song is an aggressive signal in this species. 相似文献
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Ingoglia NA Ramanathan M Zhang N Tzeng B Mathur G Opuni K Donnelly R 《Neurochemical research》2000,25(1):51-58
The N-terminal, posttranslational arginylation of proteins is ubiquitous in eukaryotic cells. Previous experiments, using purified components of the reaction incubated in the presence of exogenous substrates, have shown that only those proteins containing acidic residues at their N-terminals are arginylation substrates. However, data from experiments that used crude extracts of brain and nerve as the source of the arginylating molecules, suggest that the in vivo targets for arginylation are more complex than those demonstrated using purified components. One of the proposed functions for arginylation is as a signal for protein degradation and proteins that have undergone oxidative damage have been shown to be rapidly degraded. In the present experiments we have tested the hypothesis that the presence of an oxidatively damaged residue in a protein is a signal for its arginylation. These experiments have been performed by adding synthetic oxidized peptides to crude extracts of rat brain, incubating them with [3H]Arg and ATP and assaying for arginylated peptides using RP-HPLC. Results showed that while the oxidized A-chain of insulin was arginylated in this system, confirming previous experiments, other peptides containing oxidized residues were not. When a peptide containing Glu in the N-terminus was incubated under the same conditions it too was not a substrate for arginylation. These findings show that neither the presence of an N-terminal acidic residue nor an oxidized residue alone are sufficient to signal arginylation. Thus, another feature of the oxidized A-chain of insulin is required for arginylation. That feature remains to be identified. 相似文献
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Hala Muaddi Mithu Majumder Philippos Peidis Andreas I. Papadakis Martin Holcik Donalyn Scheuner Randal J. Kaufman Maria Hatzoglou Antonis E. Koromilas 《Molecular biology of the cell》2010,21(18):3220-3231
Various forms of stress induce pathways that converge on the phosphorylation of the alpha (α) subunit of eukaryotic translation initiation factor eIF2 at serine 51 (S51), a modification that results in a global inhibition of protein synthesis. In many cases eIF2α phosphorylation is a biological response that facilitates cells to cope with stressful environments. Glucose deficiency, an important form of stress, is associated with an induction of apoptosis. Herein, we demonstrate that eIF2α phosphorylation is a key step in maintaining a balance between the life and death of a glucose-deficient cell. That is, eIF2α phosphorylation acts as a molecular switch that shifts cells from a proapoptotic to a cytoprotective state in response to prolonged glucose deficiency. This adaptation process is associated with the timely expression of proteins and activation of pathways with significant contributions to cell survival and adaptation including the X-linked inhibitor of apoptosis protein (XIAP). We also show that among the eIF2α kinases GCN2 plays a proapoptotic role whereas PERK and PKR play a cytoprotective one in response to glucose deficiency. Our data demonstrate that eIF2α phosphorylation is a significant determinant of survival and adaptation of glucose-deficient cells with possible important implications in biological processes that interfere with glucose metabolism. 相似文献
12.
Due to the total and unexpected collapse of the Iceland scallop, Chlamys islandica, stocks around Iceland during the 2000s, a commercial fishing ban has been imposed on this valuable resource since 2003. Following the initial identification of an apicomplexan parasite in the scallops, a long-term surveillance program was established to evaluate the effect of the parasite on the population. The infections were highly prevalent in all shell sizes throughout the study. However, the parasite only impacts mature scallops where they cause severe macroscopic changes, characterized by an extensively diminished and abnormally coloured adductor muscle. A highly significant relationship was observed between infection intensity and gonad and adductor muscle indices. The first four years of the study, were characterized by high infection intensity and very poor condition of the adductor muscle and gonads, whilst during subsequent years, infections gradually decreased and the condition of the scallops improved. Histopathological changes were restricted to the presence of apicomplexan zoites which were widely distributed, causing varying degrees of pathology in all organs. In heavy infections, muscular and connective tissues were totally necrotized, destroying significant parts of numerous organs, especially the adductor muscle, digestive gland and gonads. The progression of the disease was in good synchrony with the mortality rates and the subsequent decline observed in the scallop stock and recruitment indices. Our findings strongly suggest that the apicomplexan parasite played a major role in the collapse of the Iceland scallop stock in Breidafjordur. In addition to causing mortality, the infections significantly impact gonad development which contributes further to the collapse of the stock in the form of lower larval recruitment. Furthermore, compelling evidence exists that this apicomplexan pathogen is causing serious disease outbreaks in other scallop populations. Similar abnormal adductor muscles and the parasite itself have been identified or observed in association with other mass mortality events in several different scallop species and commercial stocks in the northern hemisphere. 相似文献
13.
Hardeep Kaur Chitranshu Kumar Christophe Junot Michel B. Toledano Anand K. Bachhawat 《The Journal of biological chemistry》2009,284(21):14493-14502
GSH metabolism in yeast is carried out by the γ-glutamyl cycle as
well as by the DUG complex. One of the last steps in the
γ-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the
constitutent amino acids. Saccharomyces cerevisiae extracts carry
Cys-Gly dipeptidase activity, but the corresponding gene has not yet been
identified. We describe the isolation and characterization of a novel Cys-Gly
dipeptidase, encoded by the DUG1 gene. Dug1p had previously been
identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an
alternate GSH degradation pathway and has also been suggested to function as a
possible di- or tripeptidase based on genetic studies. We show here that Dug1p
is a homodimer that can also function in a Dug2-Dug3-independent manner as a
dipeptidase with high specificity for Cys-Gly and no activity toward tri- or
tetrapeptides in vitro. This activity requires zinc or manganese
ions. Yeast cells lacking Dug1p (dug1Δ) accumulate Cys-Gly.
Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase
M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We
also show that the Dug1p Schizosaccharomyces pombe orthologue
functions as the exclusive Cys-Gly peptidase in this organism. The human
orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by
complementation of the dug1Δ mutant and by biochemical
characterization, which revealed a high substrate specificity and affinity for
Cys-Gly. The results indicate that the Dug1p family represents a novel class
of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide
(l-γ-glutamyl-l-cysteinyl-glycine) present in
almost all eukaryotes (barring a few protozoa) and in a few prokaryotes
(1). In the cell, glutathione
exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the
millimolar range), a relatively low redox potential (-240 mV), and a high
stability conferred by the unusual peptidase-resistant γ-glutamyl bond
are three of the properties endowing GSH with the attribute of an important
cellular redox buffer. GSH also contributes to the scavenging of free radicals
and peroxides, the chelation of heavy metals, such as cadmium, the
detoxification of xenobiotics, the transport of amino acids, and the
regulation of enzyme activities through glutathionylation and serves as a
source of sulfur and nitrogen under starvation conditions
(2,
3). GSH metabolism is carried
out by the γ-glutamyl cycle, which coordinates its biosynthesis,
transport, and degradation. The six-step cycle is schematically depicted in
Fig. 1
(2).Open in a separate windowFIGURE 1.γ-Glutamyl cycle of glutathione metabolism.
γ-Glutamylcysteine synthetase and GSH synthetase carry out the first two
steps in glutathione biosynthesis. γ-glutamyltranspeptidase,
γ-glutamylcyclotransferase, 5-oxoprolinase, and Cys-Gly dipeptidase are
involved in glutathione catabolism. Activities responsible for
γ-glutamylcyclotransferase and 5-oxoprolinase have not been detected in
S. cerevisiae.In Saccharomyces cerevisiae, γ-glutamyl cyclotransferase and
5-oxoprolinase activities have not been detected, which has led to the
suggestion of the presence of an incomplete, truncated form of the
γ-glutamyl cycle (4) made
of γ-glutamyl transpeptidase
(γGT)4 and
Cys-Gly dipeptidase and only serving a GSH catabolic function. Although
γGT and Cys-Gly dipeptidase activities were detected in S.
cerevisiae cell extracts, only the γGT gene (ECM38) has
been identified so far. Cys-Gly dipeptidase activity has been identified in
humans (5,
6), rats
(7–10),
pigs (11,
12), Escherichia coli
(13,
14), and other organisms
(15,
16), and most of them belong
to the M17 or the M1 and M19 metallopeptidases gene families
(17).S. cerevisiae has an alternative γGT-independent GSH
degradation pathway (18) made
of the Dug1p, Dug2p, and Dug3p proteins that function together as a complex.
Dug1p also seem to carry nonspecific di- and tripeptidase activity, based on
genetic studies (19).We show here that Dug1p is a highly specific Cys-Gly dipeptidase, as is its
Schizosaccharomyces pombe homologue. We also show that the mammalian
orthologue of DUG1, CNDP2, can complement the defective utilization
of Cys-Gly as sulfur source of an S. cerevisiae strain lacking
DUG1 (dug1Δ). Moreover, CNDP2 has Cys-Gly dipeptidase
activity in vitro, with a strong preference for Cys-Gly over all
other dipeptides tested. CNDP2 and its homologue CNDP1 are members of the
metallopeptidases M20A family and have been known to carry carnosine
(β-alanyl-histidine) and carnosine-like (homocarnosine and anserine)
peptidase activity (20,
21). This study thus reveals
that the metallopeptidase M20A family represents a novel Cys-Gly peptidase
family, since only members of the M19, M1, and M17 family were known to carry
this function. 相似文献
14.
Jenny Erales Sabrina Lignon Brigitte Gontero 《The Journal of biological chemistry》2009,284(19):12735-12744
A new role is reported for CP12, a highly unfolded and flexible protein,
mainly known for its redox function with A4
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized
CP12 can prevent the in vitro thermal inactivation and aggregation of
GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not
redox-dependent. The protection is specific to CP12, because other proteins,
such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do
not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific
chaperone, since it does not protect other proteins, such as catalase, alcohol
dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is
necessary to prevent the aggregation and inactivation, since the mutant C66S
that does not form any complex with GAPDH cannot accomplish this protection.
Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is
partially able to protect and to slow down the inactivation and aggregation.
Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of
GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox
function but also behaves as a specific “chaperone-like protein”
for GAPDH, although a stable and not transitory interaction is observed. This
new function of CP12 may explain why it is also present in complexes involving
A2B2 GAPDHs that possess a regulatory C-terminal
extension (GapB subunit) and therefore do not require CP12 to be
redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most
photosynthetic organisms, including cyanobacteria
(1,
2), higher plants
(3), the diatom
Asterionella formosa
(4,
5), and green
(1) and red algae
(6). It allows the formation of
a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH),3 two key
enzymes of the Calvin cycle pathway, and was recently shown to interact with
fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway
(7). The
phosphoribulokinase·GAPDH·CP12 complex has been extensively
studied in Chlamydomonas reinhardtii
(8,
9) and in Arabidopsis
thaliana (10,
11). In the green alga C.
reinhardtii, the interaction between CP12 and GAPDH is strong
(8). GAPDH may exist as a
homotetramer composed of four GapA subunits (A4) in higher plants,
cyanobacteria, and green and red algae
(6,
12), but in higher plants, it
can also exist as a heterotetramer (A2B2), composed of
two subunits, GapA and GapB
(13,
14). GapB, up to now, has
exclusively been found in Streptophyta, but recently two
prasinophycean green algae, Ostreococcus tauri and Ostreococcus
lucimarinus, were also shown to possess a GapB gene, whereas
CP12 is missing (15).
The GapB subunit is similar to the GapA subunit but has a C-terminal extension
containing two redox-regulated cysteine residues
(16). Thus, although the
A4 GAPDHs lack these regulatory cysteine residues
(13,
14,
17–20),
they are also redox-regulated through its interaction with CP12, since the C
terminus of this small protein resembles the C-terminal extension of the GapB
subunit. The regulatory cysteine residues for GapA are thus supplied by CP12,
as is well documented in the literature
(1,
8,
11,
16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs)
(21–26).
The amino acid composition of these proteins causes them to have no or few
secondary structures. Their total or partial lack of structure and their high
flexibility allow them to be molecular adaptors
(27,
28). They are often able to
bind to several partners and are involved in most cellular functions
(29,
30). Recently, some IUPs have
been described in photosynthetic organisms
(31,
32).There are many functional categories of IUPs
(22,
33). They can be, for
instance, involved in permanent binding and have (i) a scavenger role,
neutralizing or storing small ligands; (ii) an assembler role by forming
complexes; and (iii) an effector role by modulating the activity of a partner
molecule (33). These functions
are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating
its redox properties (8,
34,
35), and can also bind a metal
ion (36,
37). IUPs can also bind
transiently to partners, and some of them have been found to possess a
chaperone activity (31,
38). This chaperone function
was first shown for α-synuclein
(39) and for α-casein
(40), which are fully
disordered. The amino acid composition of IUPs is less hydrophobic than those
of soluble proteins; hence, they lack hydrophobic cores and do not become
insoluble when heated. Since CP12 belongs to this family, we tested if it was
resistant to heat treatment and finally, since it is tightly bound to GAPDH,
if it could prevent aggregation of its partner, GAPDH, an enzyme well known
for its tendency to aggregate
(41–44)
and consequently a substrate commonly used in chaperone studies
(45,
46).Unlike chaperones, which form transient, dynamic complexes with their
protein substrates through hydrophobic interactions
(47,
48), CP12 forms a stable
complex with GAPDH. The interaction involves the C-terminal part of the
protein and the presence of negatively charged residues on CP12
(35). However, only a
site-directed mutagenesis has been performed to characterize the interaction
site on GAPDH. Although the mutation could have an indirect effect, the
residue Arg-197 was shown to be a good candidate for the interaction site
(49).In this report, we accordingly used proteolysis experiments coupled with
mass spectrometry to detect which regions of GAPDH are protected by its
association with CP12. To conclude, the aim of this report was to characterize
a chaperone function of CP12 that had never been described before and to map
the interaction site on GAPDH using an approach that does not involve
site-directed mutagenesis. 相似文献
15.
16.
Pamela H. Cameron Eric Chevet Olivier Pluquet David Y. Thomas John J. M. Bergeron 《The Journal of biological chemistry》2009,284(50):34570-34579
Calnexin is a type I integral membrane phosphoprotein resident of the endoplasmic reticulum. Its intraluminal domain has been deduced to function as a lectin chaperone coordinating the timing of folding of newly synthesized N-linked glycoproteins of the secretory pathway. Its C-terminal cytosolic oriented extension has an ERK1 phosphorylation site at Ser563 affecting calnexin association with the translocon. Here we find an additional function for calnexin phosphorylation at Ser563 in endoplasmic reticulum quality control. A low dose of the misfolding agent l-azetidine 2-carboxylic acid slows glycoprotein maturation and diminishes the extent and rate of secretion of newly synthesized secretory α1-antitrypsin. Under these conditions the phosphorylation of calnexin is enhanced at Ser563. Inhibition of this phosphorylation by the MEK1 inhibitor PD98059 enhanced the extent and rate of α1-antitrypsin secretion comparable with that achieved by inhibiting α-mannosidase activity with kifunensine. This is the first report in which the phosphorylation of calnexin is linked to the efficiency of secretion of a cargo glycoprotein. 相似文献
17.
18.
Hitomi Hoshino Akiko Tsuchida Kiyokazu Kametani Masako Mori Tomoko Nishizawa Takefumi Suzuki Hitomi Nakamura Heeseob Lee Yuki Ito Motohiro Kobayashi Junya Masumoto Masaya Fujita Minoru Fukuda Jun Nakayama 《The journal of histochemistry and cytochemistry》2011,59(1):98-105
Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-d-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme’s localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. 相似文献
19.
Robert W. Humble Graham Mackenzie Gordon Shaw 《Nucleosides, nucleotides & nucleic acids》2013,32(4):363-367
Abstract Several D-ribofuranosyl, D-xylofuranosyl and D-arabinofuranosyl 5-aminoimidazoles have been successfully phosphorylated to 5’ -phosphates using a phosphotransferase from wheat shoots and p-nitrophenylphosphate as a phosphate donor. 相似文献
20.
Qiyu Feng Dan Baird Sungsoo Yoo Marc Antonyak Richard A. Cerione 《The Journal of biological chemistry》2010,285(24):18806-18816
Previously we showed that Cool-1 (Cloned out of library-1)/β-Pix (Pak-interactive exchange factor) is phosphorylated at a specific tyrosine residue (Tyr-442) in a Src-dependent manner and serves as a dual function guanine nucleotide exchange factor (GEF)/signaling-effector for Cdc42 that is essential for transformation by Src. Here, we show that knocking-down Cool-1 or overexpressing a Cool-1 mutant that contains substitutions within its Dbl homology domain and is defective for GEF activity, inhibits Src-promoted cell migration. Similarly, the expression of a Cool-1 mutant containing a tyrosine to phenylalanine substitution at position 442, making it incapable of being phosphorylated in response to serum, epidermal growth factor (EGF), or Src, also causes a significant inhibition of the migration and invasive activity of cells expressing oncogenic Src. We further demonstrate that the phosphorylation of Cool-1 at Tyr-442 weakens its ability to bind to one of its primary interaction-partners, Cat-1 (Cool-associated tyrosine phosphosubstrate-1)/Git-1 (G protein-coupled receptor kinase-interactor-1), thus making Cat more accessible for binding to paxillin. This enables cells to alternate between states where they contain large numbers of focal complexes (i.e. conditions favoring Cool-1-Cat interactions) versus reduced numbers of focal complexes (conditions favoring Cat-paxillin interactions). Overall, these findings show that the phosphorylation-dephosphorylation cycle of Cool-1 at Tyr-442 can serve as a key regulatory signal for focal complex assembly-disassembly, and consequently, for the migration and invasive activity of Src-transformed cells. 相似文献