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Orwah Saleh Bertolt Gust Bj?rn Boll Hans-Peter Fiedler Lutz Heide 《The Journal of biological chemistry》2009,284(21):14439-14447
The bacterium Streptomyces anulatus 9663, isolated from the
intestine of different arthropods, produces prenylated derivatives of
phenazine 1-carboxylic acid. From this organism, we have identified the
prenyltransferase gene ppzP. ppzP resides in a gene cluster
containing orthologs of all genes known to be involved in phenazine
1-carboxylic acid biosynthesis in Pseudomonas strains as well as
genes for the six enzymes required to generate dimethylallyl diphosphate via
the mevalonate pathway. This is the first complete gene cluster of a phenazine
natural compound from streptomycetes. Heterologous expression of this cluster
in Streptomyces coelicolor M512 resulted in the formation of
prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of
ppzP, only nonprenylated phenazine 1-carboxylic acid was formed.
Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble
protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate
dimethylallyltransferase, forming a C–C bond between C-1 of the
isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many
other prenyltransferases, the reaction of PpzP is independent of the presence
of magnesium or other divalent cations. The Km value for
dimethylallyl diphosphate was determined as 116 μm. For
dihydro-PCA, half-maximal velocity was observed at 35 μm.
Kcat was calculated as 0.435 s-1. PpzP shows
obvious sequence similarity to a recently discovered family of
prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The
present finding extends the substrate range of this family, previously limited
to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives
rise to an astounding diversity of secondary metabolites in bacteria, fungi,
and plants, including many compounds that are important in pharmacotherapy.
However, surprisingly little biochemical and genetic data are available on the
enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new
family of aromatic prenyltransferases was discovered in streptomycetes
(1), Gram-positive soil
bacteria that are prolific producers of antibiotics and other biologically
active compounds (2). The
members of this enzyme family show a new type of protein fold with a unique
α-β-β-α architecture
(3) and were therefore termed
ABBA prenyltransferases (1).
Only 13 members of this family can be identified by sequence similarity
searches in the data base at present, and only four of them have been
investigated biochemically
(3–6).
Up to now, only phenolic compounds have been identified as aromatic substrates
of ABBA prenyltransferases. We now report the discovery of a new member of the
ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl
moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate
(dihydro-PCA).2
Streptomyces strains produce many of prenylated phenazines as natural
products. For the first time, the present paper reports the identification of
a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of
different arthropods, produces several prenylated phenazines, among them
endophenazine A and B (Fig.
1A) (7).
We wanted to investigate which type of prenyltransferase might catalyze the
prenylation reaction in endophenazine biosynthesis. In streptomycetes and
other microorganisms, genes involved in the biosynthesis of a secondary
metabolite are nearly always clustered in a contiguous DNA region. Therefore,
the prenyltransferase of endophenazine biosynthesis was expected to be
localized in the vicinity of the genes for the biosynthesis of the phenazine
core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663.
B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG
is responsible for the biosynthesis of PCA
(8). The enzyme PhzC catalyzes
the condensation of phosphoenolpyruvate and erythrose-4-phosphate
(i.e. the first step of the shikimate pathway), and further enzymes
of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze
the conversion of chorismate to 2-amino-2-deoxyisochorismate and the
subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively.
These reactions are well established biochemically. Fewer data are available
about the following steps (i.e. dimerization of
2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a
decarboxylation, ultimately leading to PCA via several instable
intermediates). From Pseudomonas, experimental data on the role of
PhzF and PhzA/B have been published
(8,
9), whereas the role of PhzG is
yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis
described so far from streptomycetes
(10) was found not to contain
a phzF orthologue, raising the question of whether there may be
differences in the biosynthesis of phenazines between Pseudomonas and
Streptomyces.Screening of a genomic library of the endophenazine producer strain S.
anulatus now allowed the identification of the first complete gene
cluster of a prenylated phenazine, including the structural gene of
dihydro-PCA dimethylallyltransferase. 相似文献
3.
Zhang L Kinkelaar D Huang Y Li Y Li X Wang HH 《Applied and environmental microbiology》2011,77(20):7134-7141
The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including tet(M), ermB, sul2, and bla(TEM) were detected in infant subjects. Enterococcus spp., Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Escherichia coli/Shigella spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure. 相似文献
4.
Giselle Aparecida Fagundes-Silva Gustavo Adolfo Sierra Romero Elisa Cupolillo Ellen Priscila Gadelha Yamashita Adriano Gomes-Silva Jorge Augusto de Oliveira Guerra Alda Maria Da-Cruz 《Memórias do Instituto Oswaldo Cruz》2015,110(6):797-800
In the Brazilian Amazon, American tegumentary leishmaniasis (ATL) is endemic and
presents a wide spectrum of clinical manifestations due, in part, to the circulation
of at least seven Leishmania species. Few reports of
Leishmania (Viannia) naiffi infection suggest that its occurrence
is uncommon and the reported cases present a benign clinical course and a good
response to treatment. This study aimed to strengthen the clinical and
epidemiological importance of L. (V.) naiffi in the Amazon Region
(Manaus, state of Amazonas) and to report therapeutic failure in patients infected
with this species. Thirty Leishmania spp samples isolated from
cutaneous lesions were characterised by multilocus enzyme electrophoresis. As
expected, the most common species was Leishmania (V.) guyanensis (20
cases). However, a relevant number ofL. (V.) naiffi patients (8
cases) was observed, thus demonstrating that this species is not uncommon in the
region. No patient infected withL. (V.) naiffi evolved to
spontaneous cure until the start of treatment, which indicated that this species may
not have a self-limiting nature. In addition, two of the patients experienced a poor
response to antimonial or pentamidine therapy. Thus, either ATL cases due to
L. (V.) naiffi cannot be as uncommon as previously thought or
this species is currently expanding in this region. 相似文献
5.
Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
6.
Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase
: An Enzyme That Specifically Removes the Terminal Nonreducing
Galacturonosyl Residue in Rhamnogalacturonan Regions of
Pectin1
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Margien Mutter Gerrit Beldman Stuart M. Pitson Henk A. Schols Alphons G.J. Voragen 《Plant physiology》1998,117(1):153-163
A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg−1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h). 相似文献
7.
Ruqin Kou Juliano Sartoretto Thomas Michel 《The Journal of biological chemistry》2009,284(22):14734-14743
These studies explore the connections between simvastatin, Rac1, and
AMP-activated protein kinase (AMPK) pathways in cultured vascular endothelial
cells and in arterial preparations isolated from statin-treated mice. In
addition to their prominent effects on lipoprotein metabolism, statins can
regulate the small GTPase Rac1, and may also affect the phosphorylation of the
ubiquitous AMPK. We explored pathways of statin-modulated Rac1 and AMPK
activation both in arterial preparations from statin-treated mice as well as
in cultured endothelial cells. We treated adult mice with simvastatin daily
for 2 weeks and then harvested and analyzed arterial preparations. Simvastatin
treatment of mice led to a significant increase in AMPK and LKB1
phosphorylation and to a decrease in protein kinase A activity relative to
control animals, associated with a marked increase in Rac1 activation.
Exposure of bovine aortic endothelial cells to simvastatin for 24 h strikingly
increased GTP-bound Rac1 and led to increased phosphorylation of AMPK as well
as the AMPK kinase LKB1. These responses to simvastatin were blocked by
mevalonate or geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate.
Small interfering RNA (siRNA)-mediated knockdown of AMPK abrogated
simvastatin-induced Rac1 activation and LKB1 phosphorylation. Importantly,
siRNA-mediated knockdown of the key AMPK kinase, calcium/calmodulin-dependent
protein kinase kinase β, completely blocked simvastatin-induced
endothelial cell migration and also abrogated statin-promoted phosphorylation
of AMPK and LKB1, as did pharmacological inhibition with the specific
calcium/calmodulin-dependent protein kinase β inhibitor STO-609.
Moreover, siRNA-mediated knockdown of Rac1 completely blocked
simvastatin-induced LKB1 phosphorylation, but without affecting
simvastatin-induced AMPK phosphorylation. These findings establish a key role
for simvastatin in activation of a novel Rac1-dependent signaling pathway in
the vascular wall.HMG-CoA2 reductase
inhibitors, commonly known as statins, are widely prescribed for the
prevention and treatment of hypercholesterolemia and cardiovascular diseases
(1,
2). The salutary clinical
effects of these drugs derive in part from their effects on the levels of
serum lipoproteins, yet other statin responses appear to be mediated by
alterations in vascular function involving the endothelial isoform of
nitric-oxide synthase (3) and
related signaling pathways. Inhibition of HMG-CoA reductase suppresses the
cellular levels of its enzymatic product mevalonate, thereby attenuating
formation both of cholesterol as well as the synthesis of distinct isoprenoid
compounds such as farnesyl pyrophosphate (Fpp) and geranylgeranyl
pyrophosphate (GGpp). Many key signaling proteins are covalently modified by
these isoprenoids, which are the products of a metabolic pathway that diverges
from the pathway that leads to cholesterol synthesis downstream of HMG-CoA
reductase. These isoprenoid compounds can provide lipophilic anchors that
facilitate membrane targeting and modulate protein-protein interactions of
many key signaling proteins. One such iso-prenylated signaling protein is the
GTP-binding cytoskeletonassociated protein Rac1, a member of the Rho GTPase
small G protein family that undergoes geranylgeranylation at its C terminus.
Statins also affect post-translational modification of another small GTPase,
RhoA, that, like Rac1, is a geranylgeranylated protein that is an important
determinant of vascular signaling
(4–8).
Rac1 has particularly important roles in vascular endothelial cells, where
this cytoskeleton regulatory protein modulates activity of the endothelial
isoform of nitric-oxide synthase (eNOS), a key determinant of vascular
homeostasis (9). Rac1
activation in endothelial cells is influenced by the AMP-activated protein
kinase (AMPK) (6), which itself
is phosphorylated by the protein kinase LKB1 and by the
calcium-calmodulin-dependent protein kinase β (CaMKKβ) (see review
(10)). In recent years,
numerous reports have described effects of statins on variety of these
signaling proteins in different experimental systems
(11–14).Statins have been shown to promote the phosphorylation of AMPK
(13), a heterotrimeric enzyme
involved in the modulation of cellular energy pathways that has also been
implicated in eNOS regulation
(3,
15–17).
AMPK was originally discovered and characterized as a cellular “energy
sensor” that can be activated by increases in the intracellular AMP:ATP
ratio (18). However, in recent
years, it has become clear that AMPK is also regulated through AMP-independent
pathways involving enzyme phosphorylation on threonine 172 of the enzyme''s
α subunit, leading to marked enzyme activation
(19). Protein kinases that
phosphorylate AMPK include the tumor suppressor LKB1 and the
calcium/calmodulin-dependent kinase CaMKKβ. LKB1 itself is a
phosphoprotein. The pathways that regulate LKB1 are incompletely understood,
and a variety of upstream protein kinases have been implicated in LKB1
regulation (see review (20)).
CaMKKβ is principally regulated by calcium binding, but this kinase may
also be phosphorylated by the cAMP-dependent protein kinase PKA
(21,
22). Another substrate for PKA
in vascular cells is the actin-binding phosphoprotein VASP
(23,
24); the phosphorylation state
of VASP at its PKA site can serve as a surrogate marker for the activity of
cAMP-dependent signaling pathways in the vascular wall
(25). CaMKKβ has been
shown to be involved in AMPK regulation in endothelial cells in response to
receptor tyrosine kinase activation and via G protein-coupled receptor
pathways (6). Activated AMPK
directly phosphorylates eNOS, and this kinase thereby appears be an important
determinant of NO-dependent signaling in endothelial cells. However, much
remains to be learned about the molecular mechanisms whereby statins enhance
AMPK activation.In cultured cells, statins have been shown to inhibit the
geranylgeranylation of Rac1, associated with an increase in Rac1 GTP binding
and activation (26). The
activation of Rac1 is a key step in eNOS activation: siRNA-mediated Rac1
“knockdown” in endothelial cells markedly suppresses receptor
signaling to eNOS (5,
7). siRNA-mediated AMPK
knockdown suppresses Rac1 activation, again leading to the attenuation of
receptor-dependent activation of eNOS
(6). The relationships among
these various statin-modulated signaling pathways are incompletely
characterized. The present studies identify CaMKKβ and LKB1 as critical
determinants of simvastatin-dependent activation of AMPK- and Rac1-modulated
signaling and reveal that Rac1 in turn regulates LKB1 phosphorylation. 相似文献
8.
Adrien W. Schmid Diego Chiappe V��r��ne Pignat Valerie Grimminger Ivan Hang Marc Moniatte Hilal A. Lashuel 《The Journal of biological chemistry》2009,284(19):13128-13142
Tissue transglutaminase (tTG) has been implicated in the pathogenesis of
Parkinson disease (PD). However, exactly how tTG modulates the structural and
functional properties of α-synuclein (α-syn) and contributes to
the pathogenesis of PD remains unknown. Using site-directed mutagenesis
combined with detailed biophysical and mass spectrometry analyses, we sought
to identify the exact residues involved in tTG-catalyzed cross-linking of
wild-type α-syn and α-syn mutants associated with PD. To better
understand the structural consequences of each cross-linking reaction, we
determined the effect of tTG-catalyzed cross-linking on the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Our
findings show that tTG-catalyzed cross-linking of monomeric α-syn
involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed
cross-linked monomeric α-syn composed of either wild-type or Gln →
Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping
by mass spectrometry. Using this approach, we identified the glutamine and
lysine residues involved in tTG-catalyzed intramolecular cross-linking of
α-syn. These studies demonstrate for the first time that
Gln79 and Gln109 serve as the primary tTG reactive
sites. Mutating both residues to asparagine abolishes tTG-catalyzed
cross-linking of α-syn and tTG-induced inhibition of α-syn
fibrillization in vitro. To further elucidate the sequence and
structural basis underlying these effects, we identified the lysine residues
that form isopeptide bonds with Gln79 and Gln109. This
study provides mechanistic insight into the sequence and structural basis of
the inhibitory effects of tTG on α-syn fibrillogenesis in vivo,
and it sheds light on the potential role of tTG cross-linking on modulating
the physiological and pathogenic properties of α-syn.Parkinson disease
(PD)2 is a progressive
movement disorder that is caused by the loss of dopaminergic neurons in the
substantia nigra, the part of the brain responsible for controlling movement.
Clinically, PD is manifested in symptoms that include tremors, rigidity, and
difficulty in initiating movement (bradykinesia). Pathologically, PD is
characterized by the presence of intraneuronal, cytoplasmic inclusions known
as Lewy bodies (LB), which are composed primarily of the protein
“α-synuclein” (α-syn)
(1) and are seen in the
post-mortem brains of PD patients with the sporadic or familial forms of the
disease (2). α-Syn is a
presynaptic protein of 140 residues with a “natively” unfolded
structure (3). Three missense
point mutations in α-syn (A30P, E46K, and A53T) are associated with the
early-onset, dominant, inherited form of PD
(4,
5). Moreover, duplication or
triplication of the α-syn gene has been linked to the familial
form of PD, suggesting that an increase in α-syn expression is
sufficient to cause PD. Together, these findings suggest that α-syn
plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn
oligomerization and fibrillogenesis in vivo remain poorly understood.
In vitro aggregation studies have shown that the mutations associated
with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but
only E46K and A53T α-syn show higher propensity to fibrillize than
wild-type (WT) α-syn
(6-8).
This suggests that oligomerization, rather than fibrillization, is linked to
early-onset familial PD (9).
Our understanding of the molecular composition and biochemical state of
α-syn in LBs has provided important clues about protein-protein
interactions and post-translational modifications that may play a role in
modulating oligomerization, fibrillogenesis, and LB formation of the protein.
In addition to ubiquitination
(10), phosphorylation
(11,
12), nitration
(13,
14), and C-terminal truncation
(15,
16), analysis of post-mortem
brain tissues from PD and Lewy bodies in dementia patients has confirmed the
colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked
α-syn monomers and higher molecular aggregates in LBs within
dopaminergic neurons (17,
18). Tissue transglutaminase
catalyzes a calcium-dependent transamidating reaction involving glutamine and
lysine residues, which results in the formation of a covalent cross-link via
ε-(γ-glutamyl) lysine bonds
(Fig. 2F). To date,
seven different isoforms of tTGs have been reported, of which only tTG2 seems
to be expressed in the human brain
(19), whereas tTG1 and tTG3
are more abundantly found in stratified squamous epithelia
(20). Subsequent
immuno-histochemical, colocalization, and immunoprecipitation studies have
shown that the levels of tTG and cross-linked α-syn species are
increased in the substantia nigra of PD brains
(17). These findings, combined
with the known role of tTG in cross-linking and stabilizing bimolecular
assemblies, led to the hypothesis that tTG plays an important role in the
initiation and propagation of α-syn fibril formation and that it
contributes to fibril stability in LBs. This hypothesis was initially
supported by in vitro studies demonstrating that tTG catalyzes the
polymerization of the α-syn-derived non-amyloid component (NAC) peptide
via intermolecular covalent cross-linking of residues Gln79 and
Lys80 (21) and by
other studies suggesting that tTG promotes the fibrillization of amyloidogenic
proteins implicated in the pathogenesis of other neurodegenerative diseases
such as Alzheimer disease, supranuclear palsy, Huntington disease, and other
polyglutamine diseases
(22-24).
However, recent in vitro studies with full-length α-syn have
shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn
and inhibits, rather than promotes, its fibrillization in vitro
(25,
26). The structural basis of
this inhibitory effect and the exact residues involved in tTG-mediated
cross-linking of α-syn, as well as structural and functional
consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three
intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native
(—) and cross-linked (- - -) α-syn, showing that most
tTG-catalyzed cross-linking products of WT or disease-associated mutant forms
of α-syn are intramolecularly linked (predominant peak with two
cross-links), and up to three intramolecular cross-links can occur (left
shoulder). The abbreviations M and m/cl are
used to designate native and cross-linked α-synuclein, respectively.
D and E, kinetic analysis of α-syn (A30P)
cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic
depiction of the tTG-catalyzed chemical reaction (isodipeptide formation)
between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues
involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn
and investigated how cross-linking these residues affects the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Using
single-site mutagenesis and mass spectrometry applied to exhaustive
proteolytic digests of native and cross-linked monomeric α-syn, we
identified Gln109 and Gln79 as the major tTG substrates.
We demonstrate that the altered electrophoretic mobility of the
intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of
tTG-catalyzed cross-linking of Gln109 to lysine residues in the N
terminus of α-syn, which leads to the formation of more compact
monomers. Consistent with previous studies, we show that intramolecularly
cross-linked α-syn forms off-pathway oligomers that are distinct from
those formed by the wild-type protein and that do not convert to fibrils
within the time scale of our experiments (3-5 days). We also show that
membrane-bound α-syn is a substrate of tTG and that intramolecular
cross-linking does not interfere with the ability of monomeric α-syn to
adopt an α-helical conformation upon binding to synthetic membranes.
These studies provide novel mechanistic insight into the sequence and
structural basis of events that allow tTG to inhibit α-syn
fibrillogenesis, and they shed light on the potential role of tTG-catalyzed
cross-linking in modulating the physiological and pathogenic properties of
α-syn. 相似文献
9.
Fernando Fonseca Val Vanderson Souza Sampaio Maria Belén Cassera Raquel Tapajós Andrade Pedro Luiz Tauil Wuelton Marcelo Monteiro Marcus Vinícius Guimar?es Lacerda 《Memórias do Instituto Oswaldo Cruz》2014,109(5):522-524
In the 1950s, the strategy of adding chloroquine to food salt as a prophylaxis
against malaria was considered to be a successful tool. However, with the development
of Plasmodium resistance in the Brazilian Amazon, this control
strategy was abandoned. More than 50 years later, asexual stage resistance can be
avoided by screening for antimalarial drugs that have a selective action against
gametocytes, thus old prophylactic measures can be revisited. The efficacy of the old
methods should be tested as complementary tools for the elimination of malaria. 相似文献
10.
Martin J. Sergeant Jian-Jun Li Christine Fox Nicola Brookbank Dean Rea Timothy D. H. Bugg Andrew J. Thompson 《The Journal of biological chemistry》2009,284(8):5257-5264
Members of the carotenoid cleavage dioxygenase family catalyze the
oxidative cleavage of carotenoids at various chain positions, leading to the
formation of a wide range of apocarotenoid signaling molecules. To explore the
functions of this diverse enzyme family, we have used a chemical genetic
approach to design selective inhibitors for different classes of carotenoid
cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized
in which the distance between an iron-chelating hydroxamic acid and an
aromatic ring was varied; these compounds were screened as inhibitors of four
different enzyme classes, either in vitro or in vivo. Potent
inhibitors were found that selectively inhibited enzymes that cleave
carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar
concentrations. Application of certain inhibitors at 100 μm to
Arabidopsis node explants or whole plants led to increased shoot
branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as
photoprotective molecules and are key components in animal diets, an example
being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids
occurs in plants, animals, and micro-organisms and leads to the release of a
range of apocarotenoids that function as signaling molecules with a diverse
range of functions (1). The
first gene identified as encoding a carotenoid cleavage dioxygenase
(CCD)2 was the maize
Vp14 gene that is required for the formation of abscisic acid (ABA),
an important hormone that mediates responses to drought stress and aspects of
plant development such as seed and bud dormancy
(2). The VP14 enzyme cleaves at
the 11,12 position (Fig. 1) of
the epoxycarotenoids 9′-cis-neoxanthin and/or
9-cis-violaxanthin and is now classified as a
9-cis-epoxycarotenoid dioxygenase (NCED)
(3), a subclass of the larger
CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases.
a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by
NCED; b, oxidative cleavage reactions on β-carotene and
zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be
involved in the production of a variety of apocarotenoids
(Fig. 1). In insects, the
visual pigment retinal is formed by oxidative cleavage of β-carotene by
β-carotene-15,15′-dioxygenase
(4). Retinal is produced by an
orthologous enzyme in vertebrates, where it is also converted to retinoic
acid, a regulator of differentiation during embryogenesis
(5). A distinct mammalian CCD
is believed to cleave carotenoids asymmetrically at the 9,10 position
(6) and, although its function
is unclear, recent evidence suggests a role in the metabolism of dietary
lycopene (7). The plant
volatiles β-ionone and geranylacetone are produced from an enzyme that
cleaves at the 9,10 position
(8) and the pigment
α-crocin found in the spice saffron results from an 7,8-cleavage enzyme
(9).Other CCDs have been identified where biological function is unknown, for
example, in cyanobacteria where a variety of cleavage specificities have been
described
(10-12).
In other cases, there are apocarotenoids with known functions, but the
identity or involvement of CCDs have not yet been described: grasshopper
ketone is a defensive secretion of the flightless grasshopper Romalea
microptera (13),
mycorradicin is produced by plant roots during symbiosis with arbuscular
mycorrhyza (14), and
strigolactones (15) are plant
metabolites that act as germination signals to parasitic weeds such as
Striga and Orobanche
(16).Recently it was discovered that strigolactones also function as a branching
hormone in plants (17,
18). The existence of such a
branching hormone has been known for some time, but its identity proved
elusive. However, it was known that the hormone was derived from the action of
at least two CCDs, max3 and max4 (more axillary growth)
(19), because deletion of
either of these genes in Arabidopsis thaliana, leads to a bushy
phenotype (20,
21). In Escherichia
coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position
and the apocarotenoid product (10-apo-β-carotene) is reported to be
further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene
(22). Also recent evidence
suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene
(23). How the production of
13-apo-β-carotene leads to the synthesis of the complex strigolactone is
unknown. The possibility remains that the enzymes may have different
specificities and cleavage activities in planta. In addition, a
cytochrome P450 enzyme (24) is
believed to be involved in strigolactone synthesis and acts in the pathway
downstream of the CCD genes. Strigolactone is thought to effect branching by
regulating auxin transport
(25). Because of the
involvement of CCDs in strigolactone synthesis, the possibility arises that
plant architecture and interaction with parasitic weeds and mycorrhyza could
be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to
probe function and substrate specificity of CCDs in their native biological
contexts, particularly in plant species with simple genetic systems or that
are amenable to transgenesis, there are many systems where genetic approaches
are difficult or impossible. Also, when recombinant CCDs are studied either
in vitro or in heterologous in vivo assays, such as in
E. coli strains engineered to accumulate carotenoids
(26), they are often active
against a broad range of substrates
(5,
21,
27), and in many cases the
true in vivo substrate of a particular CCD remains unknown. Therefore
additional experimental tools are needed to investigate both apocarotenoid and
CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified
that are active against known target proteins; they are then applied to a
biological system to investigate protein function in vivo
(28,
29). This approach is
complementary to conventional genetics since the small molecules can be
applied easily to a broad range of species, their application can be
controlled in dose, time, and space to provide detailed studies of biological
functions, and individual proteins or whole protein classes may be targeted by
varying the specificity of the small molecules. Notably, functions of the
plant hormones gibberellin, brassinosteroid, and abscisic acid have been
successfully probed using this approach by adapting triazoles to inhibit
specific cytochrome P450 monooxygenases involved in the metabolism of these
hormones (30).In the case of the CCD family, the tertiary amines abamine
(31) and the more active
abamineSG (32) were reported
as specific inhibitors of NCED, and abamine was used to show new functions of
abscisic acid in legume nodulation
(33). However, no selective
inhibitors for other types of CCD are known. Here we have designed a novel
class of CCD inhibitor based on hydroxamic acids, where variable chain length
was used to direct inhibition of CCD enzymes that cleave carotenoids at
specific positions. We demonstrate the use of such novel inhibitors to control
shoot branching in a model plant. 相似文献
11.
Maryline Paris Cécile Tourrel-Cuzin Cédric Plachot Alain Ktorza 《Experimental diabetes research》2004,5(2):111-121
β-cell neogenesis triggers the generation of new β-cells
from precursor cells. Neogenesis from duct epithelium is the
most currently described and the best documented process
of differentiation of precursor cells into β-cells. It is contributes
not only to β-cell mass expansion during fetal and
nonatal life but it is also involved in the maintenance of the
β-cell mass in adults. It is also required for the increase in
β-cell mass in situations of increase insulin demand (obesity,
pregnancy). A large number of factors controlling the differentiation
of β-cells has been identified. They are classified
into the following main categories: growth factors, cytokine
and inflamatory factors, and hormones such as PTHrP and
GLP-1. The fact that intestinal incretin hormone GLP-1
exerts a major trophic role on pancreatic β-cells provides
insights into the possibility to pharmacologically stimulate
β-cell neogenesis. This could have important implications
for the of treatment of type 1 and type 2 diabetes. Transdifferentiation,
that is, the differentiation of already differentiated
cells into β-cells, remains controversial.However, more
and more studies support this concept. The cells, which can
potentially “transdifferentiate” into β-cells, can belong to
the pancreas (acinar cells) and even islets, or originate from
extra-pancreatic tissues such as the liver. Neogenesis from intra-islet precursors also have been proposed and subpopulations
of cell precursors inside islets have been described
by some authors. Nestin positive cells, which have been considered
as the main candidates, appear rather as progenitors
of endothelial cells rather than β-cells and contribute to
angiogenesis rather than neogenesis. To take advantage of
the different differentiation processes may be a direction for
future cellular therapies. Ultimately, a better understanding
of the molecular mechanisms involved in β-cell neogenesis
will allow us to use any type of differentiated and/or undifferentiated
cells as a source of potential cell precursors. 相似文献
12.
13.
Jane Costa Nathália Cordeiro Correia Vanessa Lima Neiva Teresa Cristina Monte Gon?alves Márcio Felix 《Memórias do Instituto Oswaldo Cruz》2013,108(6):785-789
Triatoma brasiliensis macromelasoma is revalidated based on the
results of previous multidisciplinary studies on the Triatoma
brasiliensis complex, consisting of crossing experiments and
morphological, biological, ecological and molecular analyses. These taxonomic
tools showed the closest relationship between T. b.
macromelasoma and Triatoma brasiliensis
brasiliensis. T. b. macromelasoma is redescribed
based on specimens collected in the type locality and specimens from a F1
colony. The complex now comprises T. b. brasiliensis,
T. b. macromelasoma, Triatoma melanica,
Triatoma juazeirensis and Triatoma
sherlocki. An identification key for all members of the complex is
presented. This detailed comparative study of the morphological features of
T. b. macromelasoma and the remaining members of the
complex corroborates results from multidisciplinary analyses, suggesting that
the subspecific status is applicable. This subspecies can be distinguished by
the following combination of features: a pronotum with 1+1 narrow
brownish-yellow stripes on the submedian carinae, not attaining its apex,
hemelytra with membrane cells darkened on the central portion and legs with an
incomplete brownish-yellow ring on the apical half of the femora. Because the
T. brasiliensis complex is of distinct epidemiological
importance throughout its geographic distribution, a precise identification of
its five members is important for monitoring and controlling actions against
Chagas disease transmission. 相似文献
14.
15.
Adam Doern Xianjun Cao Arlene Sereno Christopher L. Reyes Angelina Altshuler Flora Huang Cathy Hession Albert Flavier Michael Favis Hon Tran Eric Ailor Melissa Levesque Tracey Murphy Lisa Berquist Susan Tamraz Tracey Snipas Ellen Garber William S. Shestowsky Rachel Rennard Christilyn P. Graff Xiufeng Wu William Snyder Lindsay Cole David Gregson Michael Shields Steffan N. Ho Mitchell E. Reff Scott M. Glaser Jianying Dong Stephen J. Demarest Kandasamy Hariharan 《The Journal of biological chemistry》2009,284(15):10254-10267
16.
Usha Padmanabhan D. Eric Dollins Peter C. Fridy John D. York C. Peter Downes 《The Journal of biological chemistry》2009,284(16):10571-10582
17.
18.
Yi Zhu Julius Bogomolovas Siegfried Labeit Henk Granzier 《The Journal of biological chemistry》2009,284(20):13914-13923
The small heat shock protein αB-crystallin interacts with N2B-Us, a
large unique sequence found in the N2B element of cardiac titin. Using single
molecule force spectroscopy, we studied the effect of αB-crystallin on
the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal
tandem Ig segment of titin were also studied. The effect of wild type
αB-crystallin on the single molecule force-extension curve was
determined as well as that of mutant αB-crystallins harboring the
dilated cardiomyopathy missense mutation, R157H, or the desmin-related
myopathy mutation, R120G. Results revealed that wild type αB-crystallin
decreased the persistence length of the N2B-Us (from ∼0.7 to ∼0.2 nm)
but did not alter its contour length. αB-crystallin also increased the
unfolding force of the Ig domains that flank the N2B-Us (by 51 ± 3
piconewtons); the rate constant of unfolding at zero force was estimated to be
∼17-fold lower in the presence of αB-crystallin (1.4 ×
10-4 s-1 versus 2.4 × 10-3
s-1). We also found that αB-crystallin increased the
unfolding force of Ig domains from the proximal tandem Ig segment by 28
± 6 piconewtons. The effects of αB-crystallin were attenuated by
the R157H mutation (but were still significant) and were absent when using the
R120G mutant. We conclude that αB-crystallin protects titin from damage
by lowering the persistence length of the N2B-Us and reducing the Ig domain
unfolding probability. Our finding that this effect is either attenuated
(R157H) or lost (R120G) in disease causing αB-crystallin mutations
suggests that the interaction between αB-crystallin and titin is
important for normal heart function.αB-crystallin is a member of the small heat shock protein family that
by inhibiting denaturation and aggregation of proteins functions as a
molecular chaperone (1).
Although αB-crystallin has been most intensively studied in the
vertebrate eye lens, it is also found in many other tissues
(2) with cardiac muscle
expressing αB-crystallin at 3-5% of the total soluble protein
(3). Up-regulation of
αB-crystallin occurs in a number of cardiac disorders, including
familial cardiac hypertrophy, and overexpression appears to protect the
cardiac cell from ischemia reperfusion injury (for a review see Ref.
4). An important binding
partner of αB-crystallin in cardiac muscle is titin
(5,
6). Titin is a large
filamentous protein that forms a continuous filament along the myofibril, with
single titin molecules spanning from the edge to the middle of the sarcomere,
a distance of ∼1 μm (7).
The I-band region of titin is extensible and functions as a molecular spring
that, when extended, develops force
(8,
9). This force is an important
determinant of the passive stiffness of the heart that determines the filling
characteristics during the diastolic part of the heart cycle
(10). The interaction between
αB-crystallin and titin could be important for maintaining heart
function, especially when stressed, such as during ischemia
(5), warranting studies of the
effect of αB-crystallin on the biomechanical properties of titin.The molecular spring region of titin contains three distinct spring
elements (7). The first element
is the tandem Ig segment, consisting of serially linked Ig domains that form
the so-called proximal tandem Ig segment (15 Ig domains) near the Z-disk of
the sarcomere and a distal segment (22 Ig domains) near the A-band
(11). The second spring
element is the PEVK, a unique sequence that contains largely prolines,
glutamates, valines, and lysines
(11). The third element
consists of a large unique sequence (in human 572 residues in size) named the
N2B-Us; it is heart-specific and dominates the extension of titin near the
upper limit of the physiological sarcomere length range
(12). αB-crystallin
appears to preferentially bind to the N2B-Us, although weak binding to Ig
domains has also been detected
(6). Previous studies have
shown that αB-crystallin increases the unfolding force of Ig 91-98, a
fragment that contains eight Ig domains from the distal tandem Ig segment of
titin (6). However, the
mechanical effect of αB-crystallin on the N2B-Us (its main binding
partner in titin) has not been investigated.The association between αB-crystallin and titin has prompted a search
for disease causing mutations in αB-crystallin. This revealed in
patients with dilated cardiomyopathy
(DCM),2 a missense
mutation, R157H, that affects an evolutionarily conserved amino acid residue;
the mutation decreases the binding to the N2B domain without affecting
distribution of the mutant crystallin protein in cardiomyocytes
(13). In another disease, the
desmin-related myopathy mutation R120G
(14) decreases the binding of
αB-crystallin to the N2B element and causes intracellular aggregates of
the mutant protein (13).In the present study, we used single molecule force spectroscopy and
determined the contour length (CL; end-to-end length when stretched with
infinite force) and persistence length (PL; a measure of the bending rigidity)
of the N2B-Us. We also studied the unfolding force of Ig domains, those that
flank the N2B-Us and those that make up the proximal tandem Ig segment. In
addition, we investigated the effect of wild type and R157H and R120G
αB-crystallin on the molecular mechanics of the N2B-Us, its flanking Ig
domains, and the Ig domains in the proximal tandem Ig segment. Findings
support that αB-crystallin functions as a chaperone that lowers the
probability of Ig domain unfolding and the persistence length of the titin
N2B-Us spring region. Importantly, this chaperone function is significantly
reduced by the R157H mutation and abolished by the R120G mutation. 相似文献