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1.
Yusuke Takahashi Gennadiy Moiseyev Jian-xing Ma 《The Journal of biological chemistry》2014,289(39):26743-26751
RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates'' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met1–Arg33) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations. 相似文献
2.
Marcin Golczak Philip D. Kiser David T. Lodowski Akiko Maeda Krzysztof Palczewski 《The Journal of biological chemistry》2010,285(13):9667-9682
Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment. 相似文献
3.
Carlos H Ni?o Nicolás Forero-Baena Luis E Contreras Diana Sánchez-Lancheros Katherine Figarella María H Ramírez 《Memórias do Instituto Oswaldo Cruz》2015,110(7):890-897
The intracellular parasite Trypanosoma cruzi is the aetiologicalagent of Chagas disease, a public health concern with an increasing incidence rate.This increase is due, among other reasons, to the parasite''s drug resistancemechanisms, which require nicotinamide adenine dinucleotide (NAD+).Furthermore, this molecule is involved in metabolic and intracellular signallingprocesses necessary for the survival of T. cruzi throughout its lifecycle. NAD+ biosynthesis is performed by de novo andsalvage pathways, which converge on the step that is catalysed by the enzymenicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commissionnumber: 2.7.7.1). The identification of the NMNAT of T.cruzi is important for the development of future therapeutic strategiesto treat Chagas disease. In this study, a hypothetical open reading frame (ORF) forNMNAT was identified in the genome of T. cruzi. The correspondingputative protein was analysed by simulating structural models. The ORF was amplifiedfrom genomic DNA by polymerase chain reaction and was further used for theconstruction of a corresponding recombinant expression vector. The expressedrecombinant protein was partially purified and its activity was evaluated usingenzymatic assays. These results comprise the first identification of an NMNAT inT. cruzi using bioinformatics and experimental tools and hencerepresent the first step to understanding NAD+ metabolism in theseparasites. 相似文献
4.
Thirty-five populations of Heterodera glycines and populations of 15 other Heterodera, Globodera, and Punctodera species were studied morphometrically and some were compared serologically. There was a wide range of each measurement within each nematode population. Except for one soybean cyst nematode population from Indiana, which was a tetraploid and considerably larger than the others, morphometric measurements overlapped. In a discriminant function comparison most of the populations were closely grouped but at least three were rather distinctly separated. Morphometrically H. fici, H. cruciferae, H. schachtii, and H. trifolii were closely associated with H. glycines. Serology indicated a close relationship between H. glycines, H. lespedezae, H. trifolii, H. schachtii, and the Heterodera sp. from Rumex, while H. betulae appeared to be more distantly related. 相似文献
5.
Guoyan Mo Qin Ding Zhongshan Chen Yunbo Li Ming Yan Lijing Bu Yanping Song Guohua Yin 《PloS one》2014,9(11)
The retinal pigment epithelium-specific 65 kDa protein is an isomerase encoded by the RPE65 gene (MIM 180069) that is responsible for an essential enzymatic step required for the function of the visual cycle. Mutations in the RPE65 gene cause not only subtype II of Leber congenital amaurosis (LCA) but also early-onset severe retinal dystrophy (EOSRD). This study aims to investigate a Chinese case diagnosed as EOSRD and to characterize the polymorphisms of the RPE65 gene. A seven-year-old girl with clinical symptoms of EOSRD and her parents were recruited into this study. Ophthalmologic examinations, including best-corrected visual acuity, slit-lamp, Optical coherence tomography (OCT), and fundus examination with dilated pupils, were performed to determine the clinical characteristics of the whole family. We amplified and sequenced the entire coding region and adjacent intronic sequences of the coding regions of the RPE65 gene for the whole family to explore the possible mutation. Our results demonstrate that the patient exhibited the typical clinically features of EOSRD. Her bilateral decimal visual acuity was 0.3 and 0.4 in the left and right eyes, respectively. Spectral-domain optical coherence tomography (SD-OCT) was used to assess the retinal stratification for the whole family. All together, we identified four mutations within the RPE65 gene (c.1056G>A, c.1243+2T>A, c.1338+20A>C and c.1590C>A) in the patient. Among the four mutations, c.1056G>A and c.1338+20A>C had been reported previously and another two were found for the first time in this study. Her mother also carried the novel mutation (c.1243+2T>A). Either a single or a compound heterozygous or a homozygous one mutation is expected to cause EOSRD because mutations of RPE65 gene usually cause an autosomal recessive disease. Therefore, we speculate that the c.1590C>A mutation together with the c.1243+2T>A mutation may cause the patient’s phenotype. 相似文献
6.
wrinkled1: A Novel, Low-Seed-Oil Mutant of
Arabidopsis with a Deficiency in the Seed-Specific Regulation of
Carbohydrate Metabolism 总被引:10,自引:1,他引:10 下载免费PDF全文
During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling. 相似文献
7.
Cell-Specific Production and Antimicrobial Activity of
Naphthoquinones in Roots of Lithospermum
erythrorhizon 总被引:2,自引:0,他引:2
Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere. 相似文献
8.
9.
Neetu Tewari Satyan Kalkunte David W. Murray Surendra Sharma 《The Journal of biological chemistry》2009,284(22):15224-15232
Despite serious health risks in humans and wild life, the underlying
mechanisms that explain the gene-environment effects of chemical toxicants are
largely unknown. Polychlorinated biphenyls (PCBs) are one of the most
ubiquitous environmental toxicants worldwide, with reported epidemiological
evidence for reproductive and neurocognitive anomalies in humans. Here, we
show that Aroclor 1254, a mixture of structurally distinct PCBs, causes
preterm birth in interleukin (IL)-10-/- mice at a dose that does
not show any adverse effects in wild type mice, highlighting the significance
of IL-10 as an anti-toxicant cytokine. Aroclor 1254-treated
IL-10-/- mice demonstrated increased amniotic fluid, intrauterine
growth restriction, and reduced litter size with postnatal neuromotor defects.
Further, our results identify aquaporin 1 (AQP1), a potent effector of fluid
volume regulation and angiogenic activity, as a novel placental target of
PCBs. In vivo or in vitro exposure to Aroclor 1254 coupled
with IL-10 deficiency significantly reduced the protein content of AQP1.
Reduced uterine AQP1 levels were associated with defective spiral artery
transformation. Importantly, recombinant IL-10 reversed PCB-induced in
vivo and in vitro effects. These data demonstrate for the first
time that the IL-10-AQP1 axis is a novel regulator of PCB-induced in
utero effects.The health consequences of environmental toxicants are likely to have
critical effects during in utero fetal development because of the
complex signaling cascades, high cellular proliferation rates, and
differentiation events. Mammalian reproduction involves a complex but highly
choreographed sequence of molecular processes. These processes include
interactions between the hormonally stimulated uterus and the developing
blastocyst, implantation, placental and fetal development, and parturition
(1,
2). Although the hormonal
milieu, metabolic changes, and placental microenvironment are programmed in a
pregnancy compatible manner, pregnancy presents itself as an immunological and
hormonal paradox (3,
4). The role of steroid
hormones is well known in uterine receptivity, implantation, local immune
modulation, and pregnancy success
(5). If not temporally produced
and regulated, their dysfunction lead to infertility or pregnancy loss.
Man-made chemicals like polychlorinated biphenyls
(PCBs)2 act like
hormones and interfere with their cognate receptor functions impacting normal
biological processes (6,
7). Although the genotoxic
effects of PCBs have been investigated intensively and epidemiological studies
have highlighted their health risks
(6,
7), the mechanisms responsible
for reproductive and neurodevelopmental effects still remain enigmatic. The
overarching goal of our studies is to identify unknown pathways and targets
that impart adverse effects on pregnancy. In this study, we directed our
efforts toward establishing an experimental system to evaluate the in
utero gene-environment effects of PCBs using wild type mice and their
counterparts deficient in pregnancy compatible anti-inflammatory cytokines
such as interleukin 10 (IL-10).IL-10 is a potent anti-inflammatory cytokine that controls inflammatory
insult in most organs, particularly at the maternal-fetal interface. IL-10 is
produced by gestational tissue and maternal immune cells in the intrauterine
microenvironment in humans (8,
9) and in mice
(10). We and others have
reported that IL-10-/- mice experience preterm birth and
resorptions in response to low doses of inflammatory triggers such as
lipopolysaccharide (LPS) (11,
12) or poly(I-C)
(13). Importantly, the
pregnancy outcome in treated IL-10-/- mice can be rescued by giving
an exogenous dose of IL-10
(11,
14). We have also demonstrated
poor IL-10 production in placental and decidual tissues from preterm labor
deliveries and missed abortions
(15,
16). These data suggest that
an inflammatory environment coupled with genetic stress (IL-10 deficiency) may
lead to adverse pregnancy outcomes. In consideration of these observations, we
hypothesize that exposure to toxicants such as PCBs mimics the physiological
counterpart of inflammation that predisposes to adverse pregnancy outcomes
when combined with genetic deficiency in loci crucial for pregnancy success
such as IL-10.PCBs are chlorinated aromatic hydrocarbon compounds consisting of a group
of 209 structurally diverse congeners, identified based on the position of
chlorine atoms (7). Since the
start of their manufacture in the 1920s until their ban in late 1970s, PCBs
were globally valued for their noninflammability and high heat and chemical
stability and thus were used widely in a multitude of commercial and
industrial applications (7,
17). Improper disposal and
accidental release of these compounds led to their introduction into the
environment, placing them in the list of widespread environmental
contaminants. Subsequently, their lipophilicity facilitated their
bioaccumulation in the food chain and bio-concentration at successively higher
levels (6,
18-21).
PCBs have now been detected globally, in different environmental matrices,
wild life, food, and humans (6,
18,
20). Convincing evidence exist
for their toxicity, both in humans as well as in laboratory animals
(7). From epidemiological
studies in humans it has been observed that exposure to PCBs causes various
reproductive anomalies that include irregular and shorter menstrual cycles,
delayed conception, miscarriage, reduced lactating time, low birth weight,
preterm birth, small for gestational age infants, and higher incidence of
still-births and mortality among children
(22-27).
PCB congeners may work in an aryl hydrocarbon receptor-dependent or
-independent pathway (6,
7,
28). Despite the knowledge
that PCBs affect either aryl hydrocarbon receptor or estrogen receptor
signaling, there is a paucity of molecular mechanisms underlying the most
sensitive developmental effects of PCBs, and thus new pathways and targets
need to be identified.Aroclor 1254 is a mixture of more than one hundred different PCB congeners
and may impart cumulative adverse effects on female reproductive health
(29,
30). In this study, we show
that Aroclor 1254 exposure induces preterm birth in IL-10-/- mice
with reduced litter size and birth weight, increased amniotic fluid, and
postnatal neurocognitive defects. Importantly, we have identified aquaporin 1
(AQP1) as a novel target of PCB action at the maternal-fetal interface. Our
findings for the first time provide direct experimental evidence for a
protective role of IL-10 against PCB exposure. These findings may have
implications for the understanding and management of environmental
toxicant-induced female reproductive anomalies in humans. 相似文献
10.
11.
Metabolism of
d-Glycero-d-Manno-Heptitol,
Volemitol, in Polyanthus. Discovery of a Novel Ketose
Reductase 下载免费PDF全文
Volemitol
(d-glycero-d-manno-heptitol,
α-sedoheptitol) is an unusual seven-carbon sugar alcohol that
fulfills several important physiological functions in certain species
of the genus Primula. Using the horticultural hybrid
polyanthus (Primula × polyantha) as
our model plant, we found that volemitol is the major nonstructural
carbohydrate in leaves of all stages of development, with
concentrations of up to 50 mg/g fresh weight in source leaves (about
25% of the dry weight), followed by sedoheptulose
(d-altro-2-heptulose, 36 mg/g fresh weight),
and sucrose (4 mg/g fresh weight). Volemitol was shown by the
ethylenediaminetetraacetate-exudation technique to be a prominent
phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of
the phloem sap carbohydrates, surpassed only by sucrose (63%).
Preliminary 14CO2 pulse-chase radiolabeling
experiments showed that volemitol was a major photosynthetic product,
preceded by the structurally related ketose sedoheptulose. Finally, we
present evidence for a novel NADPH-dependent ketose reductase,
tentatively called sedoheptulose reductase, in volemitol-containing
Primula species, and propose it as responsible for the
biosynthesis of volemitol in planta. Using enzyme extracts from
polyanthus leaves, we determined that sedoheptulose reductase has a pH
optimum between 7.0 and 8.0, a very high substrate specificity, and
displays saturable concentration dependence for both sedoheptulose
(apparent Km = 21 mm) and NADPH
(apparent Km = 0.4 mm). Our
results suggest that volemitol is important in certain
Primula species as a photosynthetic product, phloem
translocate, and storage carbohydrate.Alditols (sugar alcohols or acyclic polyols) may be chemically
described as reduction products of aldose or ketose sugars. The most
prevalent plant alditols are the hexitols sorbitol, mannitol, and
galactitol. However, as many as 17 different alditols occur naturally
in higher plants (for review, see Bieleski, 1982; Lewis, 1984; Loescher
and Everard, 1996). The lesser-known alditols are often restricted in
their occurrence but still fulfill important functions in those plants
where they do occur. Volemitol (Fig. (Fig.1)
1)
is a good example of a less common but important alditol. This
seven-carbon sugar alcohol seems to be confined to certain sections of
the genus Primula, so much so that it has been suggested as
a useful chemotaxonomical marker (Kremer, 1978). Very little is known
about the physiology and metabolism of volemitol in primulas, except
that it was an early photosynthetic product in cowslip (Primula
veris) and oxslip (Primula elatior) (Kremer, 1978).
Figure 1Fischer projections of volemitol and its four
structurally related seven-carbon sugars. Nomenclature follows that of
Collins (1987); trivial names are underlined.The physiological roles of alditols are manifold and largely resemble
those of disaccharides and oligosaccharides. They include
photosynthetic assimilation, translocation and storage of carbon, and
reducing power, as well as protection against different types of
stresses (for review, see Bieleski, 1982; Lewis, 1984; Loescher and
Everard, 1996; Stoop et al., 1996). The biosynthetic pathways of the
hexitols sorbitol (glucitol), mannitol, galactitol (dulcitol), and the
pentitol ribitol have been established in higher plants. They generally
use NADPH as a hydrogen donor and aldose phosphate as a hydrogen
acceptor, in concert with the corresponding phosphatases. One exception
might be galactitol, which was suggested to be formed directly from
unphosphorylated Gal (and NADPH) (Negm, 1986). Although all foliar
alditols are thought to be phloem-mobile (Lewis, 1984), this has only
been demonstrated for sorbitol, mannitol, and galactitol (Zimmermann
and Ziegler, 1975; Davis and Loescher, 1990; Moing et al., 1992; Flora
and Madore, 1993).To expand our knowledge of alditol metabolism in higher plants beyond
that of hexitols, we studied the carbohydrate metabolism of polyanthus
(Primula × polyantha). This popular
horticultural hybrid of primrose (Primula
vulgaris), oxlip, and cowslip (Mabberley, 1997) was
chosen because preliminary experiments showed that its volemitol
content is very high, similar to that of the wild-type species, and
because it may be easily grown both outdoors and indoors.We give a general overview on volemitol metabolism in polyanthus with
special emphasis on the role of volemitol in plant development and
phloem transport. We also report on a novel enzyme, a NADPH-dependent
ketose reductase, which forms volemitol by the reduction
of sedoheptulose. 相似文献
12.
The effects of oxygen and temperature on the activity and survival of infective forth-stage juveniles of Nothanguina phyllobia Thorne were examined in aqueous suspension. Rate of movement was not affected by a wide range of O₂ concentration (0.8-8.6 ppm). Activity decreased below 0.8 ppm 0 2, and at 0.15 ppm O₂ nematodes became motionless. Activity increased as a linear function of temperature up to a thermal optimum of 24 C; beyond 24 C activity decreased. Survival was greatly prolonged at low temperature. At 23 C, 50% mortality occurred within 7 d, whereas at 4 C, 70% survived after 98 d. 相似文献
13.
Amanda J. MacFarlane Cheryll A. Perry Hussein H. Girnary Dacao Gao Robert H. Allen Sally P. Stabler Barry Shane Patrick J. Stover 《The Journal of biological chemistry》2009,284(3):1533-1539
Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry
and activate single carbons for the de novo synthesis of purines,
thymidylate, and for the remethylation of homocysteine to methionine. C1
tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point
of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS)
activity that catalyzes the ATP-dependent conversion of formate and THF to
10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap
vector into the Mthfd1 gene results in embryonic lethality in mice.
Mthfd1gt/+ mice demonstrated lower hepatic
adenosylmethionine levels, which is consistent with formate serving as a
source of 1Cs for cellular methylation reactions. Surprisingly,
Mthfd1gt/+ mice exhibited decreased levels of
uracil in nuclear DNA, indicating enhanced de novo thymidylate
synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS
compete for a limiting pool of unsubstituted THF. This study demonstrates the
essentiality of the Mthfd1 gene and indicates that formate-derived
1Cs are utilized for de novo purine synthesis and the remethylation
of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity
enhances thymidylate synthesis, affirming the competition between thymidylate
synthesis and homocysteine remethylation for THF cofactors.Folate-mediated one-carbon
(1C)3 metabolism is
compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian
cells (1). In the cytoplasm, 1C
metabolism functions to carry and chemically activate single carbons for the
de novo synthesis of purines, thymidylate, and for the remethylation
of homocysteine to methionine
(2) (see
Fig. 1). Methionine can be
adenosylated to form S-adenosylmethionine (AdoMet), the major
cellular methyl group donor required for the methylation of DNA, RNA,
histones, small molecules, and lipids. Nuclear 1C metabolism functions to
synthesize thymidylate from dUMP and serine during S phase through the small
ubiquitin-like modifier-dependent translocation of cytoplasmic serine
hydroxymethyltransferase (cSHMT), dihydrofolate reductase, and thymidylate
synthase into the nucleus
(3).Open in a separate windowFIGURE 1.Folate-mediated one-carbon metabolism occurs in the mitochondria,
nucleus, and cytoplasm. Mitochondrial-derived formate traverses to the
cytoplasm where it is incorporated into the folate-activated one-carbon pool
through the activity of FTHFS and utilized in the synthesis of purines,
thymidylate, and the methylation of homocysteine to methionine. Methionine can
be converted to a methyl donor through its adenosylation to AdoMet.
Thymidylate biosynthesis occurs in the cytoplasm and nucleus. The one-carbon
unit is labeled in bold. GCS, glycine cleavage system;
mSHMT, mitochondrial serine hydroxymethyltransferase;
mMTHFD, mitochondrial methylenetetrahydrofolate dehydrogenase;
mMTHFC, mitochondrial methenyltetrahydrofolate cyclohydrolase;
mFTHFS, mitochondrial formyltetrahydrofolate synthetase;
MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC,
methenyltetrahydrofolate cyclohydrolase; FTHFS,
formyltetrahydrofolate synthetase; MTHFR, methylenetetrahydrofolate
reductase; TS, thymidylate synthase; DHFR, dihydrofolate
reductase; and cSHMT, cytoplasmic serine
hydroxymethyltransferase.Serine, through its conversion to glycine by SHMT, is a primary source of
1Cs for nucleotide and methionine synthesis
(4). SHMT generates 1Cs in the
cytoplasm, mitochondria, and nucleus, although the generation of 1Cs through
SHMT activity in the cytoplasm is not essential in mice, indicating the
essentiality of mitochondria-derived 1Cs for cytoplasmic 1C metabolism
(5). In mitochondria, the
hydroxymethyl group of serine and the C2 carbon of glycine are transferred to
tetrahydrofolate (THF) to generate 5,10-methylene-THF by the mitochondrial
isozyme of SHMT and the glycine cleavage system, respectively
(6). The 1C carried by
methylene-THF is oxidized and hydrolyzed to generate formate by the
NAD-dependent methylene-THF dehydrogenase (MTHFD) and methenyl-THF
cyclohydrolase (MTHFC) activities encoded by a single gene, Mthfd2
(7), and 10-formyl-THF
synthetase (FTHFS) activity, encoded by Mthfd1L
(8) (see
Fig. 1).In the cytoplasm, the product of the Mthfd1 gene, C1THF synthase,
is a trifunctional enzyme that contains NADP-dependent MTHFD and MTHFC
activities on the N-terminal domain of the protein, and FTHFS activity on the
C-terminal domain (9). These
three activities collectively catalyze the interconversion of THF,
10-formyl-THF, 5,10-methenyl-THF, and 5,10-methylene-THF
(10)
(Fig. 1). The ATP-dependent
FTHFS activity of C1THF synthase condenses mitochondria-derived formate with
THF to form 10-formyl-THF, which is required for the de novo
synthesis of purines (9). The
MTHFC and MTHFD activities convert 10-formyl-THF to methylene-THF
(11). Methylene-THF is
utilized in the de novo synthesis of thymidylate or, alternatively,
can be irreversibly reduced by methylene-THF reductase to 5-methyl-THF, which
is used in the remethylation of homocysteine to methionine
(12).Impairments in 1C metabolism, due to insufficient folate cofactors and/or
single nucleotide polymorphisms in genes that encode folate-dependent enzymes,
are associated with numerous pathologies and developmental anomalies,
including cancers, cardiovascular disease, and neural tube defects. The causal
mechanisms underlying the folate-pathology relationship(s) remains to be
established. However, a number of hypotheses have been proposed related to the
role of 1C metabolism in genome stability and gene expression. Decreased
thymidylate synthesis results in increased uracil misincorporation into DNA
and decreased rates of cell division, causing double strand breaks in DNA and
genomic instability (13).
Decreased AdoMet synthesis alters methylation patterns in CpG islands in DNA
and can result in histone hypomethylation, which can alter gene expression
(2). Proliferating cells also
require the de novo synthesis of purines to maintain rates of DNA
synthesis (14).It has been shown that the gene product of Mthfd2, mitochondrial
MTHFC/MTHFD is essential in mice, and Mthfd2 deficiency results in
embryonic lethality (15). This
protein is required for the generation of formate from serine in the
mitochondria of embryonic cells. Here, we have investigated the essentiality
of the Mthfd1 gene in mice and the effect of altered Mthfd1
gene expression on biomarkers of cytoplasmic 1C metabolism. Our data
demonstrate that Mthfd1 is an essential gene in mice and that
Mthfd1-deficient mice are a model for the study of folate-associated
pathologies. 相似文献
14.
15.
Meloidogyne incognita, M. arenaria, M. hapla, and M. javanica were distinguishable from each other by isoelectric focusing (IEF) of nematode egg proteins. Proteins extracted from larvae and adults of Hoplolaimus columbus and from eggs of Heterodera glycines had distinctive profiles, also. Protein profiles from eggs, preparasitic larvae and egg-laying adults of M. incognita showed differences. It was necessary to compare samples run at the same time to ensure reliability. 相似文献
16.
Luca Giliberto Roberta Borghi Alessandra Piccini Rosa Mangerini Sandro Sorbi Gabriella Cirmena Anna Garuti Bernardino Ghetti Fabrizio Tagliavini Mohamed R. Mughal Mark P. Mattson Xiongwei Zhu Xinglong Wang Michela Guglielmotto Elena Tamagno Massimo Tabaton 《The Journal of biological chemistry》2009,284(14):9027-9038
17.
18.
Despite the ever-increasing number of dementia patients worldwide, fundamental therapeutic approaches to treat this disease remain to be established. Preventive approaches such as diet, exercise and learning attract attention. Several epidemiological studies suggest that ingestion of fermented dairy products prevents cognitive decline in the elderly. These reports indicate that specific ingredients in the fermented dairy products elicit an anti-inflammatory or anti-oxidative activity that facilitates neuroprotection. The responsible components remain to be investigated. A number of studies have shown that inflammation caused by microglia is closely related to exaggeration of the pathology and cognitive decline seen in the elderly. Many researchers have proposed that controlling microglial activities could be effective in preventing and possibly curing dementia. In the present study, to elucidate specific compounds that regulate microglial activity from dairy products, repeated purification by HPLC, combined with evaluation using primary microglia, facilitated the identification of dehydroergosterol (DHE) as a novel component of the extract that enhances microglial anti-inflammatory activity. DHE contains three conjugated double bonds in a steroid ring system and is an analogue of ergosterol. Despite their related chemical structures, the anti-inflammatory activity of DHE is markedly stronger than that of ergosterol. P. candidum for camembert cheese produces DHE, but P. Roqueforti for blue cheese and Aspergillus do not. DHE also induces CD11b-positive microglia cells into CD206-positive M2 type microglia. Neurotoxicity and neuronal cell death induced by excessively activated microglia is suppressed by treatment with DHE. Thus, this is the first report to demonstrate that DHE, identified as a responsible compound in dairy products, can induce microglia into a preferable phenotype for our brain environment and can be safely introduced into the body by consumption of dairy products. We believe the uptake of DHE might help to prevent the onset of dementia. 相似文献
19.
Higher Activity of an Aldehyde Oxidase in the
Auxin-Overproducing superroot1 Mutant of
Arabidopsis thaliana 总被引:1,自引:0,他引:1 下载免费PDF全文
Mitsunori Seo Shuichi Akaba Takayuki Oritani Marianne Delarue Catherine Bellini Michel Caboche Tomokazu Koshiba 《Plant physiology》1998,116(2):687-693
Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1–3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings. 相似文献
20.
Zhonglin Xie Yunzhou Dong Junhua Zhang Roland Scholz Dietbert Neumann Ming-Hui Zou 《Molecular and cellular biology》2009,29(13):3582-3596
LKB1, a master kinase that controls at least 13 downstream protein kinases including the AMP-activated protein kinase (AMPK), resides mainly in the nucleus. A key step in LKB1 activation is its export from the nucleus to the cytoplasm. Here, we identified S307 of LKB1 as a putative novel phosphorylation site which is essential for its nucleocytoplasmic transport. In a cell-free system, recombinant PKC-ζ phosphorylates LKB1 at S307. AMPK-activating agents stimulate PKC-ζ activity and LKB1 phosphorylation at S307 in endothelial cells, hepatocytes, skeletal muscle cells, and vascular smooth muscle cells. Like the kinase-dead LKB1 D194A mutant (mutation of Asp194 to Ala), the constitutively nucleus-localized LKB1 SL26 mutant and the LKB1 S307A mutant (Ser307 to Ala) exhibit a decreased association with STRADα. Interestingly, the PKC-ζ consensus sequence surrounding LKB1 S307 is disrupted in the LKB1 SL26 mutant, thus providing a likely molecular explanation for this mutation causing LKB1 dysfunction. In addition, LKB1 nucleocytoplasmic transport and AMPK activation in response to peroxynitrite are markedly reduced by pharmacological inhibition of CRM1, which normally facilitates nuclear export of LKB1-STRAD complexes. In comparison to the LKB1 wild type, the S307A mutant complexes show reduced association with CRM1. Finally, adenoviral overexpression of wild-type LKB1 suppresses, while the LKB1 S307A mutant increases, tube formation and hydrogen peroxide-enhanced apoptosis in cultured endothelial cells. Taken together, our results suggest that, in multiple cell types the signaling pathways engaged by several physiological stimuli converge upon PKC-ζ-dependent LKB1 phosphorylation at S307, which directs the nucleocytoplasmic transport of LKB1 and consequent AMPK activation.LKB1 is a tumor suppressor (3, 25, 33, 42, 59) that is mutated in Peutz-Jeghers cancer syndrome (20, 24). This serine/threonine protein kinase phosphorylates and activates at least 13 downstream kinases, which in turn regulate multiple cellular processes, including the cell cycle, cellular proliferation, apoptosis, and energy metabolism (1, 30). One of the key downstream kinases of LKB1 is the 5′-AMP-activated protein kinase (AMPK), a serine/threonine kinase that serves as a master regulator of energy metabolism (18, 19, 28). LKB1 is ubiquitously expressed in adult and fetal tissue, particularly pancreatic, liver, testicular, cardiac, and skeletal muscle tissue (21, 25, 43, 60). In humans, LKB1 comprises 433 amino acids (436 residues in mouse LKB1) and is located predominantly in the nucleus due to its nuclear localization signal in the N-terminal noncatalytic region (residues 38 to 43) (36, 53). Paradoxically, LKB1 activation takes place predominantly in the cytoplasm, after it complexes with STRAD (STE-related adapter) and MO25 (mouse protein 25). As a result, the nucleocytoplasmic transport and subsequent association of LKB1 with STRAD and MO25 in the cytoplasm are required for full activation of LKB1 (2, 5) and its downstream kinases, including AMPK. Consistent with this theory, 12 mutants of LKB1 (including the SL26 mutants) found in patients with Peutz-Jeghers cancer syndrome are constitutively nuclear (5, 6). Further, a recent study from Macara''s group (13) shows that STRAD regulates LKB1 localization by blocking access to importin and by association with CRM1 and exportin-7, two nuclear protein exportins.LKB1 is phosphorylated at S325, T366, and S431 by upstream kinases. In addition, LKB1 autophosphorylates at S31, T185, T189, T336, and S404 (1). Mutation of any of these phosphorylation sites to Ala (to abolish phosphorylation) or Glu (to mimic phosphorylation) does not significantly affect the in vitro catalytic activity of LKB1 or its intracellular localization (5, 44, 45). Recently, we demonstrated that phosphorylation of LKB1 S428 is required for metformin-enhanced AMPK activation (56). Nevertheless, several questions such as the precise mechanism(s) underlying LKB1 activation, the relevant phosphorylation sites, and the upstream activating kinase(s) remain unclear. While it has been shown that LKB1 S428 phosphorylation is required for nucleocytoplasmic transport of LKB1, the translocation of LKB1 to the cytosol could be further regulated by unknown mechanisms. Here, we have identified S307 as a novel phosphorylation site in LKB1 and provide evidence that, in multiple cell types, phosphorylation of this site by protein kinase C ζ (PKC-ζ) induces nucleocytoplasmic transport of LKB1 and subsequent activation of AMPK and suppression of angiogenesis and apoptosis. Importantly, we provide a molecular explanation for the constitutive nuclear localization of the LKB1 SL26 mutant. Taken together, our results suggest that the phosphorylation of LKB1 S307 by PKC-ζ is essential for LKB1 regulation of cell cycle progression, proliferation, angiogenesis, and apoptosis. 相似文献