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Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal
: A Route to
4S-Hydroperoxy-2E-Nonenal and Related
Products 下载免费PDF全文
In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O2 uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer. 相似文献
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Marie Blanke Andrawes Xiang Xu Hong Liu Scott B. Ficarro Jarrod A. Marto Jon C. Aster Stephen C. Blacklow 《The Journal of biological chemistry》2013,288(35):25477-25489
Notch signaling makes critical contributions to cell fate determination in all metazoan organisms, yet remarkably little is known about the binding affinity of the four mammalian Notch receptors for their three Delta-like and two Jagged family ligands. Here, we utilized signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Systematic deletion mutagenesis of the human Notch1 ectodomain revealed that epidermal growth factor (EGF) repeats 6–15 are sufficient to maintain signaling in a reporter assay at levels comparable with the full-length receptor, and identified important contributions from EGF repeats 8–10 in conveying an activating signal in response to either Dll1 or Dll4. Truncation studies of the Dll1 and Dll4 ectodomains showed that the MNNL-EGF3 region was both necessary and sufficient for full activation. Plate-based and cell binding assays revealed a specific, calcium-dependent interaction between cell-surface and recombinant Notch receptors and ligand molecules. Finally, direct measurement of the binding affinity of Notch1 EGF repeats 6–15 for Dll1 and Dll4 revealed that Dll4 binds with at least an order of magnitude higher affinity than Dll1. Together, these studies give new insights into the features of ligand recognition by Notch1, and highlight how intrinsic differences in the biochemical behavior of receptor-ligand complexes can influence receptor-mediated responses of developmental signaling pathways. 相似文献
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Differential Expression and Internal Feedback Regulation
of
1-Aminocyclopropane-1-Carboxylate Synthase,
1-Aminocyclopropane-1-Carboxylate Oxidase, and
Ethylene Receptor
Genes in Tomato Fruit during Development and Ripening 总被引:22,自引:1,他引:21 下载免费PDF全文
Akira Nakatsuka Shiho Murachi Hironori Okunishi Shinjiro Shiomi Ryohei Nakano Yasutaka Kubo Akitsugu Inaba 《Plant physiology》1998,118(4):1295-1305
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Proper expression of the replication licensing factor Cdt1 is primarily
regulated post-translationally by ubiquitylation and proteasome degradation.
In a screen to identify novel non-histone targets of histone deacetylases
(HDACs), we found Cdt1 as a binding partner for HDAC11. Cdt1 associates
specifically and directly with HDAC11. We show that Cdt1 undergoes acetylation
and is reversibly deacetylated by HDAC11. In vitro, Cdt1 can be
acetylated at its N terminus by the lysine acetyltransferases KAT2B and KAT3B.
Acetylation protects Cdt1 from ubiquitylation and subsequent proteasomal
degradation. These results extend the list of non-histone acetylated proteins
to include a critical DNA replication factor and provide an additional level
of complexity to the regulation of Cdt1.To maintain genomic integrity, DNA replication must be tightly controlled
to ensure that each portion of the genome replicates once and only once per
cell cycle (reviewed in Ref.
1). Replication licensing
begins by the formation of the prereplication complex at multiple potential
origins of replication. This is established sequentially, with the origin
recognition complex
(ORC)2 proteins
binding first, followed by the recruitment of Cdc6 and Cdt1, which in turn
recruit the MCM2–7 proteins. MCM proteins act as the replicative
helicase. The licensed replication origins are activated by cyclin-dependent
kinases at the start of S phase. Licensing occurs throughout the cell cycle
once S phase is complete.Cdt1 levels fluctuate throughout the cell cycle. It is destabilized at
G1/S transition, and then levels begin to climb again upon S phase
completion. To prevent licensing at inappropriate times, two separate
processes regulate the inactivation or destruction of Cdt1. First, geminin
negatively regulates Cdt1 function by prevention of the association of Cdt1
with MCM2–7 via steric hindrance
(2). Interestingly, geminin
also positively regulates Cdt1 by preventing its ubiquitylation, perhaps by
prevention of its interaction with an E3 ligase. This allows Cdt1 to
accumulate in G2 and M phases, to ensure adequate pools of Cdt1 to
license the next cycle of replication
(3). The ratio of geminin to
Cdt1 likely determines whether geminin positively or negatively regulates Cdt1
(4). Second, Cdt1 is targeted
for proteolysis by two distinct ubiquitin E3 ligases: the SCF-Skp2 complex and
the DDB1-Cul4 complex (5).
Phosphorylation by cyclin A/Cdk2 promotes interaction of Cdt1 with Skp2,
leading to Cdt1 degradation during S phase
(6–8).
In addition, DDB1-Cul4 utilizes proliferating cell nuclear antigen as a
binding platform to contact Cdt1, targeting the destruction of Cdt1 in S phase
or following DNA damage (9,
10). Ubiquitylation by either
of these E3 ligases promotes degradation of Cdt1 by the proteasome.Ubiquitylation occurs primarily (but not exclusively) on the ε-amino
group of lysine residues. Another prominent post-translational modification
that occurs on that residue is acetylation. Acetylation and, correspondingly,
deacetylation can modulate the function and activity of a variety of proteins
(see Ref. 11 for review).
Here, we report that Cdt1 physically interacts with HDAC11, a class IV histone
deacetylase (12,
13), as well as with several
lysine acetyltransferases (KATs). We show that Cdt1 is an acetylated protein
and further show that acetylation protects Cdt1 from ubiquitylation and
subsequent proteasomal degradation. This study uncovers yet another layer of
complexity to the regulation of the critical licensing factor Cdt1. 相似文献
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Zemfira Karamysheva Laura A. Diaz-Martinez Sara E. Crow Bing Li Hongtao Yu 《The Journal of biological chemistry》2009,284(3):1772-1780
Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early
mitosis and, thus, prevents premature sister-chromatid separation. The protein
level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is
down-regulated in G1/S. Here we show that Sgo1 is degraded during
the exit from mitosis, and its degradation depends on the anaphase-promoting
complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels
of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C
bound to Cdh1 (APC/CCdh1) in vitro. We have further
identified two functional degradation motifs in Sgo1; that is, a KEN
(Lys-Glu-Asn) box and a destruction box (D box). Although removal of either
motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box
deleted is stable in cells. Surprisingly, mitosis progresses normally in the
presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not
required for sister-chromatid separation or mitotic exit. Finally, we show
that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1
steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in
mitosis and occurs in two steps in vertebrate cells
(1-3).
In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and
removed from chromosome arms
(1,
4). Then, cleavage of
centromeric cohesin by separase takes place at the metaphase-to-anaphase
transition to allow sister-chromatid separation
(5). The shugoshin (Sgo) family
of proteins plays an important role in the protection of centromeric cohesion
(6,
7). Human cells depleted of
Sgo1 by RNAi undergo massive chromosome missegregation
(8-11).
In cells with compromised Sgo1 function, centromeric cohesin is improperly
phosphorylated and removed (4,
11), resulting in premature
sister-chromatid separation. It has been shown recently that Sgo1 collaborates
with PP2A to counteract the action of Plk1 and other mitotic kinases and to
protect centromeric cohesin from premature removal
(12-14).
In addition, Sgo1 has also been shown to promote stable
kinetochore-microtubule attachment and sense tension across sister
kinetochores (8,
15). Thus, Sgo1 is crucial for
mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting
complex/cyclosome
(APC/C),2 a large
multiprotein ubiquitin ligase that targets key mitotic regulators for
destruction by the proteasome
(16). APC/C selects substrates
for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize
specific sequences called APC/C degrons within target proteins
(17). Several APC/C degrons
have been characterized, including the destruction box (D box) and the
Lys-Glu-Asn box (KEN box) (18,
19). The D box, with the
consensus amino acid sequence of RXXLXXXN(X
indicates any amino acid), are found in many APC/C substrates, including
mitotic cyclins and are essential for their ubiquitin-mediated destruction.
The KEN box, which contains a consensus KEN motif, is also found in several
APC/C substrates and is preferentially but not exclusively recognized by
APC/CCdh1. When APC/C is active, it directs progression through and
exit from mitosis by catalyzing the ubiquitination and timely destruction of
mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor
securin (16). The APC/C
activity needs to be tightly controlled to prevent unscheduled substrate
degradation. An important mechanism for APC/C regulation is the spindle
checkpoint, which prevents the activation of APC/C and destruction of its
substrates in response to kinetochores that have not properly attached to the
mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein
levels oscillate during the cell cycle
(8,
9). In this article we study
the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during
mitotic exit, and this degradation depends on APC/CCdh1. We further
show that both KEN and D boxes are required for Sgo1 degradation in
vivo and ubiquitination in vitro. Removal of these motifs
stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1
protein in human cells does not change the kinetics of chromosome segregation
and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes
place but is not required for mitotic exit. Finally, we show that Bub1
regulates Sgo1 protein levels through a mechanism that does not involve
APC/C-mediated degradation. 相似文献
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Regulation and Functional Expression of
Cinnamate
4-Hydroxylase from Parsley 总被引:2,自引:0,他引:2 下载免费PDF全文
A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit. 相似文献
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Shunichi Nakayama Keiji Kiyoshi Toshimori Kadokura Atsumi Nakazato 《Applied and environmental microbiology》2011,77(18):6470-6475
We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system. 相似文献
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Roberto Zenteno-Cuevas Francisco X Silva-Hernández Fabiola Mendoza-Damián Maria Dolores Ramírez-Hernández Karen Vázquez-Medina Lorena Widrobo-García Aremy Cuellar-Sanchez Raquel Mu?íz-Salazar Leonor Enciso-Moreno Lucia Monserrat Pérez-Navarro José Antonio Enciso-Moreno 《Memórias do Instituto Oswaldo Cruz》2013,108(6):718-723
Tuberculosis (TB) is an infectocontagious respiratory disease caused by members
of the Mycobacterium tuberculosis complex. A 7 base pair (bp)
deletion in the locus polyketide synthase
(pks)15/1 is described as polymorphic among members of the
M. tuberculosis complex, enabling the identification of
Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to
characterise this locus in TB isolates from Mexico. One hundred
twenty clinical isolates were recovered from the states of Veracruz and Estado
de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the
locus pks15/1, while genotypic characterisation was
performed by spoligotyping. One hundred and fifty isolates contained the 7 bp
deletion, while five had the wild type locus. Lineages X (22%),
LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates
were identified as Beijing and two (1%) EAI-Manila. The wild type
pks15/1 locus was observed in all Asian lineage isolates
tested. Our results confirm the utility of locus pks15/1 as a
molecular marker for identifying Asian lineages of the M.
tuberculosis complex. This marker could be of great value in the
epidemiological surveillance of TB, especially in countries like Mexico, where
the prevalence of such lineages is unknown. 相似文献
20.
Oxygen Requirement and Inhibition of C4
Photosynthesis
: An Analysis of C4 Plants Deficient in the
C3 and C4 Cycles 总被引:2,自引:0,他引:2 下载免费PDF全文
Jo?o P. Maroco Maurice S.B. Ku Peter J. Lea Louisa V. Dever Richard C. Leegood Robert T. Furbank Gerald E. Edwards 《Plant physiology》1998,116(2):823-832
The basis for O2 sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3 plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency. 相似文献