ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals.
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Among higher plants graminaceous species have the unique ability to efficiently acquire iron from alkaline soils with low iron solubility by secreting phytosiderophores, which are hexadentate metal chelators with high affinity for Fe(III). Iron(III)-phytosiderophores are subsequently taken up by roots via YS1 transporters, that belong to the OPT oligopeptide transporter family. Despite its physiological importance at alkaline pH, uptake of Fe-phytosiderophores into roots of wild-type maize plants was greater at acidic pH and sensitive to the proton uncoupler CCCP. To access the mechanism of Fe-phytosiderophore acquisition, ZmYS1 was expressed in an iron uptake-defective yeast mutant and in Xenopus oocytes, where ZmYS1-dependent Fe-phytosiderophore transport was stimulated at acidic pH and sensitive to CCCP. Electrophysiological analysis in oocytes demonstrated that Fephytosiderophore transport depends on proton cotransport and on the membrane potential, which allows ZmYS1-mediated transport even at alkaline pH. We further investigated substrate specificity and observed that ZmYS1 complemented the growth defect of the zinc uptake-defective yeast mutant zap1 and transported various phytosiderophore-bound metals into oocytes, including zinc, copper, nickel, and, at a lower rate, also manganese and cadmium. Unexpectedly, ZmYS1 also transported Ni(II), Fe(II), and Fe(III) complexes with nicotianamine, a structural analog of phytosiderophores, which has been shown to act as an intracellular metal chelator in all higher plants. Our results show that ZmYS1 encodes a proton-coupled broad-range metal-phytosiderophore transporter that additionally transports Fe- and Ni-nicotianamine. These biochemical properties indicate a novel role of YS1 transporters for heavy metal homeostasis in plants. (+info)
The present studies were undertaken to investigate the potential effect of a calcium channel blocker (CCB) to enhance the inhibitory effect of an angiotensin (Ang) II type 1 (AT1) receptor blocker (ARB) on vascular injury and the cellular mechanism of the effect of CCB on vascular remodeling. In polyethylene cuff-induced vascular injury of the mouse femoral artery, proliferation of vascular smooth muscle cells (VSMCs) and neointimal formation associated with activation of extracellular signal-regulated kinase (ERK), and tyrosine-phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3, inflammatory response assessed by monocyte chemoattractant protein-1 and tumor necrosis factor-alpha expression, as well as oxidative stress such as expression of NADH/NADPH oxidase p22(phox) subunit and superoxide production, were less in AT1a receptor null mice. Administration of nonhypotensive doses of a CCB, azelnidipine (0.5 or 1 mg/kg per day) attenuated these parameters in wild-type and AT1a receptor null mice. Coadministration of lower doses of an ARB, olmesartan (0.5 mg/kg per day), and azelnidipine (0.1 mg/kg per day), which did not affect vascular remodeling, significantly inhibited these parameters in wild-type mice. Moreover, the effective dose of azelnidipine (1 mg/kg per day) exaggerated the inhibitory action of olmesartan at effective doses of 1 or 3 mg/kg per day on VSMC proliferation in the injured arteries. These results suggest that azelnidipine could inhibit vascular injury at least partly independent of the inhibition of AT1 receptor activation and that azelnidipine could exaggerate the vascular protective effects of olmesartan, suggesting clinical possibility that the combination of CCB and ARB could be more effective in the treatment of vascular diseases. (+info)
Synthesis of the proline analogue [2,3-3H]azetidine-2-carboxylic acid. Uptake and incorporation in Arabidopsis thaliana and Escherichia coli.
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Azetidine-2-carboxylic acid, the 4-membered ring noranalogue of proline, is regularly used in the study of proline metabolism as well as the study of protein conformation. We prepared D,L-[2,3-3H]azetidine-2-carboxylic acid with an optimized 10% yield from commercially available 4-amino-[2,3-3H]butyric acid. Purification was performed by fast-protein liquid chromatography. The biological activity was checked in both Arabidopsis thaliana and Escherichia coli. The obtained specific activity of 10 mCi/mmol was sufficient for most uptake and incorporation studies. (+info)
Nck-dependent activation of extracellular signal-regulated kinase-1 and regulation of cell survival during endoplasmic reticulum stress.
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In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1alpha-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival. (+info)
Role of the yeast acetyltransferase Mpr1 in oxidative stress: regulation of oxygen reactive species caused by a toxic proline catabolism intermediate.
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The MPR1 gene, which is found in the Sigma1278b strain but is not present in the sequenced laboratory strain S288C, of the budding yeast Saccharomyces cerevisiae encodes a previously uncharacterized N-acetyltransferase that detoxifies the proline analogue azetidine-2-carboxylate (AZC). However, it is unlikely that AZC is a natural substrate of Mpr1 because AZC is found only in some plant species. In our search for the physiological function of Mpr1, we found that mpr1-disrupted cells were hypersensitive to oxidative stresses and contained increased levels of reactive oxygen species (ROS). In contrast, overexpression of MPR1 leads to an increase in cell viability and a decrease in ROS level after oxidative treatments. These results indicate that Mpr1 can reduce intracellular oxidation levels. Because put2-disrupted yeast cells lacking Delta(1)-pyrroline-5-carboxylate (P5C) dehydrogenase have increased ROS, we examined the role of Mpr1 in put2-disrupted strains. When grown on media containing urea and proline as the nitrogen source, put2-disrupted cells did not grow as well as WT cells and accumulated intracellular levels of P5C that were first detected in yeast cells and ROS. On the other hand, put2-disrupted cells that overexpressed MPR1 had considerably lower ROS levels. In vitro studies with bacterially expressed Mpr1 demonstrated that Mpr1 can acetylate P5C, or, more likely, its equilibrium compound glutamate-gamma-semialdehyde, at neutral pH. These results suggest that the proline catabolism intermediate P5C is toxic to yeast cells because of the formation of ROS, and Mpr1 regulates the ROS level under P5C-induced oxidative stress. (+info)
Many amino acid substitutions in a hypoxia-inducible transcription factor (HIF)-1alpha-like peptide cause only minor changes in its hydroxylation by the HIF prolyl 4-hydroxylases: substitution of 3,4-dehydroproline or azetidine-2-carboxylic acid for the proline leads to a high rate of uncoupled 2-oxoglutarate decarboxylation.
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Three human prolyl 4-hydroxylases (P4Hs) regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylating a Leu-Xaa-Xaa-Leu-Ala-Pro motif. We report here that the two leucines in the Leu-Glu-Met-Leu-Ala-Pro core motif of a 20-residue peptide corresponding to the sequence around Pro(564) in HIF-1alpha can be replaced by many residues with no or only a modest decrease in its substrate properties or in some cases even a slight increase. The glutamate and methionine could be substituted by almost any residue, eight amino acids in the former position and four in the latter being even better for HIF-P4H-3 than the wild-type residues. Alanine was by far the strictest requirement, because no residue could fully substitute for it in the case of HIF-P4H-1, and only serine or isoleucine, valine, and serine did this in the cases of HIF-P4Hs 2 and 3. Peptides with more than one substitution, having the core sequences Trp-Glu-Met-Val-Ala-Pro, Tyr-Glu-Met-Ile-Ala-Pro, Ile-Glu-Met-Ile-Ala-Pro, Trp-Glu-Met-Val-Ser-Pro, and Trp-Glu-Ala-Val-Ser-Pro were in most cases equally as good or almost as good substrates as the wild-type peptide. The acidic residues present in the 20-residue peptide also played a distinct role, but alanine substitution for any six of them, and in some combinations even three of them, had no negative effects. Substitution of the proline by 3,4-dehydroproline or l-azetidine-2-carboxylic acid, but not any other residue, led to a high rate of uncoupled 2-oxoglutarate decarboxylation with no hydroxylation. The data obtained for the three HIF-P4Hs in various experiments were in most cases similar, but in some cases HIF-P4H-3 showed distinctly different properties. (+info)
Effect of azelnidipine on angiotensin II-mediated growth-promoting signaling in vascular smooth muscle cells.
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The detailed mechanism of the effects of extracellular Ca2+ entry blockade on angiotensin II (Ang II) type 1 (AT1) receptor-mediated growth-promoting signals in vascular smooth muscle cells (VSMCs) is not fully understood. Ang II stimulation caused biphasic activation of growth-promoting signals, reaching a peak at 5 to 10 min followed by a decrease and a second peak at around 2 to 4 h. Addition of PD98059 (2'-amino-3'-methoxyflavone), a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, or AG490 [alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide], a Janus-activated kinase 2 (Jak2) inhibitor, even 4 h after Ang II treatment inhibited [3H]thymidine incorporation. The calcium channel blocker azelnidipine attenuated the later peaks of extracellular signal-regulated kinase (ERK), tyrosine kinase 2, Jak2 activation, and phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3. Interestingly, azelnidipine increased rather than decreased the later ERK peaks in cells treated with small interfering RNA against mitogen-activated protein kinase phosphatase-1. Ang II-mediated [3H]thymidine incorporation was inhibited dose dependently by azelnidipine and also by azelnidipine, plus olmesartan, whereas olmesartan or azelnidipine alone at such lower doses did not affect [3H]thymidine incorporation. These data provide new insight into the manner in which calcium channels exert an essential action in the AT1 receptor-mediated growth-promoting actions in VSMCs. (+info)
Additive beneficial effects of the combination of a calcium channel blocker and an angiotensin blocker on a hypertensive rat-heart failure model.
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The present study was undertaken to examine the effects of a calcium channel blocker, azelnidipine (1 mg/kg/day), an angiotensin converting enzyme (ACE) inhibitor, temocapril (10 mg/kg/day), an angiotensin II type 1 (AT1) receptor blocker (ARB), olmesartan (5 mg/kg/day), and their combination on Dahl salt-sensitive rats (DS rats) developing heart failure with preserved systolic function. DS rats were fed a high-salt diet (8% NaCl) from 7 weeks of age and progressively developed hypertension. Although monotherapy with azelnidipine lowered the blood pressure of DS rats to a greater extent than monotherapy with temocapril or olmesartan, the three drugs had similar effects on cardiac hypertrophy, cardiac fibrosis, the expressions of brain natriuretic peptide, transforming growth factor-beta1, collagen I, collagen III and monocyte chemoattractant protein-1 mRNA (as estimated by Northern blot analysis), and cardiac diastolic dysfunction (as estimated by echocardiography). These results show that ACE and AT1 receptor, as well as hypertension, are involved in the development of heart failure with preserved systolic function in DS rats. The combination of azelnidipine with olmesartan or temocapril produced no additive hypotensive effect in DS rats and no additive effect on cardiac hypertrophy or gene expressions. However, the combination therapy prolonged the survival rate of DS rats more than azelnidipine (p <0.01) or temocapril alone (p <0.05), and this additive beneficial effect by the combination therapy was associated with a greater reduction of cardiac fibrosis, urinary albumin excretion and serum creatinine. Our results thus showed that the combination of a calcium channel blocker with an ARB or an ACE inhibitor had additive preventive effects on a rat model of hypertensive heart failure with preserved systolic function. Thus, combination therapy with these agents seems to be a useful therapeutic strategy for the prevention of hypertensive heart failure. (+info)