A possible role of rabbit heart cytosol tocopherol binding in the transfer of tocopherol into nuclei.
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ABSTRACT:
SUBMITTER: Guarnieri C
PROVIDER: S-EPMC1162115 | biostudies-other | 1980 Aug
REPOSITORIES: biostudies-other
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Project description:The concept that heat stress (HS) causes a large accumulation of reactive oxygen species (ROS) is widely accepted. However, the intracellular compartmentation of ROS accumulation has been poorly characterized. We therefore used redox-sensitive green fluorescent protein (roGFP2) to provide compartment-specific information on heat-induced redox changes of the nuclei and cytosol of Arabidopsis leaf epidermal and stomatal guard cells. We show that HS causes a large increase in the degree of oxidation of both compartments, causing large shifts in the glutathione redox potentials of the cells. Heat-induced increases in the levels of the marker transcripts, heat shock protein (HSP)101, and ascorbate peroxidase (APX)2 were maximal after 15 min of the onset of the heat treatment. RNAseq analysis of the transcript profiles of the control and heat-treated seedlings revealed large changes in transcripts encoding HSPs, mitochondrial proteins, transcription factors, and other nuclear localized components. We conclude that HS causes extensive oxidation of the nucleus as well as the cytosol. We propose that the heat-induced changes in the nuclear redox state are central to both genetic and epigenetic control of plant responses to HS.
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Project description:Previous work has shown that the α-tocopherol transfer protein (α-TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which α-TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of α-TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of α-TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired α-TTP-assisted secretion of α-tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.
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Project description:Tocopherols (vitamin E) are lipid-soluble antioxidants produced by all plants and algae, and many cyanobacteria, yet their functions in these photosynthetic organisms are still not fully understood. We have previously reported that the vitamin E deficient 2 (vte2) mutant of Arabidopsis thaliana is sensitive to low temperature (LT) due to impaired transfer cell wall (TCW) development and photoassimilate export associated with massive callose deposition in transfer cells of the phloem. To further understand the roles of tocopherols in LT induced TCW development we compared the global transcript profiles of vte2 and wild-type leaves during LT treatment. Tocopherol deficiency had no significant impact on global gene expression in permissive conditions, but significantly affected expression of 77 genes after 48 h of LT treatment. In vte2 relative to wild type, genes associated with solute transport were repressed, while those involved in various pathogen responses and cell wall modifications, including two members of callose synthase gene family, GLUCAN SYNTHASE LIKE 4 (GSL4) and GSL11, were induced. However, introduction of gsl4 or gsl11 mutations individually into the vte2 background did not suppress callose deposition or the overall LT-induced phenotypes of vte2. Intriguingly, introduction of a mutation disrupting GSL5, the major GSL responsible for pathogen-induced callose deposition, into vte2 substantially reduced vascular callose deposition at LT, but again had no effect on the photoassimilate export phenotype of LT-treated vte2. These results suggest that GSL5 plays a major role in TCW callose deposition in LT-treated vte2 but that this GSL5-dependent callose deposition is not the primary cause of the impaired photoassimilate export phenotype.
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Project description:alpha-Tocopherol is a member of the vitamin E family that functions as the principal fat-soluble antioxidant in vertebrates. Body-wide distribution of tocopherol is regulated by the hepatic alpha-tocopherol transfer protein (alphaTTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of alphaTTP is thought to stem from its ability to facilitate the transfer of vitamin E between membranes, but the mechanism by which the protein exerts this activity remains poorly understood. Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to alphaTTP increases with increasing alphaTTP concentration. This concentration dependence indicates that ligand transfer by alphaTTP involves direct protein-membrane interaction. In support of this notion, equilibrium analyses employing filtration, dual polarization interferometry, and tryptophan fluorescence demonstrated the presence of a stable alphaTTP-bilayer complex. The physical association of alphaTTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in alphaTTP that cause the hereditary disorder ataxia with vitamin E deficiency diminish the effect of tocopherol on the protein-membrane association, suggesting a possible mechanism for the accompanying pathology.
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Project description:Chloroquine (CQ) is a widely prescribed anti-malarial agent and is also prescribed to treat autoimmune diseases. Clinical treatment with CQ is often accompanied by serious side effects such as hepatitis and retinopathy. As a weak base, CQ accumulates in intracellular acidic organelles, raises the pH, and induces osmotic swelling and permeabilization of acidic organelles, which account for CQ-induced cytotoxicity. We reported previously that CQ treatment caused α-tocopherol transfer protein (α-TTP), a gene product of familial vitamin E deficiency, to change its location from the cytosol to the surface of acidic organelles. Here we show that α-TTP plays a novel role in protecting against CQ toxicity both in vitro and in vivo. In the presence of CQ, rat hepatoma McARH7777 cells, which do not express α-TTP endogenously, showed more severe cytotoxicity, such as larger vacuolation of acidic organelles and caspase activation, than α-TTP transfectant cells. Similarly, α-TTP knockout mice showed more severe CQ toxicity, such as hepatotoxicity and retinopathy, than wild-type mice. These effects were not ameliorated by vitamin E supplementation. In contrast to bafilomycin A1 treatment, which prevents CQ accumulation in cells by raising the pH of acidic organelles, α-TTP expression prevented CQ accumulation without affecting the pH of acidic organelles. Taken together, our data suggest that α-TTP protects against CQ toxicity by preventing CQ accumulation in acidic organelles through a mechanism distinct from vitamin E transport.
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Project description:In plants, lipid trafficking within and inside the cell is carried out by lipid-binding and transfer proteins. Ligands for these proteins are building and signaling lipid molecules, secondary metabolites with different biological activities due to which they perform diverse functions in plants. Many different classes of such lipid-binding and transfer proteins have been found, but the most common and represented in plants are lipid transfer proteins (LTPs), pathogenesis-related class 10 (PR-10) proteins, acyl-CoA-binding proteins (ACBPs), and puroindolines (PINs). A low degree of amino acid sequence homology but similar spatial structures containing an internal hydrophobic cavity are common features of these classes of proteins. In this review, we summarize the latest known data on the features of these protein classes with particular focus on their ability to bind and transfer lipid ligands. We analyzed the structural features of these proteins, the diversity of their possible ligands, the key amino acids participating in ligand binding, the currently known mechanisms of ligand binding and transferring, as well as prospects for possible application.
| S-EPMC9866449 | biostudies-literature
Project description:α-tocopherol transfer protein (TTP) was previously reported to self-aggregate into 24-meric spheres (α-TTPS) and to possess transcytotic potency across mono-layers of human umbilical vein endothelial cells (HUVECs). In this work, we describe the characterisation of a functional TTP variant with its vitamer selectivity shifted towards γ-tocopherol. The shift was obtained by introducing an alanine to leucine substitution into the substrate-binding pocket at position 156 through site directed mutagenesis. We report here the X-ray crystal structure of the γ-tocopherol specific particle (γ-TTPS) at 2.24 Å resolution. γ-TTPS features full functionality compared to its α-tocopherol specific parent including self-aggregation potency and transcytotic activity in trans-well experiments using primary HUVEC cells. The impact of the A156L mutation on TTP function is quantified in vitro by measuring the affinity towards γ-tocopherol through micro-differential scanning calorimetry and by determining its ligand-transfer activity. Finally, cell culture experiments using adherently grown HUVEC cells indicate that the protomers of γ-TTP, in contrast to α-TTP, do not counteract cytokine-mediated inflammation at a transcriptional level. Our results suggest that the A156L substitution in TTP is fully functional and has the potential to pave the way for further experiments towards the understanding of α-tocopherol homeostasis in humans.
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Project description:The hepatic ?-tocopherol transfer protein (TTP) is required for optimal ?-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP's significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of ?-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early development of the vertebrate central nervous system.
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Project description:alpha-Tocopherol transfer protein (alpha-TTP) supplements nascent very-low-density lipoprotein (VLDL) preferentially with alpha-tocopherol by selecting the alpha-isomers against other stereoisomers of tocopherol. It is exclusively expressed in liver. We investigated whether the expression of the hepatic alpha-TTP can be induced by dietary tocopherols. Vitamin E-depleted rats were fed with a diet containing alpha- and delta-tocopherol (ratio 1:3). The expression of alpha-TTP mRNA was measured in liver tissue. The ratio of tocopherol stereoisomers was determined in plasma, plasma lipoproteins and tissues to measure the metabolic action of alpha-TTP. Refeeding a diet containing either alpha- or delta-tocopherol, or both, caused a steady increase of the expression of alpha-TTP mRNA. In parallel the alpha/delta-tocopherol ratio increased in plasma, VLDL, high-density lipoprotein and low-density lipoprotein as well as in liver tissue, when the diet was fed containing both isomers. The alpha-tocopherol/delta-tocopherol ratio of heart, kidney, lung, lamellar bodies of lung and in lung lavage showed no or a comparatively low increase. The data show that both tocopherol isomers were able to induce alpha-TTP mRNA in rat liver and, thus, the ability of liver to select for the alpha-isomer. On the other hand, tocopherol depletion did not change the expression of hepatic alpha-TTP mRNA in the rat.
| S-EPMC1219391 | biostudies-other
Project description:There are eight naturally occurring forms of the dietary antioxidant vitamin E. Of these, only ?-tocopherol is retained at high levels in vertebrate plasma and tissues. This selectivity is achieved in part by the action of the hepatic ?-tocopherol transfer protein (TTP), which facilitates the selective incorporation of dietary ?-tocopherol into circulating lipoproteins. We examined the effects of vitamin E on TTP expression in cultured hepatocytes. Treatment with vitamin E precipitated a time- and dose-dependent increase in the steady-state levels of TTP. This stabilization was caused by ?-tocopherol-induced attenuation of the ubiquitination of TTP and its subsequent degradation by the proteasome. In vitro, vitamin E protected TTP from proteolytic degradation by trypsin, suggesting ligand-induced changes in protein conformation. Cell fractionation studies showed that TTP is distributed between the cytosolic and membranous organelle fraction, and that tocopherol induced the translocation of some TTP from the cytosol to the organelle fraction. Furthermore, vitamin E markedly attenuated the degradation of organelle-bound TTP. These findings suggest that vitamin E imparts a distinct conformation on TTP that is associated with localization to a specific cellular compartment, where the protein is less susceptible to proteasomal degradation.
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