| 1. |
- Sengottaiyan, Palanivelu, et al.
(författare)
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Characterization of the biochemical and biophysical properties of the Saccharomyces cerevisiae phosphate transporter Pho89
- 2013
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 436:3, s. 551-556
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na+/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.
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| 2. |
- Lagerstedt, Jens O., 1975, et al.
(författare)
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Mapping the structural transition in an amyloidogenic apolipoprotein A-I
- 2007
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Ingår i: Biochemistry. - 0006-2960. ; 46:34, s. 9693-9
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Tidskriftsartikel (refereegranskat)abstract
- The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIOWA) leads to the formation of beta-secondary structure rich amyloid fibrils in vivo. Here we show that full-length apoA-IIOWA has a decreased lipid-binding capability, an increased amino-terminal sensitivity to protease, and a propensity to form annular protofibrils visible by electron microscopy. The molecular basis for the conversion of apolipoprotein A-I to a proamyloidogenic form was examined by electron paramagnetic resonance spectroscopy. Our recent findings [Lagerstedt, J. O., Budamagunta, M. S., Oda, M. N., and Voss, J. C. (2007) J. Biol. Chem. 282, 9143-9149] indicate that Gly26 in the native apoprotein separates a preceding beta-strand structure (residues 20-25) from a downstream largely alpha-helical region. The current study demonstrates that the G26R variant promotes a structural transition of positions 27-56 to a mixture of coil and beta-strand secondary structure. Microscopy and staining by amyloidophilic dyes suggest that this alteration extends throughout the protein within 1 week of incubation in vitro, leading to insoluble aggregates of distinct morphology. The severe consequences of the Iowa mutation likely arise from the combination of losing the contribution of the native Gly residue in terminating beta-strand propagation and the promotion of beta-structure when an Arg is introduced adjacent to the succeeding residue of identical charge and size, Arg27.
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| 3. |
- Thuswaldner, Sophie, 1976-, et al.
(författare)
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Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis
- 2007
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Ingår i: J Biol Chem. - 0021-9258 .- 1083-351X. ; 282:12, s. 8848-59
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Tidskriftsartikel (refereegranskat)abstract
- In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.
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| 4. |
- Petrlova, Jitka, et al.
(författare)
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Conformational and aggregation properties of the 1-93 fragment of apolipoprotein A-I
- 2014
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 23:11, s. 71-1559
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Tidskriftsartikel (refereegranskat)abstract
- Several disease-linked mutations of apolipoprotein A-I, the major protein in high-density lipoprotein (HDL), are known to be amyloidogenic, and the fibrils often contain N-terminal fragments of the protein. Here, we present a combined computational and experimental study of the fibril-associated disordered 1-93 fragment of this protein, in wild-type and mutated (G26R, S36A, K40L, W50R) forms. In atomic-level Monte Carlo simulations of the free monomer, validated by circular dichroism spectroscopy, we observe changes in the position-dependent β-strand probability induced by mutations. We find that these conformational shifts match well with the effects of these mutations in thioflavin T fluorescence and transmission electron microscopy experiments. Together, our results point to molecular mechanisms that may have a key role in disease-linked aggregation of apolipoprotein A-I.
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| 5. |
- Pratt, J. R., et al.
(författare)
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Effects of methylphosphonate, a phosphate analogue, on the expression and degradation of the high-affinity phosphate transporter Pho84, in Saccharomyces cerevisiae
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:45, s. 14444-53
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, the Pho84 high-affinity transport system is the major phosphate transporter activated when the cells experience a limitation in external phosphate. In this study, we have compared the phosphate-responsive mechanism of cells expressing PHO84 with a Deltapho84 strain by use of a phosphate analogue, methylphosphonate, which was judged to be suitable for assessment of phosphate homeostasis in the cells. Intracellular levels of the analogue, which in several respects mimicks phosphate, were monitored by (31)P NMR spectroscopy. Results show that methylphosphonate is a nonhydrolyzable and nonutilizable analogue that cannot be used to replenish phosphate or polyphosphate in yeast cells grown under conditions of phosphate limitation. However, the presence of methylphosphonate under such conditions represses the Pho5 acidic phosphatase activity of PHO84 cells, a finding that implies a direct role of the analogue in the regulation of phosphate-responsive genes and/or proteins. Likewise, accumulation of the Pho84 protein at the plasma membrane of the same cells is inhibited by methylphosphonate, although the derepressive expression of the PHO84 gene is unperturbed. Thus, a post-transcriptional regulation is suggested. Supportive of this suggestion is the fact that addition of methylphosphonate to cells with abundant and active Pho84 at the plasma membrane causes enhanced internalization of the Pho84 protein. Altogether, these observations suggest that the Pho84 transporter is regulated not only at the transcriptional level but also by a direct molecule-sensing mechanism at the protein level.
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| 6. |
- Lagerstedt, Jens O., 1975, et al.
(författare)
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Structure and function of the GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae
- 2005
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:2, s. 511-7
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Tidskriftsartikel (refereegranskat)abstract
- The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Deltagtr1 similar to the Deltapho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84-green fluorescent protein or Pho84-myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1-185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186-310) displayed no evidence of conformational changes upon GTP addition.
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| 7. |
- Domingo-Espín, Joan, et al.
(författare)
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Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism
- 2018
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Ingår i: Biochimica et Biophysica Acta - Molecular Basis of Disease. - : Elsevier BV. - 0925-4439. ; 1864:9, s. 2822-2834
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Tidskriftsartikel (refereegranskat)abstract
- Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated. ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion. Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.
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| 8. |
- Lagerstedt, Jens O., 1975, et al.
(författare)
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Electron paramagnetic resonance spectroscopy of site-directed spin labels reveals the structural heterogeneity in the N-terminal domain of apoA-I in solution
- 2007
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Ingår i: J Biol Chem. - 0021-9258. ; 282:12, s. 9143-9
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.
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| 9. |
- Lagerstedt, Jens O., 1975, et al.
(författare)
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Structural modeling of dual-affinity purified Pho84 phosphate transporter
- 2004
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Ingår i: FEBS Lett. - : Wiley. - 0014-5793 .- 1873-3468. ; 578:3, s. 262-8
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Tidskriftsartikel (refereegranskat)abstract
- The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.
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| 10. |
- Petrlova, Jitka, et al.
(författare)
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Molecular crowding impacts the structure of apolipoprotein A-I with potential implications on in vivo metabolism and function
- 2016
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Ingår i: Biopolymers. - : Wiley. - 0006-3525. ; 105:10, s. 683-692
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Tidskriftsartikel (refereegranskat)abstract
- The effect molecular crowding, defined as the volume exclusion exerted by one soluble inert molecule upon another soluble molecule, has on the structure and self-interaction of lipid-free apoA-I were explored. The influence of molecular crowding on lipid-free apoA-I oligomerization and internal dynamics has been analyzed using electron paramagnetic resonance (EPR) spectroscopy measurements of nitroxide spin label at selected positions throughout the protein sequence and at varying concentrations of the crowding agent Ficoll-70. The targeted positions include sites previously shown to be sensitive for detecting intermolecular interaction via spin–spin coupling. Circular dichroism was used to study secondary structural changes in lipid-free apoA-I imposed by increasing concentrations of the crowding agent. Crosslinking and SDS-PAGE gel analysis was employed to further characterize the role molecular crowding plays in inducing apoA-I oligomerization. It was concluded that the dynamic apoA-I structure and oligomeric state was altered in the presence of the crowding agent. It was also found that the C-terminal was slightly more sensitive to molecular crowding. Finally, the data described the region around residue 217 in the C-terminal domain of apoA-I as the most sensitive reporter of the crowding-induced self-association of apoA-I. The implications of this behavior to in vivo functionality are discussed.
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