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Träfflista för sökning "WFRF:(Lagerstedt Jens O.) "

Sökning: WFRF:(Lagerstedt Jens O.)

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  • [1]234Nästa
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1.
  • Sengottaiyan, Palanivelu, et al. (författare)
  • The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae
  • 2012
  • Ingår i: BMC Biochemistry. - : BioMed Central. - 1471-2091 .- 1471-2091. ; 13
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundThe Gtr1 protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. Gtr1 has been implicated in various cellular processes. Particularly, the Switch regions in the GTPase domain of Gtr1 are essential for TORC1 activation and amino acid signaling [R. Gong, L. Li, Y. Liu, P. Wang, H. Yang, L. Wang, J. Cheng, K.L. Guan, Y. Xu, Genes Dev. 25 (2011) 1668–1673]. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation.ResultsBy employing tryptophan fluorescence analysis and radioactive GTPase assays, we demonstrate that Gtr1 can adopt two distinct GDP- and GTP-bound conformations, and that it hydrolyses GTP much slower than Ras proteins. Using cysteine mutagenesis of Arginine-37 and Valine-67, residues at the Switch I and II regions, respectively, we show altered GTPase activity and associated conformational changes as compared to the wild type protein and the cysteine-less mutant.ConclusionsThe extremely low intrinsic GTPase activity of Gtr1 implies requirement for interaction with activating proteins to support its physiological function. These findings as well as the altered properties obtained by mutagenesis in the Switch regions provide insights into the function of Gtr1 and its homologues in yeast and mammals.
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2.
  • Thuswaldner, S., et al. (författare)
  • Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis
  • 2007
  • Ingår i: J Biol Chem. - 0021-9258 .- 1083-351X. ; 282:12, s. 8848-59
  • 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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  • Lagerstedt, Jens O., 1975, et al. (författare)
  • Structural modeling of dual-affinity purified Pho84 phosphate transporter
  • 2004
  • Ingår i: FEBS Lett. - 0014-5793 .- 1873-3468. ; 578:3, s. 262-8
  • 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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5.
  • Petrlova, Jitka, et al. (författare)
  • Conformational and aggregation properties of the 1-93 fragment of apolipoprotein A-I
  • 2014
  • Ingår i: Protein Science. - : The Protein Society. - 1469-896X. ; 23:11, s. 71-1559
  • 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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6.
  • Sengottaiyan, Palanivelu, et al. (författare)
  • Characterization of the biochemical and biophysical properties of the Saccharomyces cerevisiae phosphate transporter Pho89
  • 2013
  • Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 436:3, s. 551-556
  • 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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7.
  • Azhar, Salman, et al. (författare)
  • Novel ABCA1 peptide agonists with antidiabetic action
  • 2019
  • Ingår i: Molecular and Cellular Endocrinology. - : Elsevier. - 0303-7207. ; 480, s. 1-11
  • Tidskriftsartikel (refereegranskat)abstract
    • Previously, apoE-derived ABCA1 agonist peptides have been shown to possess anti-atherosclerotic and possibly antidiabetic properties. Here we assessed the in vitro and in vivo actions of a second generation of ABCA1 peptide agonists, CS6253 and T6991-2, on glucose homeostasis. The results show that these two peptides improve glucose tolerance in a prediabetic diet-induced obesity mouse model by enhancing insulin secretion. It was further demonstrated that T6991-2 also improved glucose tolerance in leptin-deficient (ob/ob) mice. CS6253 increased insulin secretion both under basal conditions and in response to high glucose stimulation in pancreatic INS-1 β-cells rendered leptin receptor deficient with specific siRNA. Additional in vitro cell studies suggest that the CS6253 agonist attenuates hepatic gluconeogenesis and glucose transport. It also potentiates insulin-stimulated glucose uptake and utilization. These observed anti-diabetic actions suggest additional benefits of the CS6253 and T6991-2 ABCA1 peptide agonists for cardiovascular disease beyond their direct anti-atherosclerosis properties previously described.
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