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  • 1.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Meng, Meng
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Gornicka, Agnieszka
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase2012Ingår i: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 79, s. 39-45Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

  • 2. Fa, M.
    et al.
    Bergström, F.
    Hägglöf, P.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, M.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Johansson, L. B.-A.
    Ny, T.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites2000Ingår i: Structure, with Folding & Design, Vol. 8, nr 4, s. 397-405Artikel i tidskrift (Refereegranskat)
  • 3.
    Fa, Ming
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Bergström, Fredrik
    Teknisk-naturvetenskaplig fakultet, Kemi.
    Hägglöf, Peter
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Johansson, Lennart B-Å
    Teknisk-naturvetenskaplig fakultet, Kemi.
    Ny, Tor
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites2000Ingår i: Structure, Vol. 8, nr 4, s. 397-405Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin–protease complex have been proposed. In the absence of a spatial structure of a serpin–protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial.

    Results: We used a recently developed method for making precise distance measurements, based on donor–donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1′) were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into β sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease–inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker.

    Conclusions: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

  • 4.
    Fallah, Mahsa
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Shen, Yue
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brodén, Jessica
    Bäckman, Assar
    Lundskog, Bertil
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Johansson, Mikael
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper.
    Blomqvist, Michael
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper.
    Liu, Kui
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Plasminogen activation is required for the development of radiation-induced dermatitis2018Ingår i: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 9, nr 11, artikel-id 1051Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild type (plg+/+) mice, the plasminogen level increased in the radiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg−/−) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/ mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

     

     

  • 5.
    Fallah, Mahsa
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Viklund, Emil
    Shen, Yue
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bäckman, Assar
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lundskog, Bertil
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Johansson, Mikael
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper.
    Blomqvist, Michael
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper.
    Liu, Kui
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Plasminogen enhances the healing of radiation-induced wounds via decreased expression of pro-inflammatory and pro-fibrotic factorsManuskript (preprint) (Övrigt vetenskapligt)
  • 6. Geisler, Matt
    et al.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Karpinski, Stanislaw
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses.2004Ingår i: Plant Molecular Biology, ISSN 0167-4412, Vol. 56, nr 5, s. 783-94Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

  • 7.
    Hägglöf, Peter
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bergström, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Johansson, Lennart B-Å
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted2004Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, nr 3, s. 823-832Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1′ residues acting as a “bait” for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into β-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor–donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in β-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into β-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

  • 8.
    Kleczkowski, Leszek A
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Decker, Daniel
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    UDP-sugar pyrophosphorylase: a new old mechanism for sugar activation2011Ingår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 156, nr 1, s. 3-10Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recent developments in studies on properties and functions of UDP-sugar pyrophosphorylase (USPase) in metabolism are presented. The protein was characterized from plants and protozoans (Leishmania, Trypanosoma), but apparently it is also present in bacteria. In plants, USPase deficiency leads to male-sterility. USPase produces a variety of UDP-sugars and their analogs required for cell wall biosynthesis as well as for protein and lipid glycosylation, among other functions. Substrate specificity of USPases from different sources is reviewed, and their function/ structure properties are discussed, based on recent crystallization of the protein, with emphasis on common structural blueprint with some other pyrophosphorylases. Some strategies for future research on USPase are discussed.

  • 9.
    Kleczkowski, Leszek A
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Geisler, Matt
    Fitzek, Elisabeth
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.2011Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, nr 3, s. 375-379Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

  • 10.
    Kleczkowski, Leszek A
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Kunz, Sabine
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mechanisms of UDP-glucose synthesis in plants2010Ingår i: Critical reviews in plant sciences, ISSN 0735-2689, E-ISSN 1549-7836, Vol. 29, nr 4, s. 191-203Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Substantial progress has been made in studies on enzymes synthesizing UDP-glucose (UDPG) which is essential for sucrose and cell wall biosynthesis, and in an array of other processes, e.g. glycosylation of proteins and lipids. The enzymes include UDPG pyrophosphorylase, UDP-sugar pyrophosphorylase (USPase) and sucrose synthase (SuSy). Genes coding for those proteins are under complex spatial and temporal regulation, and are frequently coexpressed. Recent evidence for regulation of some of the UDPG-synthesizing proteins by posttranslational modifications and oligomerization, together with discoveries of novel isozymes and unexpected locations within a cell (including chloroplasts and mitochondria) have made the studies exciting, but complex. The enzymes differ in specificity for sugar and nucleotide portions of their substrates/products, and may be involved in distinct metabolic pathways, but also in signaling. Homology models for USPase and SuSy structures are presented, based on recent crystallization of structurally related proteins. Future challenges in research on UDPG-producing enzymes are underlined.

  • 11.
    Kleczkowski, Leszek
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Martz, Francoise
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Factors affecting oligomerization status of UDP-glucose pyrophosphorylase.2005Ingår i: Phytochemistry, ISSN 0031-9422, Vol. 66, nr 24, s. 2815-21Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6–9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.

  • 12. Lobov, S
    et al.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ranson, M
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Conformational rearrangements of plasminogen activator inhibitor type 22009Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 66, nr 10, s. 1782-3; author reply 1784Artikel i tidskrift (Refereegranskat)
  • 13.
    Lobov, Sergei
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Bergström, Fredrik
    Teknisk-naturvetenskaplig fakultet, Kemi.
    Johansson, Lennart B-Å
    Teknisk-naturvetenskaplig fakultet, Kemi.
    Ny, Tor
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Structural Bases of the Redox-dependent Conformational Switch in the Serpin PAI-22004Ingår i: Journal of Molecular Biology, Vol. 344, nr 5, s. 1359-68Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open β-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the β-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54 Å to the bottom of PAI-2 so that the C79–C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.

  • 14.
    Martz, Françoise
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Wilczynska, Malgorzata
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase2002Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 367, nr 1, s. 295-300Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Barley UDP-glucose pyrophosphorylase (UGPase), a key enzyme for the synthesis of sucrose, cellulose and other saccharides, was expressed in Escherichia coli and purified. Using both native electrophoresis and gel filtration, the recombinant and crude leaf UGPase proteins were found to exist as a mixture of monomers, dimers and higher-order polymers. In order to understand the molecular basis for the oligomerization of UGPase, a conserved Cys residue was replaced (C99S mutant) and several amino acids were substituted (LIV to NIN, KK to LL and LLL to NNN) in a conserved hydrophobic domain (amino acids 117-138). The C99S mutant had about half the V (max) of the wild-type and a 12-fold higher K (m) for PP(i), whereas NIN and LL mutations lowered the V (max) by 12- and 2-fold, respectively, with relatively small effects on substrate K (m) values (the NNN mutant was insoluble/inactive). The NIN mutation resulted in a low-activity oligomerized enzyme form, with very little monomer formation. Activity staining on native PAGE gels as well as gel-filtration studies demonstrated that the monomer was the sole enzymically active form. Possible implications of the oligomerization status of UGPase for post-translational regulation of the enzyme are discussed.

  • 15.
    Meng, Meng
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Fitzek, Elisabeth
    Gajowniczek, Agnieszka
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Domain-specific determinants of catalysis/ substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase2009Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1794, s. 1734-1742Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region ("NB loop"). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called "I-loop" region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K(m) with PPi (up to 33mM), whereas Ncut deletions had greatly increased K(m) with UDPG (up to 57mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K(m) values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.

  • 16.
    Meng, Meng
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis2008Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1784, nr 6, s. 967-972Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10–30% lower than that of UGPase-2, depending on direction of the reaction, whereas its Km values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03–0.14 mM vs. 0.07–0.36 mM, respectively). Both proteins were “true” UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.

  • 17.
    Przygodzka, Patrycja
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ramstedt, Björn
    Tengel, Tobias
    Larsson, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment2010Ingår i: BMC Cell Biology, ISSN 1471-2121, E-ISSN 1471-2121, Vol. 11, s. 30-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function.

    Results: We found that the majority of naturally expressed bomapin was located in the nucleus. Both the natural and recombinant bomapin had a disulfide bond which linked the only two bomapin cysteines: one located in the CD-loop and the other near the C-terminus. Computer modelling showed that the cysteines are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of the overall bomapin structure. Low-level ectopic expression of bomapin in bomapin-deficient K562 cells resulted in about 90% increased cell proliferation under normal growth conditions. On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation. Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important. The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation. In contrast to the survival-promoting activity of bomapin in cells cultured under optimal growth conditions, bomapin enhanced cell apoptosis following growth factor withdrawal.

    Conclusions: We propose that bomapin is a redox-sensitive nuclear serpin that augments proliferation or apoptosis of leukaemia cells, depending on growth factors availability.

  • 18.
    Shen, Yue
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Guo, Yongzhi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Du, Chun
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hellström, Sten
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mice deficient in urokinase-type plasminogen activator have delayed healing of tympanic membrane perforations2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 12, s. e51303-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mice deficient in plasminogen, the precursor of plasmin, show completely arrested healing of tympanic membrane (TM) perforations, indicating that plasmin plays an essential role in TM healing. The activation of plasminogen to plasmin is performed by two plasminogen activators (PAs), urokinase-type PA (uPA) and tissue-type PA (tPA). To elucidate the functional roles of PAs in the healing of TM perforations, we investigated the phenotypes of single gene-deficient mice lacking uPA (uPA(-/-)) or tPA (tPA(-/-)) after TM perforation. Delayed healing of TM perforations was observed in uPA(-/-) mice but not tPA(-/-) mice. The migration of keratinocytes was clearly delayed and seemed to be misoriented in uPA(-/-) mice. Furthermore, fibrin deposition and the inflammatory response were persistent in these mice. Our findings demonstrate that uPA plays a role in the healing of TM perforations. The observed phenotypes in uPA(-/-) mice are most likely due to the reduced generation of plasmin.

  • 19.
    Shen, Yue
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Guo, Yongzhi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mikus, Peter
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sulniute, Rima
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Li, Jinan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Plasminogen is a key proinflammatory regulator that accelerates the healing of acute and diabetic wounds2012Ingår i: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 119, nr 24, s. 5879-5887Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Despite decades of research on wound healing, effective biologic agents for the treatment of chronic wounds, especially diabetic wounds, are still lacking. In the present study, we report that the inert plasma protein plasminogen (plg) acts as a key regulatory molecule that potentiates wound healing in mice. Early in the healing process, plg bound to inflammatory cells is transported to the wound area, where the level of plg is increased locally, leading to the induction of cytokines and intracellular signaling events and to a potentiation of the early inflammatory response. Systemic administration of additional plg not only accelerates the healing of acute burn wounds in wild-type mice, but also improves the healing of chronic diabetic wounds in a mouse model of diabetes. Our results suggest that the administration of plg may be a novel therapeutic strategy to treat many different types of wounds, especially chronic wounds such as those caused by diabetes. (Blood. 2012; 119(24):5879-5887)

  • 20.
    Shen, Yue
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Guo, Yongzhi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Li, Jinan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hellström, Sten
    Department of Audiology and Neurotology, Karolinska University Hospital.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Plasminogen initiates and potentiates the healing of acute and chronic tympanic membrane perforations in mice2014Ingår i: Journal of Translational Medicine, ISSN 1479-5876, E-ISSN 1479-5876, Vol. 12, artikel-id 5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Most tympanic membrane (TM) perforations heal spontaneously, but approximately 10-20% remain open as chronic TM perforations. Chronic perforations can lead to an impaired hearing ability and recurrent middle ear infections. Traditionally, these perforations must be surgically closed, which is costly and time consuming. Therefore, there is a need for simpler therapeutic strategies. Previous studies by us have shown that plasminogen (plg) is a potent pro-inflammatory regulator that accelerates cutaneous wound healing in mice. We have also shown that the healing of TM perforations is completely arrested in plg-deficient (plg(-/-)) mice and that these mice develop chronic TM perforations. In the present study, we investigated the therapeutic potential of local plg injection in acute and chronic TM perforation mice models. Methods: Plg(-/-) mice and wild-type mice were subjected to standardized TM perforations followed by local injection of plg into the soft tissue surrounding the TM. TM perforations with chronic characteristics were induced by leaving TM perforations in plg(-/-) mice untreated for 9 days before treatment. The healing process was observed through otomicroscope and finally confirmed by immunostaining. The quality of TM healing was evaluated based on the morphology of the TM. Result: Daily local injections of plg into the soft tissue surrounding the TM restored the ability to heal TM perforations in plg(-/-) mice in a dose-dependent manner, and potentiated the healing rate and quality in wild-type mice. A single local injection of plg initiated the healing of the chronic-like TM perforations in these mice, resulting in a closed TM with a continuous but rather thick outer keratinocyte layer. However, three plg injections led to a completely healed TM with a thin keratinizing squamous epithelium covering a connective tissue layer. Conclusion: Our data suggests that plg is a promising drug candidate for the treatment of chronic TM perforations in humans.

  • 21.
    Sulniute, Rima
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lindh, Tomas
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Protetik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Li, Jinan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Plasmin is essential in preventing periodontitis in mice2011Ingår i: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 179, nr 2, s. 819-828Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Periodontitis involves bacterial infection, inflammation of the periodontium, degradation of gum tissue, and alveolar bone resorption, which eventually leads to loss of teeth. To study the role of the broad-spectrum protease plasmin in periodontitis, we examined the oral health of plasminogen (Plg)-deficient mice. In wild-type mice, the periodontium was unaffected at all time points studied; in Plg-deficient mice, periodontitis progressed rapidly, within 20 weeks. Morphological study results of Plg-deficient mice revealed detachment of gingival tissue, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. IHC staining showed massive infiltration of neutrophils in the periodontal tissues. Interestingly, doubly deficient mice, lacking both tissue- and urokinase-type plasminogen activators, developed periodontal disease similar to that in Pig-deficient mice; however, mice lacking only tissue- or urokinase-type plasminogen activator remained healthy. Supplementation by injection of Pig-deficient mice with human plasminogen for 10 days led to necrotic tissue absorption, inflammation subsidence, and full regeneration of gum tissues. Notably, there was also partial regrowth of degraded alveolar bone. Taken together, our results show that plasminogen is essential for the maintenance of a healthy periodontium and plays an important role in combating the spontaneous development of chronic periodontitis. Moreover, reversal to healthy status after supplementation of Pig-deficient mice with plasminogen suggests the possibility of using plasminogen for therapy of periodontal diseases. (Am J Pathol 2011, 179:819-828; DOI: 10.1016/j.ajpath.2011.05.003)

  • 22.
    Sulniute, Rima
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Shen, Yue
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Guo, Yongzhi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ahlskog, Nina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Li, Jinan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Plasminogen is a critical regulator of cutaneous wound healingManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Wound healing is a well-orchestrated, complex process leading to the repair of injured tissues. Two major proteolytic systems, the matrix metalloproteases and the plasminogen activator system, are involved in this process. The lack of plasminogen (plg) has previously been reported to cause a delay in wound closure in mice, and to be complemented by matrix metalloproteases. However, our previous finding that tympanic membrane perforations in plgdeficient mice do not heal indicated that plg has more important function in wound healing than previously regarded. In later studies, we have found that plg accumulates in the wound early during the healing process and potentiates the inflammatory response and the healing. In the present study, we have used incision and burn wound models in wild-type and plgdeficient mice to further investigate the role of plg in the later phases of the healing process, including its role after re-epithelization. In addition to the earlier observed delay of wound reepithelizationin plg-deficient mice, we have found that the tissue remodeling processes that take place after re-epithelization is also impaired in these mice. By morphological and immunohistochemical analyses, we found that plg-deficient mice had delayed granulationtissue formation, and were unable to clear the provisional matrix. Extensive fibrin deposition and persistent neutrophil infiltration even at day 60 post-wounding indicate that the inflammation was present subcutaneously in plg-deficient mice even at later time points. Importantly, intravenous or subcutaneous supplementation of plg-deficient mice by human plg led to a restored healing rate, and a healing pattern that was comparable to that in wildtype mice. Therefore, in addition to its important function in early stages of cutaneous wound healing, plg is also crucial for later phases, by clearing fibrin deposits and resolving inflammation after full re-epithelization of the wound. Our results suggest that plg may be a potential therapeutic agent for improving the healing of different types of skin wounds.

  • 23.
    Sulniute, Rima
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Shen, Yue
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Guo, Yong-Zhi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Fallah, Mahsa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ahlskog, Nina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Lina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rakhimova, Olena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brodén, Jessica
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Boija, Hege
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Moghaddam, Aliyeh
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Li, Jinan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Plasminogen is a critical regulator of cutaneous wound healing2016Ingår i: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 115, nr 5, s. 1001-1009Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Wound healing is a complicated biological process that consist of partially overlapping inflammatory, proliferation and tissue remodelling phases. A successful wound healing depends on a proper activation and subsequent termination of the inflammatory phase. The failure to terminate the inflammation halts the completion of wound healing and is a known reason for formation of chronic wounds. Previous studies have shown that wound closure is delayed in plasminogen deficient mice, and a role for plasminogen in dissection of extracellular matrix was suggested. However, our finding that plasminogen is transported to the wound by inflammatory cells early during the healing process, where it potentiates inflammation, indicates that plasminogen may also have other roles in the wound healing process. Here we report that plasminogen-deficient mice have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialisation, indicating inefficient debridement and chronic inflammation. Delayed formation of granulation tissue suggests that fibroblast function is impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for subsequent steps, including resolution of inflammation and activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing process that is comparable to that in wild-type mice. Besides of being an activator of the inflammatory phase during wound healing, plasminogen is also required for the subsequent termination of inflammation. Based on these results, we propose that plasminogen may be an important future therapeutic agent for wound treatment.

  • 24. Wielkoszynski, Tomasz
    et al.
    Moghaddam, Aliyeh
    Backman, Assar
    Broden, Jessica
    Piotrowski, Rafal
    Mond-Paszek, Renata
    Kozarenko, Alexander
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Omnio AB, Umeå, Sweden.
    Novel diagnostic ELISA test for discrimination between infections with Yersinia enterocolitica and Yersinia pseudotuberculosis2018Ingår i: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 37, nr 12, s. 2301-2306Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Yersiniosis is a foodborne infection caused by Yersinia enterocolitica or Yersinia pseudotuberculosis. Although yersiniosis is most often self-limiting, some patients develop chronic infections, such as reactive arthritis, glomerulonephritis, or myocarditis, which require an antibiotic treatment. Whereas early infections can be diagnosed by direct detection of bacteria, chronic infections can only be identified by serological tests. At this point, a serological method for differentiation between infections with the two Yersinia species is important since antibiotic susceptibility of these bacteria is different. Traditional immunoassays do not distinguish between infections with Y. enterocolitica and Y. pseudotuberculosis. The only test that allows for this differentiation is Mikrogen's strip test where discrimination between the two types of infection is based on two recombinant bacterial proteins, MyfA and PsaA (specific for Y. enterocolitica and Y. pseudotuberculosis, respectively). Here, we show that Y. enterocolitica and Y. pseudotuberculosis, cultured under the conditions that mimic the natural rout of infection, express surface antigens different from MyfA and PsaA that can also be used in a discrimination test. Further, we describe a new ELISA that is based on the whole bacteria and recombinant MyfA and PsaA as antigens, and that allows the differentiation between infections with Y. enterocolitica and Y. pseudotuberculosis and simultaneous detection of yersiniosis.

  • 25.
    Wilczynska, Malgorzata
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Fa, M
    Karolin, J
    Ohlsson, P I
    Johansson, L B
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Structural insights into serpin-protease complexes reveal the inhibitory mechanism of serpins.1997Ingår i: Nature Structural Biology, ISSN 1072-8368, Vol. 4, nr 5, s. 354-7Artikel i tidskrift (Refereegranskat)
  • 26.
    Wilczynska, Malgorzata
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Fa, M
    Ohlsson, P I
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The inhibition mechanism of serpins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex.1995Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 270, nr 50, s. 29652-5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.

  • 27.
    Wilczynska, Malgorzata
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lobov, Sergei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The spontaneous polymerization of plasminogen activator inhibitor type-2 and Z-antitrypsin are due to different molecular aberrations2003Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 537, nr 1-3, s. 11-16Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The wild-type form of plasminogen activator inhibitor type-2 (PAI-2) and the pathogenic Z-mutant of alpha(1)-antitrypsin (alpha(1)AT) are serpins that spontaneously polymerize by the loop-sheet mechanism. Compared to the consensus serpin sequence, both PAI-2 and Z-alpha(1)AT have deviations in the so-called breach region located at the top of the A beta-sheet. In the case of Z-alpha(1)AT, conformational perturbations caused by a single amino acid substitution result in polymerization in vivo and predisposes to disease. To test whether the polymerization of PAI-2 is due to aberrations in the breach region, we constructed substitution mutants of PAI-2 with conserved residues in this region. Analysis of the mutants revealed that deviations in the breach region modulate but are not the major cause of PAI-2 polymerization. Rather, PAI-2 exists in a highly polymerogenic conformation and does not require conformational rearrangements before polymerization can take place.

  • 28.
    Wilczynska, Malgorzata
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lobov, Sergei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ohlsson, Per-Ingvar
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization.2003Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, nr 8, s. 1753-1761Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.

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