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  • 1.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mitochondria and Alzheimer's disease: amyloid-beta peptide uptake and degradation by the presequence protease, hPreP2009In: Journal of Bioenergetics and Biomembranes, ISSN 0145-479X, E-ISSN 1573-6881, Vol. 41, no 5, p. 447-451Article in journal (Refereed)
    Abstract [en]

    Several lines of evidence suggest mitochondrial dysfunction as a possible underlying mechanism of Alzheimer's disease (AD). Accumulation of the amyloid-beta peptide (Abeta), a neurotoxic peptide implicated in the pathogenesis of AD, has been detected in brain mitochondria of AD patients and AD transgenic mouse models. In vitro evidence suggests that the Abeta causes mitochondrial dysfunction e.g. oxidative stress, mitochondrial fragmentation and decreased activity of cytochrome c oxidase and TCA cycle enzymes. Here we review the link between mitochondrial dysfunctions and AD. In particular we focus on the mechanism for Abeta uptake by mitochondria and on the recently identified Abeta degrading protease in human brain mitochondria.

  • 2.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Engmann, Tanja
    Pavlov, Pavel
    Langer, Thomas
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Matrix localized AtPreP complements intermembrane space located homologue, MOP112, in Saccaromyces cerevisiaeManuscript (preprint) (Other academic)
  • 3.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Engmann, Tanja
    Spånning, Erika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Voegtle, F. -Nora
    Pavlov, Pavel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meisinger, Chris
    Langer, Thomas
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 410, no 3, p. 400-410Article in journal (Refereed)
    Abstract [en]

    Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.

  • 4.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guo, Lan
    Pinho, Catarina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yan, Shi Du
    Decreased proteolytic activity of the PreP peptidasome in Alzheimer disease brain mitochondria and transgenic modelsManuscript (preprint) (Other academic)
  • 5.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guo, Lan
    Yan, Shiqiang
    Du, Heng
    Pinho, Catarina Moreira
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chen, John Xi
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yan, Shirley ShiDu
    Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria2011In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 27, no 1, p. 75-87Article in journal (Refereed)
    Abstract [en]

    Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolytic activity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.

  • 6.
    Berglund, Anna-Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pujol, Calire
    Duchene, Anne-Marie
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 393, no 4, p. 803-814Article in journal (Refereed)
    Abstract [en]

    Most of the organellar amino acyl-tRNA synthetases (aaRSs) are dually targeted to both mitochondria and chloroplasts using dual targeting peptides (dTPs). We have investigated the targeting properties and domain structure of dTPs of seven aaRSs by studying the in vitro and in vivo import of N-terminal deleted constructs of dTPs fused to green fluorescent protein. The deletion constructs were designed based on prediction programs, TargetP and Predotar, as well as LogoPlots derived from organellar proteomes in Arabidopsis thaliana. In vitro import was performed either into a single isolated organelle or as dual import (i.e., into a mixture of isolated mitochondria and chloroplasts followed by reisolation of the organelles). In vivo import was investigated as transient expression of the green fluorescent protein constructs in Nicotiana benthamiana protoplasts. Characterization of recognition determinants showed that the N-terminal portions of TyrRS-, ValRS- and ThrRS-dTPs (27, 22 and 23 amino acids, respectively) are required for targeting into both mitochondria and chloroplasts. Surprisingly, these N-terminal portions contain no or very few arginines (or lysines) but very high number of hydroxylated residues (26–51%). For two aaRSs, a domain structure of the dTP became evident. Removal of 20 residues from the dTP of ProRS abolished chloroplastic import, indicating that the N-terminal region was required for chloroplast targeting, whereas deletion of 16 N-terminal amino acids from AspRS-dTP inhibited the mitochondrial import, showing that in this case, the N-terminal portion was required for the mitochondrial import. Finally, deletion of N-terminal regions of dTPs for IleRS and LysRS did not affect dual targeting. In summary, it can be concluded that there is no general rule for how the determinants for dual targeting are distributed within dTPs; in most cases, the N-terminal portion is essential for import into both organelles, but in a few cases, a domain structure was observed.

  • 7.
    Berglund, Anna-Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Spånning, Erika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biverståhl, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Maddalo, Gianluca
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Tellgren-Roth, Christian
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dual targeting to Mitochondria and Chloroplasts: Characterization of Thr-tRNA Synthetase Targeting Peptide2009In: Molecular Plant, ISSN 1674-2052, Vol. 6, no 2, p. 1298-1309Article in journal (Refereed)
    Abstract [en]

    There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins of Arabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides (dTPs). The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr–tRNA synthetase (ThrRS–dTP) studying organellar import of N- and C-terminal deletion constructs of ThrRS–dTP coupled to GFP. These results show that the 23 amino acid long N-terminal portion of ThrRS–dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS–dTP(2–60) to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS–dTP(2–60) inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS–dTP(2–60) indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

  • 8.
    Bhushan, Shashi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johnson, Kenneth A
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proteolytic mechanism of a novel mitochondrial and chloroplastic PreP peptidasome.2006In: Biol Chem, ISSN 1431-6730, Vol. 387, no 8, p. 1087-90Article in journal (Refereed)
    Abstract [en]

    The 2.1-A-resolution crystal structure of the novel mitochondrial and chloroplastic metalloendopeptidase, AtPreP1, revealed a unique peptidasome structure, in which the two halves of the enzyme completely enfold a huge proteolytic cavity. Based on the structure, we proposed a novel mechanism for proteolysis involving hinge-bending motions, which cause the protease to open and close in response to substrate binding. We generated four double-mutants of AtPreP1 by introducing cysteines at positions where disulfide bonds can be formed in order to lock and unlock the protease and tested the activity under oxidizing and reducing conditions. The overall results support the proposed mechanism.

  • 9.
    Bhushan, Shashi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kuhn, Claus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Roth, Christian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting2006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 16, p. 3966-3972Article in journal (Refereed)
    Abstract [en]

    We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.

  • 10.
    Bhushan, Shashi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pavlov, Pavel F
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rudhe, Charlotta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    In vitro and in vivo methods to study protein import into plant mitochondria.2007In: Methods Mol Biol, ISSN 1064-3745, Vol. 390, p. 131-50Article in journal (Refereed)
    Abstract [en]

    Plant mitochondria contain about 1000 proteins, 90-99% of which in different plant species are nuclear encoded, synthesized on cytosolic polyribosomes, and imported into the organelle. Most of the nuclear-encoded proteins are synthesized as precursors containing an N-terminal extension called a presequence or targeting peptide that directs the protein to the mitochondria. Here we describe in vitro and in vivo methods to study mitochondrial protein import in plants. In vitro synthesized precursor proteins can be imported in vitro into isolated mitochondria (single organelle import). However, missorting of chloroplast precursors in vitro into isolated mitochondria has been observed. A novel dual import system for simultaneous import of proteins into isolated mitochondria and chloroplasts followed by reisolation of the organelles is superior over the single import system as it abolishes the mistargeting. Precursor proteins can also be imported into the mitochondria in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a presequence or a full-length precursor protein fused to a reporter gene, most commonly the green fluorescence protein (GFP) in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.

  • 11. Björk, Behnosh
    et al.
    Pinho, Catarina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bäckman, Hans
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fratiglioni, Laura
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Graff, Caroline
    Genetic and biochemical studies of SNPs in the mitochondrial Aβ-degrading protease, hPrePManuscript (preprint) (Other academic)
  • 12. Brunetti, Dario
    et al.
    Torsvik, Janniche
    Dallabona, Cristina
    Teixeira, Pedro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sztromwasser, Pawel
    Fernandez-Vizarra, Erika
    Cerutti, Raffaele
    Reyes, Aurelio
    Preziuso, Carmela
    D'Amati, Giulia
    Baruffini, Enrico
    Goffrini, Paola
    Viscomi, Carlo
    Ferrero, Ileana
    Boman, Helge
    Telstad, Wenche
    Johansson, Stefan
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Knappskog, Per M.
    Zeviani, Massimo
    Bindoff, Laurence A.
    Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration2016In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 8, no 3, p. 176-190Article in journal (Refereed)
    Abstract [en]

    Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (A). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested invitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1(+/-) heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of A-positive amyloid deposits. Our results show that PITRM1 is responsible for significant A degradation and that impairment of its activity results in A accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.

  • 13.
    Bäckman, Hans G
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pessoa, João
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP.2009In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 583, no 17, p. 2727-33Article in journal (Refereed)
    Abstract [en]

    The dual-targeted mitochondrial and chloroplastic zinc metallooligopeptidase from Arabidopsis, AtPreP, functions as a peptidasome that degrades targeting peptides and other small unstructured peptides. In addition to Zn located in the catalytic site, AtPreP also contains two Mg-binding sites. We have investigated the role of Mg-binding using AtPreP variants, in which one or both sites were rendered unable to bind Mg(2+). Our results show that metal binding besides that of the active site is crucial for AtPreP proteolysis, particularly the inner site appears essential for normal proteolytic function. This is also supported by its evolutionary conservation among all plant species of PreP.

  • 14. Chen, Jue
    et al.
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Levine, Rodney L.
    Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide2014In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 77, p. 57-63Article in journal (Refereed)
    Abstract [en]

    The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-beta peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.

  • 15.
    Falkevall, Annelie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bhushan, Shashi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pavlov, Pavel F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Busch, Katrin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johnson, Kenneth A.
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tjernberg, Lars
    Ankarcrona, Maria
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 39, p. 29096-29104Article in journal (Refereed)
    Abstract [en]

    Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease.

  • 16. Fang, Du
    et al.
    Wang, Yongfu
    Zhang, Zhihua
    Du, Heng
    Yan, Shiqiang
    Sun, Qinru
    Zhong, Changjia
    Wu, Long
    Vangavaragu, Jhansi Rani
    Yan, Shijun
    Hu, Gang
    Guo, Lan
    Rabinowitz, Molly
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Arancio, Ottavio
    Sosunov, Alexander A.
    McKhann, Guy M.
    Chen, John Xi
    Yan, Shirley ShiDu
    Increased neuronal PreP activity reduces A beta accumulation, attenuates neuroinflammation and improves mitochondrial and synaptic function in Alzheimer disease's mouse model2015In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 24, no 18, p. 5198-5210Article in journal (Refereed)
    Abstract [en]

    Accumulation of amyloid-beta (A beta) in synaptic mitochondria is associated with mitochondrial and synaptic injury. The underlying mechanisms and strategies to eliminate A beta and rescue mitochondrial and synaptic defects remain elusive. Presequence protease (PreP), a mitochondrial peptidasome, is a novel mitochondrial A beta degrading enzyme. Here, we demonstrate for the first time that increased expression of active human PreP in cortical neurons attenuates Alzheimer disease's (AD)-like mitochondrial amyloid pathology and synaptic mitochondrial dysfunction, and suppresses mitochondrial oxidative stress. Notably, PreP-overexpressed AD mice show significant reduction in the production of proinflammatory mediators. Accordingly, increased neuronal PreP expression improves learning and memory and synaptic function in vivo AD mice, and alleviates A beta-mediated reduction of long-term potentiation (LTP). Our results provide in vivo evidence that PreP may play an important role in maintaining mitochondrial integrity and function by clearance and degradation of mitochondrial A beta along with the improvement in synaptic and behavioral function in AD mouse model. Thus, enhancing PreP activity/expression may be a new therapeutic avenue for treatment of AD.

  • 17.
    Ge, Changrong
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Spånning, Erika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana2014In: Molecular Plant, ISSN 1674-2052, Vol. 7, no 1, p. 121-136Article in journal (Refereed)
    Abstract [en]

    Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide (TP) directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis (MVDA) with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed. dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for the chloroplast import.

  • 18.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The organellar peptidasome, PreP: a journey from Arabidopsis to Alzheimer's disease2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 1076-1080Article in journal (Refereed)
    Abstract [en]

    The novel peptidasome, called presequence protease, PreP, was originally identified and characterized in Arabidopsis thaliana as a mitochondrial matrix and chloroplast stroma localized metalloprotease. PreP has a function as the organellar peptide clearing protease and is responsible for degrading free targeting peptides and also other unstructured peptides up to 65 amino acid residues that might be toxic to organellar functions. PreP contains an inverted Zn-binding motif and belongs to the pitrilysin protease family. The crystal structure of AtPreP refined at 2.1 A demonstrated a unique totally enclosed large cavity of 10000 A3 that opens and closes in response to peptide binding, revealing a novel catalytic mechanism for proteolysis. Homologues of PreP have been found in yeast and human mitochondria. Interestingly, the human PreP, hPreP, is the protease that is responsible for clearing the human brain mitochondria from the toxic amyloid-beta peptide (Abeta) associated with Alzheimer's disease (AD). Accumulation of Abeta has been shown in the brain mitochondria from AD patients and mutant transgenic mice overexpressing Abeta. Here, we present a review of our present knowledge on structural and functional characteristics of PreP and discuss its mitochondrial Abeta-degrading activity in the human brain mitochondria in relation to AD.

  • 19.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Inoue, Kentaro
    Processing and Degradation of Chloroplast Extension Peptides2014In: Plastid Biology / [ed] Theg, S. M.; Wollman, F. A., New York: Springer, 2014, p. 305-323Chapter in book (Refereed)
    Abstract [en]

    Most chloroplast proteins are synthesized as larger precursors with cleavable extension peptides. These extensions include import signals called transit peptides, export signals for thylakoid transfer, and the C-terminal extension of the chloroplast-encoded D1 subunit of the photosystem II. Transit peptides are necessary for transport of nuclear-encoded proteins from the cytoplasm across the double-membrane envelope, and are cleaved off by Stromal Processing Peptidase (SPP) in the stroma. Further degradation of transit peptides involves SPP and Presequence Protease (PreP). Thylakoid-transfer sequences are required for correct intraorganellar protein sorting and cleaved by Thylakoidal Processing Peptidase (TPP) in the thylakoid lumen. The C-terminal extension of the D1 protein is not required for precursor targeting and integration into the protein complex; however its removal by Carboxyl-terminal peptidase called CtpA in the thylakoid lumen is needed for proper formation of the photosystem II Mn4CaO5 cluster. Biochemical studies in the 1980s-1990s defined basic properties of SPP, TPP and CtpA, while PreP was discovered in the early 2000s. Recent molecular genetic studies demonstrated physiological importance as well as some unprecedented functions of these enzymes. This chapter gives a comprehensive survey on processing and degradation of chloroplast extension peptides. The emphasis is on biochemical, molecular and evolutionary aspects of proteases. The significances of the presence and processing of these extension peptides are also discussed.

  • 20.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mitochondrial and Chloroplastic Targeting Peptides Peptidase, PreP2013In: Handbook of proteolytic enzymes, vols 1 and 2, 3rd edition / [ed] Rawlings, ND; Salvesen, GS, AMSTERDAM: ELSEVIER SCIENCE BV , 2013, 3rd, p. 1426-1430Chapter in book (Refereed)
  • 21.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bhushan, Shashi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Two novel mitochondrial and chloroplastic targeting-peptide-degrading peptidasomes in A. thaliana, AtPreP1 and AtPreP2.2006In: Biol Chem, ISSN 1431-6730, Vol. 387, no 10-11, p. 1441-7Article in journal (Refereed)
    Abstract [en]

    Two novel metalloendopeptidases in Arabidopsis thaliana, AtPreP1 and AtPreP2, are responsible for the degradation of targeting peptides in mitochondria and chloroplasts. Both AtPreP1 and AtPreP2 contain ambiguous targeting peptides and are dually targeted to both organelles. The proteases also have the capacity to degrade unstructured peptides of up to 65 amino acid residues, but not small proteins. The catalysis occurs in a huge catalytic chamber revealed by the crystal structure of AtPreP1 at 2.1 A. The enzymes show a preference for basic and small uncharged amino acids or serines at the cleavage sites. Despite similarities in cleavage specificities, cleavage-site recognition differs for both proteases and is context- and structure-dependent. The AtPreP1 and AtPreP2 genes are differentially expressed in Arabidopsis.

  • 22.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Whelan, James
    Plant mitochondrial protein import2007In: Plant mitochondria, 2007Chapter in book (Other academic)
  • 23.
    Glaser, Elzbieta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Whelan, James
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Protein import into plant mitochondria2011In: Plant Mitochondria / [ed] Frank Kempken, Springer , 2011, p. 261-288Chapter in book (Refereed)
    Abstract [en]

    The presence of plastids in plant cells requires a higher level of precursor recognition by the mitochondrial protein import apparatus than in nonplant organisms. Although the plant presequences display the overall features observed in yeast and mammals, they are generally longer and more hydrophilic. Most of them are highly organelle specific, but some have ambiguous targeting specificity delive-ring a protein to both mitochondria and chloroplasts. Many components of plant protein import apparatus appear different to that in yeast and mammalian systems. The three outer membrane mitochondrial proteins characterized to play role as receptors in plants – Tom20, OM64, and metaxin – are plant specific. However, the channel forming units of the TOM and SAM complexes, Tom40 and Sam50, respectively, are orthologous to these components in yeast. While components of the MIA and TIM complexes also display high levels of orthology, functional studies indicate divergences in function and mechanism. Differences exist also in terms of intraorganellar localization of proteolytic events, e.g., the location of the mitochondrial processing peptidase, MPP, involved in removing targeting signals is different, whereas the function and location of the presequence protease, PreP, degrading targeting peptides, is well conserved. Overall, although the protein import machinery of mitochondria from all organisms appears to have coopted and uses the channel forming subunits from the endosymbiont that gave rise to mitochondria, there is a greater diversity in plant components in comparison to those from nonplant species

  • 24. Hansson Petersen, Camilla A.
    et al.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Behbahani, Homira
    Wiehager, Birgitta
    Pavlov, Pavel F
    Alafuzoff, Irina
    Leinonen, Ville
    Ito, Akira
    Winblad, Bengt
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 35, p. 13145-13150Article in journal (Refereed)
    Abstract [en]

    The amyloid beta-peptide (Abeta) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Abeta and accumulation of Abeta has been detected in mitochondria. Because Abeta is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Abeta uptake. Our results from rat mitochondria show that Abeta is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Abeta association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Abeta to mitochondrial cristae. A similar distribution pattern of Abeta in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Abeta in mitochondria and demonstrate both in vitro and in vivo that Abeta is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Abeta can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Abeta, a peptide considered to be of major significance in Alzheimer's disease.

  • 25. Haussuehl, Kirsten
    et al.
    Huesgen, Pitter F
    Meier, Marc
    Dessi, Patrick
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Adamski, Jerzy
    Adamska, Iwona
    Eukaryotic GCP1 is a conserved mitochondrial protein required for progression of embryo development beyond the globular stage in Arabidopsis thaliana.2009In: The Biochemical journal, ISSN 1470-8728, Vol. 423, no 3, p. 333-41Article in journal (Refereed)
    Abstract [en]

    GCPs (glycoproteases) are members of the HSP70 (heat-shock protein 70)/actin ATPase superfamily that are highly conserved in taxonomically diverse species from bacteria to man, suggesting an essential physiological role. Although originally identified and annotated as putative endopeptidases, a proteolytic activity could not be confirmed for these proteins. Our survey of genome databases revealed that all eukaryotic organisms contain two GCP genes [called GCP1 and GCP2/Kae1 (kinase-associated endopeptidase 1)], whereas prokaryotes have only one, either of the GCP1- (Bacteria) or the GCP2/Kae1- (Archaea) type. GCP2/Kae1 is essential for telomere elongation and transcription of essential genes, although little is known about the localization, expression and physiological role of GCP1. In the present study on GCP1-type proteins from eukaryotic organisms we demonstrated that GCP1 is a mitochondrial protein in Homo sapiens [called here GCP1/OSGEPL1 (O-sialoglycoprotein endopeptidase)] and Arabidopsis thaliana, which is located/anchored to the mitochondrial inner membrane. Analysis of mRNA and protein levels revealed that the expression of GCP1/OSGEPL1 in A. thaliana and H. sapiens is tissue- and organ-specific and depends on the developmental stage, suggesting a more specialized function for this protein. We showed that homozygous A. thaliana GCP1 T-DNA (transferred DNA) insertion lines were embryonic lethal. Embryos in homozygous seeds were arrested at the globular stage and failed to undergo the transition into the heart stage. On the basis of these data we propose that the mitochondrial GCP1 is essential for embryonic development in plants.

  • 26. Hedskog, Louise
    et al.
    Brohede, Jesper
    Wiehager, Birgitta
    Pinho, Catarina Moreira
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Revathikumar, Priya
    Lilius, Lena
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Graff, Caroline
    Karlstrom, Helena
    Ankarcrona, Maria
    Biochemical studies of poly t variants in the alzheimer's disease associated tomm40 gene2012In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 31, no 3, p. 527-536Article in journal (Refereed)
    Abstract [en]

    The apolipoprotein E (APOE) gene remains the most strongly established risk factor for late onset Alzheimer's disease (LOAD). Recently the gene, TOMM40, which is in linkage disequilibrium with APOE, was identified to be associated with LOAD in genome-wide association studies. One of the identified polymorphisms in TOMM40 is rs10524523, which is located in intron 6 and composed of thymidine repeats varying between 14 to 36 base-pairs in length. Reported results are contradictory in regard to the very long poly-T variant that has been associated with both increased and decreased risk of LOAD. Our study aimed to elucidate the functional implication of rs10524523 in an in vitro model of human fibroblast cells obtained from cognitively healthy APOE epsilon 3/epsilon 4 carriers harboring very long or short poly-T variants coupled to their APOE epsilon 3 allele. We have studied (i) expression levels of TOM40 protein and mRNA, (ii) TOM40 mRNA splicing, and (iii) mitochondrial function and morphology; and we have found no significant differences in regards to very long or short poly-T variant.

  • 27. Hedskog, Louise
    et al.
    Pinho, Catarina Moreira
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Filadi, Riccardo
    Rönnbäck, Annica
    Hertwig, Laura
    Wiehager, Birgitta
    Larssen, Pia
    Gellhaar, Sandra
    Sandebring, Anna
    Westerlund, Marie
    Graff, Caroline
    Winblad, Bengt
    Galter, Dagmar
    Behbahani, Homira
    Pizzo, Paola
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 19, p. 7916-7921Article in journal (Refereed)
    Abstract [en]

    It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and sigma 1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)(Swe/Lon) mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid beta-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.

  • 28. Johnson, Kenneth A
    et al.
    Bhushan, Shashi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ståhl, Annelie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hallberg, B Martin
    Frohn, Anne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The closed structure of presequence protease PreP forms a unique 10,000 Angstroms3 chamber for proteolysis.2006In: EMBO J, ISSN 0261-4189, Vol. 25, no 9, p. 1977-86Article in journal (Refereed)
    Abstract [en]

    Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Angstroms resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10,000 Angstroms(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.

  • 29.
    Kmiec, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Branca, Rui M. M.
    Berkowitz, Oliver
    Li, Lu
    Wang, Yan
    Murcha, Monika W.
    Whelan, James
    Lehtiö, Janne
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Accumulation of endogenous peptides triggers a pathogen stress response in Arabidopsis thaliana2018In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 96, no 4, p. 705-715Article in journal (Refereed)
    Abstract [en]

    The stepwise degradation of peptides to amino acids in plant mitochondria and chloroplasts is catalyzed by a network of oligopeptidases (presequence protease PreP, organellar oligopeptidase OOP) and aminopeptidases. In the present report, we show that the lack of oligopeptidase activity in Arabidopsis thaliana results in the accumulation of endogenous free peptides, mostly of chloroplastic origin (targeting peptides and degradation products). Using mRNA sequencing and deep coverage proteomics, allowing for the identification of 17 000 transcripts and 11 000 proteins, respectively, we uncover a peptide-stress response occurring in plants lacking PreP and OOP oligopeptidase activity. The peptide-stress response results in the activation of the classical plant defense pathways in the absence of pathogenic challenge. The constitutive activation of the pathogen-defense pathways imposes a strong growth penalty and a reduction of the plants reproductive fitness. Our results indicate that the absence of organellar oligopeptidases PreP1/2 and OOP results in the accumulation of peptides that are perceived as pathogenic effectors and activate the signaling pathways of plant-defense response.

  • 30.
    Kmiec, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Branca, Rui M. M.
    Murcha, Monika W.
    Lehtiö, Janne
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A Common Peptidolytic Mechanism for Targeting Peptide Degradation in Mitochondria and Chloroplasts2018In: Molecular Plant, ISSN 1674-2052, E-ISSN 1752-9867, Vol. 11, no 2, p. 342-345Article in journal (Refereed)
  • 31.
    Kmiec, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berntsson, Ronnie P. -A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Murcha, Monika W.
    Branca, Rui M. M.
    Radomiljac, Jordan D.
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bakali, Amin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehtio, Janne
    Whelan, James
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 40, p. E3761-E3769Article in journal (Refereed)
    Abstract [en]

    Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 angstrom, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 angstrom(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.

  • 32.
    Kmiec, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Phenotypical consequences of expressing the dually targeted Presequence Protease, AtPreP, exclusively in mitochondria2014In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 100C, p. 167-170Article in journal (Refereed)
    Abstract [en]

    Endosymbiotic organelles, mitochondria and chloroplasts, are sites of an intensive protein synthesis and degradation. A consequence of these processes is production of both free targeting peptides, i.e. mitochondrial presequences and chloroplastic transit peptides, and other short unstructured peptides. Mitochondrial, as well as chloroplastic peptides are degraded by Presequence Protease (Prep), which is dually targeted to mitochondrial matrix and chloroplastic stroma. Elimination of PreP in Arabidopsis thaliana leads to growth retardation, chlorosis and impairment of mitochondrial functions potentially due to the accumulation of targeting peptides. In this work we analyzed the influence of the restoration of mitochondrial peptide degradation by AtPreP on plant phenotype. We showed that exclusive mitochondrial expression of AtPreP results in total restoration of the proteolytic activity, but it does not restore the wild-type phenotype. The plants grow shorter roots and smaller rosettes compared to the plants expressing AtPreP1 in both mitochondria and chloroplasts. With this analysis we are aiming at understanding the physiological impact of the role of the dually targeted AtPreP in single type of destination organelle.

  • 33.
    Kmiec, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Shredding the signal: targeting peptide degradation in mitochondria and chloroplasts2014In: Trends in Plant Science, ISSN 1360-1385, E-ISSN 1878-4372, Vol. 19, no 12, p. 771-778Article, review/survey (Refereed)
    Abstract [en]

    The biogenesis and functionality of mitochondria and chloroplasts depend on the constant turnover of their proteins. The majority of mitochondrial and chloroplastic proteins are imported as precursors via their N-terminal targeting peptides. After import, the targeting peptides are cleaved off and degraded. Recent work has elucidated a pathway involved in the degradation of targeting peptides in mitochondria and chloroplasts, with two proteolytic components: the presequence protease (PreP) and the organellar oligopeptidase (OOP). PreP and OOP are specialized in degrading peptides of different lengths, with the substrate restriction being dictated by the structure of their proteolytic cavities. The importance of the intraorganellar peptide degradation is highlighted by the fact that elimination of both oligopeptidases affects growth and development of Arabidopsis thaliana.

  • 34.
    Kmiec, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Murcha, Monika W.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Divergent evolution of the M3A family of metallopeptidases in plants2016In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 157, no 3, p. 380-388Article in journal (Refereed)
    Abstract [en]

    Plants, as stationary organisms, have developed mechanisms allowing them efficient resource reallocation and a response to changing environmental conditions. One of these mechanisms is proteome remodeling via a broad peptidase network present in various cellular compartments including mitochondria and chloroplasts. The genome of the model plant Arabidopsis thaliana encodes as many as 616 putative peptidase-coding genes organized in 55 peptidase families. In this study, we describe the M3A family of peptidases, which comprises four members: mitochondrial and chloroplastic oligopeptidase (OOP), cytosolic oligopeptidase (CyOP), mitochondrial octapeptidyl aminopeptidase 1 (Oct1) and plant-specific protein of M3 family (PSPM3) of unknown function. We have analyzed the evolutionary conservation of M3A peptidases across plant species and the functional specialization of the three distinct subfamilies. We found that the subfamily-containing OOP and CyOP-like peptidases, responsible for oligopeptide degradation in the endosymbiotic organelles (OOP) or in the cytosol (CyOP), are highly conserved in all kingdoms of life. The Oct1-like peptidase subfamily involved in pre-protein maturation in mitochondria is conserved in all eukaryotes, whereas the PSPM3-like protein subfamily is strictly conserved in higher plants only and is of unknown function. Specific characteristics within PSPM3 sequences, i.e. occurrence of a N-terminal transmembrane domain and amino acid changes in distal substrate-binding motif, distinguish PSPM3 proteins from other members of M3A family. We performed peptidase activity measurements to analyze the role of substrate-binding residues in the different Arabidopsis M3A paralogs.

  • 35.
    Kmiec-Wisniewska, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A novel mitochondrial and chloroplast peptidasome, PreP2012In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 145, no 1, p. 180-186Article in journal (Refereed)
    Abstract [en]

    A novel mitochondrial and chloroplast peptidasome, the Presequence Protease (PreP) degrades organellar targeting peptides as well as other unstructured peptides up to 65 amino acid residues in length. PreP belongs to the pitrilysin oligopeptidase family (M16C) containing an inverted zinc-binding motif. The crystal structure of Arabidopsis thaliana PreP, AtPreP, refined at 2.1 angstrom, revealed a novel mechanism of proteolysis in which two halves of the enzyme connected by a hinge region enclose a large catalytic chamber opening and closing in response to peptide binding. Double knock-out mutant of AtPreP1 and AtPreP2 results in a severe phenotype, including decreased size and growth rate, chlorosis and organellar abnormalities, such as altered chloroplast starch content, partial loss of the integrity of the inner mitochondrial membrane and reduced mitochondrial respiration. PreP homologues are also present in yeast and humans. Interestingly, human PreP has been associated with Alzheimer's disease as it is responsible for degradation of amyloid-beta peptide in brain mitochondria.

  • 36.
    Kmiec-Wisniewska, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Duncan, Owen
    Whelan, James
    Murcha, Monika W.
    Evolution of protein import pathways2012In: Mitochondrial genome evolution / [ed] Laurence Maréchal-Drouard, London: Academic Press, 2012, p. 315-346Chapter in book (Refereed)
    Abstract [en]

    Although a single endosymbiotic event is accepted to have led to the evolution of mitochondria, the machinery that is necessary to import the hundreds of cytosol-synthesized precursor proteins has diverged between various lineages. Plants have the additional requirement to sort protein between mitochondria and plastids, and yet there are also many cases of dual-targeted proteins. The machinery that achieved this process, the mitochondrial protein import apparatus, is composed of a variety of multi-subunit protein complexes on the outer and inner mitochondrial membranes. In plant mitochondria, there are differences in protein composition and/or function in the translocase of the outer membrane and in the intermembrane space. Furthermore, whereas the inner membrane translocases appear more conserved, there is an expansion in the preprotein and amino acid transporter (PRAT) family of proteins that suggest that neofunctionalization has occurred within this family. Our understanding of the processing and degradation of mitochondrial targeting signals in all systems is based on the intensive studies in plants.

  • 37. Langer, Yeshaya
    et al.
    Aran, Adi
    Gulsuner, Suleyman
    Abu Libdeh, Bassam
    Renbaum, Paul
    Brunetti, Dario
    Teixeira, Pedro-Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Walsh, Tom
    Zeligson, Sharon
    Ruotolo, Roberta
    Beeri, Rachel
    Dweikat, Imad
    Shahrour, Maher
    Weinberg-Shukron, Ariella
    Zandeh, Fouad
    Baruffini, Enrico
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    King, Mary-Claire
    Levy-Lahad, Ephrat
    Zeviani, Massimo
    Segel, Reeval
    Mitochondrial PITRM1 peptidase loss-of-function in childhood cerebellar atrophy2018In: Journal of Medical Genetics, ISSN 0022-2593, E-ISSN 1468-6244, Vol. 55, no 9, p. 599-606Article in journal (Refereed)
    Abstract [en]

    Objective To identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy. Methods Identification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients' cells and in yeast. Results Two brothers from a consanguineous Palestinian family presented with progressive spinocerebellar ataxia, mental retardation and psychotic episodes. Serial brain imaging showed severe progressive cerebellar atrophy. Whole exome sequencing revealed a novel mutation: pitrilysin metallopeptidase 1 (PITRM1) c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in PITRM1 protein. Whole genome sequencing revealed a chromosome X structural rearrangement that also segregated with the disease. Independently, two siblings from a second Palestinian family presented with similar, somewhat milder symptoms and the same PITRM1 mutation on a shared haplotype. PITRM1T931M carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. PITRM1 is a mitochondrial matrix enzyme that degrades 10-65 amino acid oligopeptides, including the mitochondrial fraction of amyloid-beta peptide. Analysis of peptide cleavage activity by the PITRM1T931M protein revealed a significant decrease in the degradation capacity specifically of peptides >= 40 amino acids. Conclusion PITRM1T931M results in childhood-onset recessive cerebellar pathology. Severity of PITRM1-related disease may be affected by the degree of impairment in cleavage of mitochondrial long peptides. Disruption and deletion of X linked regulatory segments may also contribute to severity.

  • 38. Mossmann, Dirk
    et al.
    Voegtle, F-Nora
    Taskin, Asli Aras
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ring, Julia
    Burkhart, Julia M.
    Burger, Nils
    Pinho, Catarina Moreira
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tadic, Jelena
    Loreth, Desiree
    Graff, Caroline
    Metzger, Friedrich
    Sickmann, Albert
    Kretz, Oliver
    Wiedemann, Nils
    Zahedi, Rene P.
    Madeo, Frank
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meisinger, Chris
    Amyloid-beta Peptide Induces Mitochondrial Dysfunction by Inhibition of Preprotein Maturation2014In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 20, no 4, p. 662-669Article in journal (Refereed)
    Abstract [en]

    Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (A beta). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, A beta inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which A beta peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease.

  • 39. Murcha, Monika W.
    et al.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kubiszewski-Jakubiak, Szymon
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Whelan, James
    Protein import into plant mitochondria: signals, machinery, processing, and regulation2014In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 65, no 22, p. 6301-6335Article, review/survey (Refereed)
    Abstract [en]

    The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis.

  • 40. Murcha, Monika W.
    et al.
    Kubiszewski-Jakubiak, Szymon
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guegel, Irene L.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Narsai, Reena
    Ivanova, Aneta
    Megel, Cyrille
    Schock, Annette
    Kraus, Sabrina
    Berkowitz, Oliver
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Philippar, Katrin
    Marechal-Drouard, Laurence
    Soll, Juergen
    Whelan, James
    Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria2016In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 172, no 4, p. 2471-2490Article in journal (Refereed)
    Abstract [en]

    A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-a-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.

  • 41.
    Nilsson, Stefan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bäckman, Hans G.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pesaresi, Paolo
    Leister, Dario
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana2009In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 71, no 4-5, p. 497-508Article in journal (Refereed)
    Abstract [en]

    A novel peptidasome PreP is responsible for degradation of targeting peptides and other unstructured peptides in mitochondria and chloroplasts. Arabidopsis thaliana contains two PreP isoforms, AtPreP1, and AtPreP2. Here we have characterized single and double prep knockout mutants. Immunoblot analysis of atprep1 and atprep2 mutants showed that both isoforms are expressed in all tissues with the highest expression in flowers and siliques; additionally, AtPreP1 accumulated to a much higher level in comparison to AtPreP2. The atprep2 mutant behaved like wild type, whereas deletion of AtPreP1 resulted in slightly pale-green seedlings. Analysis of the atprep1 atprep2 double mutant revealed a chlorotic phenotype in true leaves with diminished chlorophyll a and b content, but unchanged Chl a/b ratio indicating a proportional decrease of both PSI and PSII complexes. Mitochondrial respiratory rates (state 3) were lower and the mitochondria were partially uncoupled. EM pictures on cross sections of the first true leaves showed aberrant chloroplasts, including less grana stacking and less starch granules. Mitochondria with extremely variable size could also be observed. At later developmental stages the plants appeared almost normal. However, all through the development there was a statistically significant decrease of ~40% in the accumulated biomass in the double mutant plants in comparison to wild type. In mitochondria, deletion of AtPreP was not compensated by activation of any peptidolytic activity, whereas chloroplast membranes contained a minor peptidolytic activity not related to AtPreP. In summary, the AtPreP peptidasome is required for efficient plant growth and organelle function particularly during early development.

  • 42.
    Pavlov, Pavel F
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hansson Petersen, Camilla
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    Mitochondrial accumulation of APP and Abeta: significance for Alzheimer disease pathogenesis.2009In: Journal of cellular and molecular medicine, ISSN 1582-4934, Vol. 13, no 10, p. 4137-45Article in journal (Refereed)
    Abstract [en]

    Accumulating evidence suggest that alterations in energy metabolism are among the earliest events that occur in the Alzheimer disease (AD) affected brain. Energy consumption is drastically decreased in the AD-affected regions of cerebral cortex and hippocampus pointing towards compromised mitochondrial function of neurons within specific brain regions. This is accompanied by an elevated production of reactive oxygen species contributing to increased rates of neuronal loss in the AD-affected brain regions. In this review, we will discuss the role of mitochondrial function and dysfunction in AD. We will focus on the consequences of amyloid precursor protein and amyloid-beta peptide accumulation in mitochondria and their involvement in AD pathogenesis.

  • 43. Pesaresi, Paolo
    et al.
    Masiero, Simona
    Eubel, Holger
    Braun, Hans-Peter
    Bhushan, Shashi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Salamini, Francesco
    Leister, Dario
    Nuclear photosynthetic gene expression is synergistically modulated by rates of protein synthesis in chloroplasts and mitochondria.2006In: Plant Cell, ISSN 1040-4651, Vol. 18, no 4, p. 970-91Article in journal (Refereed)
    Abstract [en]

    Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5'-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression.

  • 44.
    Pinho, Catarina Moreira
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bjork, Behnosh F.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bäckman, Hans G.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fratiglioni, Laura
    Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI).
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Graff, Caroline
    Genetic and biochemical studies of SNPs of the mitochondrial A beta-degrading protease, hPreP2010In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 469, no 2, p. 204-208Article in journal (Refereed)
    Abstract [en]

    Several studies suggest mitochondrial dysfunction as a possible mechanism underlying the development of Alzheimer disease (AD). There is data showing that amyloid-beta (A beta) peptide is present in AD brain mitochondria. The human presequence protease (hPreP) was recently shown to be the major mitochondrial A beta-degrading enzyme. We investigated if there is an increased susceptibility to AD, which can be attributed to genetic variation in the hPreP gene PITRM1 and if the proteolytic efficiency of recombinant hPreP variants is affected. When a total of 673 AD cases and 649 controls were genotyped for 18 single nucleotide polymorphisms (SNPs), no genetic association between any of the SNPs and the risk for AD was found. In contrast, functional analysis of four non-synonymous SNPs in hPreP revealed a decreased activity compared to wild type hPreP. Using A beta, the presequence of ATP synthase F-1 beta subunit and a fluorescent peptide as substrates, the lowest activity was observed for the hPreP(A525D) variant, corresponding to rs1224893, which displayed only 20-30% of wild type activity. Furthermore, the activity of all variants was restored by the addition of Mg2+, suggesting an important role for this metal during proteolysis. In conclusion, our data suggest that genetic variation in the hPreP gene PITRM1 may potentially contribute to mitochondrial dysfunctions.

  • 45.
    Pinho, Catarina Moreira
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mitochondrial import and degradation of amyloid-beta peptide2014In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1837, no 7, p. 1069-1074Article, review/survey (Refereed)
    Abstract [en]

    Mitochondrial dysfunctions associated with amyloid-beta peptide (A beta) accumulation in mitochondria have been observed in Alzheimer's disease (AD) patients' brains and in AD mice models. A beta is produced by sequential action of beta- and gamma-secretases cleaving the amyloid precursor protein (APP). The gamma-secretase complex was found in mitochondria-associated endoplasmic reticulum membranes (MAM) suggesting that this could be a potential site of A beta production, from which A beta is further transported into the mitochondria. In vitro, A beta was shown to be imported into the mitochondria through the translocase of the outer membrane (TOM) complex. The mitochondrial presequence protease (Prep) is responsible for A beta degradation reducing toxic effects of A beta on mitochondrial functions. The proteolytic activity of PreP is, however, lower in AD brain temporal lobe mitochondria and in AD transgenic mice models, possibly due to an increased reactive oxygen species (ROS) production. Here, we review the intracellular mechanisms of A beta production, its mitochondrial import and the intra-mitochondrial degradation. We also discuss the implications of a reduced efficiency of mitochondrial A beta clearance for AD. Understanding the underlying mechanisms may provide new insights into mitochondria related pathogenesis of AD and development of drug therapy against AD. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  • 46.
    Pinho, Catarina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Viera da Silva, Diogo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedskog, Louise
    Karolinska Institutet.
    Ankarcrona, Maria
    Karolinska Institutet.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Presequence processing increases the stability of the human Presequence Protease, hPrePManuscript (preprint) (Other academic)
    Abstract [en]

    Most of the mitochondrial matrix proteins are nuclear encoded, synthesized in the cytoplasm and have to be targeted to the mitochondria. For matrix proteins, this is generally achieved due to the presence of a N-terminal sequence, called presequence. After reaching the mitochondrial matrix, the presequence is cleaved off by the mitochondrial processing peptidase, MPP, giving rise to the mature protein and the presequence. Free presequences are degraded in the mitochondrial matrix by the Presequence Protease, PreP. Previous studies demonstrated that the correct maturation of mitochondrial proteins is important either for stability or catalytic activity of the protein.

    In the present study, we estimated the presequence length of the human PreP, hPreP, to be 28 amino acids long, using HEK293T cells and recombinant MPP. Furthermore, we analyzed the activity of the recombinant hPreP precursor and its mature form using two peptides, amyloid-β (1-40) peptide or the synthetic peptide substrate V, and we observed that the proteolytic maturation does not affect hPreP enzymatic activity. However, we detected a significantly lower stability for the hPreP precursor in comparison to the mature form of the enzyme, through pulse-chase experiments using vaccinia virus expression system in mammalian cells. These results show that the mitochondrial processing is required for the hPreP stability.

  • 47. Salinas, Thalia
    et al.
    Duchêne, Anne-Marie
    Delage, Ludovic
    Nilsson, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zaepfel, Marlyse
    Maréchal-Drouard, Laurence
    The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria.2006In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 103, no 48, p. 18362-7Article in journal (Refereed)
    Abstract [en]

    In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

  • 48.
    Ståhl, Annelie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Bhushan, Shashi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biverståhl, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Moberg, Per
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Morisett, Magali
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vener, Alexander
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Two Novel Targeting Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, no 4, p. 847-860Article in journal (Refereed)
    Abstract [en]

    Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F1β pre-sequence, peptides derived from the F1β pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F1β pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P1 position and small, uncharged amino acids or serine residues in the P1P′1

    position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the α-helical elements of the F1β pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic α-helix and positively charged amino acid residues and degraded the F1β pre-sequence into 10–16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F1β pre-sequence into 10–23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.

  • 49.
    Teixeira, Pedro F.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Branca, Rui M.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A Flowchart to Analyze Protease Activity in Plant Mitochondria2015In: Plant Mitochondria: Methods and Protocols / [ed] James Whelan, Monika W. Murcha, Springer-Verlag New York, 2015, Vol. 1305, p. 123-130Chapter in book (Refereed)
    Abstract [en]

    Proteases are one of the most abundant classes of enzymes and are involved in a plethora of biological processes in many cellular compartments, including the mitochondria. To understand the role of proteases is essential to determine their substrate repertoire, preferably in an in vivo setting. In this chapter we describe general guidelines to analyze protease activity using several strategies, from in-gel analysis to mass spectrometry mapping of the cleavage site(s) and fluorogenic probes that can easily be used in vivo. To exemplify this flowchart, we used the recently characterized organellar oligopeptidase of Arabidopsis (Arabidopsis thaliana), an enzyme that takes part in degradation of short peptides within mitochondria and chloroplasts.

  • 50.
    Teixeira, Pedro F.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Branca, Rui M. M.
    Murcha, Monika W.
    Byzia, Anna
    Ivanova, Aneta
    Whelan, James
    Drag, Marcin
    Lehtio, Janne
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A multi-step peptidolytic cascade for amino acid recovery in chloroplasts2017In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 13, no 1, p. 15-17Article in journal (Refereed)
    Abstract [en]

    Plastids (including chloroplasts) are subcellular sites for a plethora of proteolytic reactions, required in functions ranging from protein biogenesis to quality control. Here we show that peptides generated from pre-protein maturation within chloroplasts of Arabidopsis thaliana are degraded to amino acids by a multi-step peptidolytic cascade consisting of oligopeptidases and aminopeptidases, effectively allowing the recovery of single amino acids within these organelles.

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