Publications by authors named "Elzbieta Glaser"

63 Publications

A mitochondrial prolyl aminopeptidase PAP2 releases N-terminal proline and regulates proline homeostasis during stress response.

Plant J 2020 12 10;104(5):1182-1194. Epub 2020 Oct 10.

School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009, Australia.

Most mitochondrial proteins are synthesised in the cytosol and targeted into the organelle via N-terminal targeting peptides that are cleaved upon import. The free targeting peptide is subsequently processed in a stepwise manner, with single amino acids released as final products. Here, we have characterised a proline-cleaving aminopeptidase in Arabidopsis thaliana, prolyl aminopeptidase-2 (PAP2, At3g61540). Activity assays show that PAP2 has a preferred activity to hydrolyse N-terminal proline. Protein localisation studies revealed that PAP2 is exclusively targeted to mitochondria. Characterisation of pap2 mutants show defective pollen, enhanced dark-induced senescence and increased susceptibility to abiotic stresses, which are likely attributed to a reduced level of accumulated free proline. Taken together, these results demonstrate the role of PAP2 in proline cleavage from mitochondrial peptides and proline homeostasis, which is required for the development of male gametophyte, tolerance to abiotic stresses, and leaf senescence.
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http://dx.doi.org/10.1111/tpj.14987DOI Listing
December 2020

Accumulation of endogenous peptides triggers a pathogen stress response in Arabidopsis thaliana.

Plant J 2018 11;96(4):705-715

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1111/tpj.14100DOI Listing
November 2018

Mitochondrial peptidase loss-of-function in childhood cerebellar atrophy.

J Med Genet 2018 09 15;55(9):599-606. Epub 2018 May 15.

Department of Pediatrics, Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel.

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 () c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in 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 mutation on a shared haplotype. carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. 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 protein revealed a significant decrease in the degradation capacity specifically of peptides ≥40 amino acids.

Conclusion: results in childhood-onset recessive cerebellar pathology. Severity of -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.
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http://dx.doi.org/10.1136/jmedgenet-2018-105330DOI Listing
September 2018

Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin.

J Mol Biol 2018 02 26;430(3):348-362. Epub 2017 Nov 26.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden. Electronic address:

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN in complex with the products of neurotensin cleavage at 2.7Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.
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http://dx.doi.org/10.1016/j.jmb.2017.11.011DOI Listing
February 2018

A Common Peptidolytic Mechanism for Targeting Peptide Degradation in Mitochondria and Chloroplasts.

Mol Plant 2018 02 26;11(2):342-345. Epub 2017 Nov 26.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, 106 91 Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.molp.2017.11.008DOI Listing
February 2018

A multi-step peptidolytic cascade for amino acid recovery in chloroplasts.

Nat Chem Biol 2017 Jan 31;13(1):15-17. Epub 2016 Oct 31.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden.

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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http://dx.doi.org/10.1038/nchembio.2227DOI Listing
January 2017

Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria.

Plant Physiol 2016 12 27;172(4):2471-2490. Epub 2016 Oct 27.

Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Western Australia 6009, Australia (M.W.M., S.K.-J., A.I.);

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-α-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.
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http://dx.doi.org/10.1104/pp.16.01519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129730PMC
December 2016

Divergent evolution of the M3A family of metallopeptidases in plants.

Physiol Plant 2016 Jul 3;157(3):380-8. Epub 2016 Jun 3.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1111/ppl.12457DOI Listing
July 2016

Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function.

Mol Plant 2016 05 29;9(5):696-710. Epub 2016 Jan 29.

Department of Animal, Plant and Soil Science, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Science, La Trobe University, Bundoora, VIC 3086, Australia. Electronic address:

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1:At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.
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http://dx.doi.org/10.1016/j.molp.2016.01.009DOI Listing
May 2016

Defective PITRM1 mitochondrial peptidase is associated with Aβ amyloidotic neurodegeneration.

EMBO Mol Med 2016 Mar;8(3):176-90

Department of Neurology, Haukeland University Hospital, Bergen, Norway Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway

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 (Aβ). 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 in vitro, 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 Aβ-positive amyloid deposits. Our results show that PITRM1 is responsible for significant Aβ degradation and that impairment of its activity results in Aβ accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.
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http://dx.doi.org/10.15252/emmm.201505894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772954PMC
March 2016

Increased neuronal PreP activity reduces Aβ accumulation, attenuates neuroinflammation and improves mitochondrial and synaptic function in Alzheimer disease's mouse model.

Hum Mol Genet 2015 Sep 29;24(18):5198-210. Epub 2015 Jun 29.

Department of Pharmacology and Toxicology, Higuchi Bioscience Center, School of Pharmacy, University of Kansas, Lawrence, KS 66047, USA,

Accumulation of amyloid-β (Aβ) in synaptic mitochondria is associated with mitochondrial and synaptic injury. The underlying mechanisms and strategies to eliminate Aβ and rescue mitochondrial and synaptic defects remain elusive. Presequence protease (PreP), a mitochondrial peptidasome, is a novel mitochondrial Aβ 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 Aβ-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 Aβ 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.
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http://dx.doi.org/10.1093/hmg/ddv241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550821PMC
September 2015

Interaction of the dual targeting peptide of Thr-tRNA synthetase with the chloroplastic receptor Toc34 in Arabidopsis thaliana.

FEBS Open Bio 2015 30;5:405-12. Epub 2015 Apr 30.

Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden.

Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl-tRNA synthetase in Arabidopsis thaliana (AtThrRS-dTP) interacts with the mitochondrial receptor AtTom20 mainly through its N-terminal part. Here we report on the interaction of AtThrRS-dTP with the chloroplastic receptor AtToc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in (15)N HSQC spectra of AtThrRS-dTP as a function of AtToc34 concentration. Line broadening shows that the interaction with AtToc34 involves residues along the entire sequence, which is not the case for AtTom20. The N-terminal φχχφφ motif, which plays an important role in AtTom20 recognition, shows no specificity for AtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of AtThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of AtThrRS-dTP is essential for import into both organelles, while the C-terminal L30-P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide of AtThr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors.
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http://dx.doi.org/10.1016/j.fob.2015.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430637PMC
June 2015

A flowchart to analyze protease activity in plant mitochondria.

Methods Mol Biol 2015 ;1305:123-30

Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Svante Arrhenius väg 16C, Stockholm, 106 91, Sweden,

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.
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http://dx.doi.org/10.1007/978-1-4939-2639-8_8DOI Listing
January 2016

Protein import into plant mitochondria: signals, machinery, processing, and regulation.

J Exp Bot 2014 Dec 16;65(22):6301-35. Epub 2014 Oct 16.

Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Science, La Trobe University, Bundoora, Victoria, 3086, Australia

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.
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http://dx.doi.org/10.1093/jxb/eru399DOI Listing
December 2014

Shredding the signal: targeting peptide degradation in mitochondria and chloroplasts.

Trends Plant Sci 2014 Dec 7;19(12):771-8. Epub 2014 Oct 7.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, SE-106 91 Stockholm, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tplants.2014.09.004DOI Listing
December 2014

Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide.

Free Radic Biol Med 2014 Dec 16;77:57-63. Epub 2014 Sep 16.

Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA. Electronic address:

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-β 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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2014.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258540PMC
December 2014

Amyloid-β peptide induces mitochondrial dysfunction by inhibition of preprotein maturation.

Cell Metab 2014 Oct 28;20(4):662-9. Epub 2014 Aug 28.

Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, 79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany. Electronic address:

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 (Aβ). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aβ 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 Aβ peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease.
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http://dx.doi.org/10.1016/j.cmet.2014.07.024DOI Listing
October 2014

Mitochondrial import and degradation of amyloid-β peptide.

Biochim Biophys Acta 2014 Jul 18;1837(7):1069-74. Epub 2014 Feb 18.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, SE-106 91 Stockholm, Sweden. Electronic address:

Mitochondrial dysfunctions associated with amyloid-β peptide (Aβ) accumulation in mitochondria have been observed in Alzheimer's disease (AD) patients' brains and in AD mice models. Aβ is produced by sequential action of β- and γ-secretases cleaving the amyloid precursor protein (APP). The γ-secretase complex was found in mitochondria-associated endoplasmic reticulum membranes (MAM) suggesting that this could be a potential site of Aβ production, from which Aβ is further transported into the mitochondria. In vitro, Aβ 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 Aβ degradation reducing toxic effects of Aβ 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 Aβ production, its mitochondrial import and the intra-mitochondrial degradation. We also discuss the implications of a reduced efficiency of mitochondrial Aβ 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.
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http://dx.doi.org/10.1016/j.bbabio.2014.02.007DOI Listing
July 2014

Phenotypical consequences of expressing the dually targeted Presequence Protease, AtPreP, exclusively in mitochondria.

Biochimie 2014 May 25;100:167-70. Epub 2013 Dec 25.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, SE-106 91 Stockholm, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biochi.2013.12.012DOI Listing
May 2014

Import determinants of organelle-specific and dual targeting peptides of mitochondria and chloroplasts in Arabidopsis thaliana.

Mol Plant 2014 Jan 8;7(1):121-36. Epub 2013 Nov 8.

Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-106 91 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1093/mp/sst148DOI Listing
January 2014

Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts.

Proc Natl Acad Sci U S A 2013 Oct 16;110(40):E3761-9. Epub 2013 Sep 16.

Departments of Biochemistry and Biophysics and Neurochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.

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 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å(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.
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http://dx.doi.org/10.1073/pnas.1307637110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791733PMC
October 2013

Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models.

Proc Natl Acad Sci U S A 2013 May 25;110(19):7916-21. Epub 2013 Apr 25.

Department of Neurobiology, Care Sciences and Society, Karolinska Institutet-Alzheimer's Disease Research Center, Karolinska Institutet, 141 86 Stockholm, Sweden.

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 σ1 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 β-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.
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http://dx.doi.org/10.1073/pnas.1300677110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651455PMC
May 2013

In vitro oxidative inactivation of human presequence protease (hPreP).

Free Radic Biol Med 2012 Dec 3;53(11):2188-95. Epub 2012 Oct 3.

Arrhenius Laboratories for Natural Sciences, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.

The mitochondrial peptidasome called presequence protease (PreP) is responsible for the degradation of presequences and other unstructured peptides including the amyloid-β peptide, whose accumulation may have deleterious effects on mitochondrial function. Recent studies showed that PreP activity is reduced in Alzheimer disease (AD) patients and AD mouse models compared to controls, which correlated with an enhanced reactive oxygen species production in mitochondria. In this study, we have investigated the effects of a biologically relevant oxidant, hydrogen peroxide (H(2)O(2)), on the activity of recombinant human PreP (hPreP). H(2)O(2) inhibited hPreP activity in a concentration-dependent manner, resulting in oxidation of amino acid residues (detected by carbonylation) and lowered protein stability. Substitution of the evolutionarily conserved methionine 206 for leucine resulted in increased sensitivity of hPreP to oxidation, indicating a possible protective role of M206 as internal antioxidant. The activity of hPreP oxidized at low concentrations of H(2)O(2) could be restored by methionine sulfoxide reductase A (MsrA), an enzyme that localizes to the mitochondrial matrix, suggesting that hPreP constitutes a substrate for MsrA. In summary, our in vitro results suggest a possible redox control of hPreP in the mitochondrial matrix and support the protective role of the conserved methionine 206 residue as an internal antioxidant.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.09.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589710PMC
December 2012

NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana.

FEBS J 2012 Oct 31;279(19):3738-3748. Epub 2012 Aug 31.

Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, Sweden.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide.
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http://dx.doi.org/10.1111/j.1742-4658.2012.08735.xDOI Listing
October 2012

Biochemical studies of poly-T variants in the Alzheimer's disease associated TOMM40 gene.

J Alzheimers Dis 2012 ;31(3):527-36

KI-Alzheimer's Disease Research Center, Karolinska Institutet, Department of Neurobiology, Care Sciences and Society (NVS), Stockholm, Sweden.

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 ε3/ε4 carriers harboring very long or short poly-T variants coupled to their APOE ε3 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.
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http://dx.doi.org/10.3233/JAD-2012-120580DOI Listing
May 2013

Processing peptidases in mitochondria and chloroplasts.

Biochim Biophys Acta 2013 Feb 1;1833(2):360-70. Epub 2012 Apr 1.

Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, SE-106 91 Stockholm, Sweden.

Most of the mitochondrial and chloroplastic proteins are nuclear encoded and synthesized in the cytosol as precursor proteins with N-terminal extensions called targeting peptides. Targeting peptides function as organellar import signals, they are recognized by the import receptors and route precursors through the protein translocons across the organellar membranes. After the fulfilled function, targeting peptides are proteolytically cleaved off inside the organelles by different processing peptidases. The processing of mitochondrial precursors is catalyzed in the matrix by the Mitochondrial Processing Peptidase, MPP, the Mitochondrial Intermediate Peptidase, MIP (recently called Octapeptidyl aminopeptidase 1, Oct1) and the Intermediate cleaving peptidase of 55kDa, Icp55. Furthermore, different inner membrane peptidases (Inner Membrane Proteases, IMPs, Atp23, rhomboids and AAA proteases) catalyze additional processing functions, resulting in intra-mitochondrial sorting of proteins, the targeting to the intermembrane space or in the assembly of proteins into inner membrane complexes. Chloroplast targeting peptides are cleaved off in the stroma by the Stromal Processing Peptidase, SPP. If the protein is further translocated to the thylakoid lumen, an additional thylakoid-transfer sequence is removed by the Thylakoidal Processing Peptidase, TPP. Proper function of the D1 protein of Photosystem II reaction center requires its C-terminal processing by Carboxy-terminal processing protease, CtpA. Both in mitochondria and in chloroplasts, the cleaved targeting peptides are finally degraded by the Presequence Protease, PreP. The organellar proteases involved in precursor processing and targeting peptide degradation constitute themselves a quality control system ensuring the correct maturation and localization of proteins as well as assembly of protein complexes, contributing to sustenance of organelle functions. Dysfunctions of several mitochondrial processing proteases have been shown to be associated with human diseases. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.
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http://dx.doi.org/10.1016/j.bbamcr.2012.03.012DOI Listing
February 2013

A novel mitochondrial and chloroplast peptidasome, PreP.

Physiol Plant 2012 May 21;145(1):180-6. Epub 2011 Nov 21.

Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Science, Stockholm University, SE-10691 Stockholm, Sweden.

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 Å, 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-β peptide in brain mitochondria.
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http://dx.doi.org/10.1111/j.1399-3054.2011.01531.xDOI Listing
May 2012

Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria.

J Alzheimers Dis 2011 ;27(1):75-87

Department of Surgery, College of Physicians and Surgeons of Columbia University, New York, NY, USA.

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.
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http://dx.doi.org/10.3233/JAD-2011-101716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381900PMC
February 2012

Targeting capacity and conservation of PreP homologues localization in mitochondria of different species.

J Mol Biol 2011 Jul 23;410(3):400-10. Epub 2011 May 23.

Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Science, Stockholm University, SE-10691 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.jmb.2011.05.009DOI Listing
July 2011

The organellar peptidasome, PreP: a journey from Arabidopsis to Alzheimer's disease.

Biochim Biophys Acta 2010 Jun-Jul;1797(6-7):1076-80. Epub 2009 Dec 28.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.bbabio.2009.12.016DOI Listing
January 2011