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Functional and structural studies of the Presequence protease, PreP
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Elzbieta Glaser)
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

AtPreP (Arabidopsis thaliana Presequence Protease) is a zink metallooligopeptidase that is dually targeted to both mitochondria and chloroplasts. In these organelles it functions as a peptidasome that degrades the N-terminal targeting peptides that are cleaved off from the mature protein after protein import, as well as other unstructured peptides. In A. thaliana there are two isoforms of PreP, AtPreP1 and AtPreP2. 

We have performed characterization studies of single and double prep knockout plants. Immunoblot analysis revealed that both PreP isoforms are expressed in all tissues with highest expression levels in flowers and siliques. Furthermore, AtPreP1 was shown to be the most abundant isoform of the two. When comparing phenotype, the atprep2 mutant was similar to wild type, whereas the atprep1 mutant had a slight pale-green phenotype in the early developmental stages. The atprep1 atprep2 double knockout plants showed a chlorotic phenotype in true leaves, especially prominent during the early developmental stages. When analysing the first true leaves of double knockout plants, we found a significant decrease in chlorophyll a and b content. Mitochondrial respiratory rates measurements showed partially uncoupled mitochondria. Ultrastructure analysis using electron microscopy on double knockout plants showed aberrant chloroplasts with altered grana stacking and clearly fewer starch granules. Older plants showed less altered  phenotype, although there was a significant decrease in the accumulated biomass of about 40% compared to wild type. Peptidolytic activity studies showed no sign of compensatory mechanisms in the absence of AtPreP in mitochondria; in contrast we found a peptidolytic activity in the chloroplast membranes not related to AtPreP.

In addition to zinc located in the catalytic site, crystallographic data revealed two Mg-binding sites in the AtPreP structure. To further investigate the role of these Mg-binding sites, we have made AtPreP variants that are unable to bind metal ions. Our data shows that one of these sites located close to the catalytic site is important for the activity of AtPreP.

We also measured proteolytic activity of four human PreP-SNP variants and observed that the activity of all the hPreP-SNPs variants was lower; especially the hPreP-SNP (A525D) variant that displayed only 20-30 % of wild type activity. Interestingly, the activity was fully restored for all SNP-variants by addition of Mg2+

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2014. , 37 p.
Keyword [en]
Presequence Protease, PreP, AtPreP, hPreP
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-102310OAI: oai:DiVA.org:su-102310DiVA: diva2:709100
Presentation
2014-04-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrheniusväg 16 B, Stockholm, 15:00 (English)
Opponent
Supervisors
Available from: 2014-04-01 Created: 2014-03-31 Last updated: 2014-04-01Bibliographically approved
List of papers
1. Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana
Open this publication in new window or tab >>Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana
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2009 (English)In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 71, no 4-5, 497-508 p.Article in journal (Refereed) Published
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.

Keyword
PreP, Protease, Knockout, Targeting peptide, Mitochondria, Chloroplast
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-24762 (URN)10.1007/s11103-009-9534-6 (DOI)
Available from: 2008-03-19 Created: 2008-03-19 Last updated: 2014-04-01Bibliographically approved
2. Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP.
Open this publication in new window or tab >>Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP.
2009 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 583, no 17, 2727-33 p.Article in journal (Refereed) Published
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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:su:diva-34682 (URN)10.1016/j.febslet.2009.07.040 (DOI)000270149800005 ()19646442 (PubMedID)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2017-12-12Bibliographically approved
3. Genetic and biochemical studies of SNPs of the mitochondrial A beta-degrading protease, hPreP
Open this publication in new window or tab >>Genetic and biochemical studies of SNPs of the mitochondrial A beta-degrading protease, hPreP
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2010 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 469, no 2, 204-208 p.Article in journal (Refereed) Published
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.

Keyword
Amyloid-beta peptide, Alzheimer disease, SNP, Mitochondria, Proteolysis, PreP
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-50536 (URN)10.1016/j.neulet.2009.11.075 (DOI)000274508700006 ()
Note

authorCount :8

Available from: 2010-12-28 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved

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