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Genetic and biochemical studies of SNPs of the mitochondrial A beta-degrading protease, hPreP
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2010. Vol. 469, no 2, 204-208 p.
Keyword [en]
Amyloid-beta peptide, Alzheimer disease, SNP, Mitochondria, Proteolysis, PreP
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-50536DOI: 10.1016/j.neulet.2009.11.075ISI: 000274508700006OAI: oai:DiVA.org:su-50536DiVA: diva2:381604
Note

authorCount :8

Available from: 2010-12-28 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Mitochondria in Alzheimer's Disease: The Presequence Protease and Mitochondria-Associated ER Membranes
Open this publication in new window or tab >>Mitochondria in Alzheimer's Disease: The Presequence Protease and Mitochondria-Associated ER Membranes
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease (AD) is one of the most prevalent age-related neurodegenerative disorders and the accumulation of the amyloid-β peptide (Aβ) in the temporal lobe has been implicated in the pathology of AD. Synaptic transmission in neuronal cells is a highly energy dependent process, which relies on the presence and proper function of mitochondria. A growing number of studies have analyzed the roles of mitochondria in AD. Interestingly, Aβ accumulation in mitochondria was detected in AD patient brains and in AD mouse models, which was associated with the formation of reactive oxygen species (ROS) and neuronal death. In mitochondria, the only protease capable of clearing Aβ is the Presequence Protease, PreP.

The aim of this thesis was to study the involvement of mitochondria and hPreP in AD. We investigated how the mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), which are involved in the regulation of Ca2+ signaling, phospholipids synthesis and apoptosis, are affected in AD. We observed MAM at synapses and found that these structures are essential for neuronal and astrocytic survival. We detected altered MAM protein levels in AD patient brains and in AD mouse models in early stages of the disease and found that MAM can be functionally modulated by Aβ. We analyzed human PreP (hPreP) activity in brain extracts from AD patients and different factors that can affect hPreP function. Interestingly, we detected low hPreP activity in AD patient brains and in AD mouse models, which were associated with increased ROS levels and lower cytochrome c oxidase activity. This suggested that protein oxidation could contribute to impaired activity. Furthermore, we investigated a potential correlation between 18 single nucleotide polymorphisms (SNPs) in the PITRM1 gene, encoding hPreP, and the risk for developing AD. Even though we could not find any genetic correlation in the Swedish population examined, biochemical analysis of four non-synonymous hPreP-SNPs, selected on the basis of their location in hPreP structure, showed lower hPreP activity. Furthermore, we demonstrated in vivo and in vitro that the hPreP presequence is processed at amino acid 28 by mitochondrial processing peptidase (MPP) and that inefficient processing does not affect the enzymatic activity of hPreP but it decreases the stability of the protein.

Together, these results indicate that MAM dysfunctions, inefficient Aβ clearance in mitochondria by hPreP, hPreP mutations or inefficient processing, may contribute to the development of AD.  

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. 79 p.
Keyword
Alzheimer's Disease, Mitochondria, Mitochondria-associated ER membranes, human Presequence Protease
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-93285 (URN)978-91-7447-733-7 (ISBN)
Public defence
2013-10-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2013-09-19 Created: 2013-09-06 Last updated: 2013-09-11Bibliographically approved
2. Functional and structural studies of the Presequence protease, PreP
Open this publication in new window or tab >>Functional and structural studies of the Presequence protease, PreP
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
Presequence Protease, PreP, AtPreP, hPreP
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-102310 (URN)
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

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