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Presequence processing increases the stability of the human Presequence 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.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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(English)Manuscript (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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-93283OAI: oai:DiVA.org:su-93283DiVA: diva2:646097
Available from: 2013-09-06 Created: 2013-09-06 Last updated: 2013-09-10Bibliographically 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)
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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

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