| Abstract |
Mitochondria are essential organelles of most eukaryotic cells and their biogenesis relies on mitochondrial protein import pathways. Glutamate dehydrogenase (GDH), an enzyme that plays a central role in cellular metabolism, is present in almost all living organisms. In mammals, GDH makes up, in some instances, more than 10% of the mitochondrial matrix proteins. In human and in great apes it exists in two isoforms, hGDH1 and hGDH2, which both have an unusually large mitochondrial targeting signal (MTS) consisting of 53 amino acid residues (N53). Their N53 peptide has the tendency to form two amphipathic α-helices (α1, α2). In this PhD thesis, the N53 peptides of hGDHs were characterized. Moreover, the evolution of the GDH MTS among organisms was studied.
In the first section we have found that the in vitro synthesized hGDHs can be efficiently imported, proteolytically processed into isolated yeast mitochondria and they form hexamers. The mitochondrial import of hGDHs process depends on the TIM23 complex, on the mitochondrial inner membrane potential and on the concentration of divalent metal ions. Although the N53 peptides of hGDHs share 83% homology, we observed that the isoform hGDH2 is imported or/and processed faster in mitochondria than hGDH1. We observed that the mutation G35R in hGDH2, that arises from the common polymorphism GLUD2(c.G103A) (Plaitakis et al, 2010), does not affect its mitochondrial targeting capacity. Furthermore, in order to make a construct that can be used to search new molecular protein partners of hGDH1, we found that the addition of the epitope His at the C-terminus of hGDH1 (hGDH1-His) does not interrupt either the mitochondrial targeting efficiency or hexameric formation.
We studied the N53 peptide in portions and separate from the rest hGDH, in order to understand its properties. Import experiments in already isolated mitochondria showed that the N53 peptides of hGDHs are essential for mitochondrial localization of hGDHs and they are able to target the non-mitochondrial protein DHFR into mitochondria. Additionally, deletion of the α1 helix of hGDH2 abolishes its mitochondrial import. The α1 helix of hGDH2 is sufficient to target the DHFR protein into mitochondria, when it is fused N-terminally (α1-DHFR) but not, when it is fused C-terminally (DHFR-α1). However, the α2 does not seem to have an autonomous mitochondrial targeting capacity. We found also that the fused peptide α1α2, but not the α1 alone, is able to target the mature hGDH2 protein into mitochondria. Our findings are in agreement with experiments in mammalian cell lines with the epitope EGFP from our collaborators at the Neurology Laboratory of the University of Crete. Therefore, we found that the mitochondrial targeting of hGDH2 relies on the synergistic effect of the two α-helical structures, with the first one having the leading role.
It is generally believed that the MTS do not have a common motif. However, the MTS are rich in positively charged amino acids and are characterized by amphipathicity and tendency for α-helix formation. Our data suggest that the net positive charge of the N-terminal part of the N53 of hGDH2 rather than the net positive charge of the C-terminal part of the N53 or the amphipathicity and propensity for α-helix formation is the main determinant for their mitochondrial import. Moreover, the proteolytic removal of the N53 in the mitochondrial matrix is not essential for the import of hGDHs in mitochondria.
In the second section, we have studied the evolution of the MTS of GDH among organisms using in silico prediction programs for ~170 distinct GDH proteins. This study suggested that the GDH cleavable MTS first arose in the kingdom of ciliophora protista and then evolved step by step into highly efficient, substantially complex and more positive charged MTS in mammals, with potential exceptions the GDH in birds and reptiles. Moreover, the plants are predicted to have GDH with non-cleaved MTS and we failed to predict MTS in the GDH in fungi. In addition, our analyses showed that in mammals, the α1 helix is evolutionary more conserved than the α2 helix
Experimentally, in vivo and in organello studies carried out in collaboration with the Neurology Laboratory showed that the MTS of GDH from T. thermophila (ciliate), C. elegans (roundworm), D. melanogaster (fly) and X. laevis (frog) alone are able to target DHFR and EGFP into mitochondria. However, the MTS of GDH from T. thermophila seems not to be sufficient to target efficiently the mature hGDH2 into mitochondria.
The findings of the present study are an important step towards revealing the full molecular details underlying the functional characteristics and the evolution of the mitochondrial targeting signals for mitochondrial biogenesis.
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Μιτοχονδριακή στόχευση της ανθρώπινης γλουταμικής αφυδρογονάσης : δομή σινιάλου στόχευσης, λειτουργία και εξέλιξη
