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Identifier

000347786

Title

Μελέτη δομής και λειτουργίας γλουταμικής αφυδρογονάσης ειδικής για το νευρικό σύστημα

Author

Μαστοροδήμος, Βασίλειος Χρήστου

Thesis advisor

Πλαϊτάκης, Ανδρέας

Reviewer

Θερμού, Κυριακή
Καραγωγέως, Δόμνα

Abstract

Glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is located in the mitochondria. In addition, there is evidence for extramitochondrial localization of GDH. Human GDH exists in housekeeping (hGDH1) and nerve tissue-specific (hGDH2) isoenzymes encoded by the GLUD1 and the GLUD2 genes respectively, which differ markedly in their basal activity, allosteric regulation and thermal stability. hGDH2 is thermolabile, shows low basal activity that is fully restored by ADP albeit at higher concentrations than hGDH1, is more sensitive to L-leucine activation and to the synergistic effect of ADP and L-leucine and resistant to GTP inhibition. Site directed mutagenesis of GLUD1 revealed that replacement of Gly456 by Ala made the enzyme resistant to GTP without altering its regulation by ADP. In addition substitution of Ser for Arg443 (which lies in the antenna region of GDH) virtually abolished basal activity, made the enzyme extremely sensitive to heat inactivation and totally abrogated the activation of the enzyme by L-Leucine. However the presence of low concentrations of ADP (0.025- 0.1 mM) permitted the activation of the mutant by L-leucine. Structural modeling implicated that the replacement of Arg443 by Ser may disrupt the H-bond(s) between Arg443 and Ser409 (part of the ascending strand of the antenna) from an adjacent subunit and thus may result in closure of the active catalytic cleft. Substitution of the Ser409 for Arg rendered the enzyme virtually inactive at baseline and resistant to Lleucine activation. ADP fully restored the activity of mutant and made it sensitive to L-leucine activation. On the other hand, replacement of Ser409 by Asp had a lesser effect on basal activity and to L-leucine activation than Arg substitution. Hence, these data confirm that interaction of adjacent subunits through the Arg443 and Ser409 residues plays a crucial role in setting the basal activity levels. Abrogation of Lleucine activation by R443S, S409R or S409D mutants is consistent with the concept that these mutations lead to a closed conformation, which hinders access of L-leucine to the catalytic site for it action. Since the S409R hGDH1 mutant was insensitive to heat denaturation, the interaction between the two residues does not seem to be responsible for heat sensitivity. In addition both S409R and S409D hGDH1 mutants showed a differential response to GTP inhibition, with the former being more sensitive and the latter more resistant to GTP in comparison to the wt hGDH1. Moreover, we created a double hGDH1 mutant that had both Arg443Ser and Gly456Ala in the same polypeptide chain. Functional analyses revealed that the doubly mutated enzyme had similar properties but did not acquire all the characteristics of the wild-type hGDH2. Subcellular localization studies were performed using expression vectors for hGDH1 and hGDH2 fused with the enhanced green fluorescence protein (EGFP) that transiently transfected COS 7, HeLa, CHO, HEK 293 and neuroblastoma cell lines. When the cultured cells were transfected with EGFP-tagged hGDH1 or hGDH2, confocal microscopy demonstrated localization of the fluorescence in the cytoplasmic region within coarse structures resembling mitochondria. Co-transfection experiments using a mixture of hGDH1-EGFP (or hGDH2-EGFP) vector and the pDsRed2-Mito vector (a mitochondrial marker) showed an identical fluorescence pattern in merged pictures, thus confirming the mitochondrial localization of both human GDHs. In addition, a small fraction of the hGDH1 (or hGDH2) was found to co-localize with endoplasmic reticular marker pDsRed2-ER. There was no evidence for the nuclear or cytoplasmic localization of either GDH isoforms. Our results elucidate further the properties that allow the brain isoenzyme to function well under the special conditions prevailing in the human Central Nervous System.

Physical description

214 σ. : πιν. ; 30 εκ.

Language

Greek

Subject

Glutamate Dehydrogenase

Nervous System

Δεϋδρογονάση γλουταμικού

Νευρικό σύστημα

Issue date

2007-12-14

Collection