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Identifier 000403389
Title Functional structural and genetic studies on human glutamate dehydrogenases
Alternative Title Λειτουργικές δομικές και γενετικές μελέτες των ανθρώπινων γλουταμικών αφυδρογονασών
Author Δημοβασίλη, Χριστίνα
Thesis advisor Ζαγανάς, Ιωάννης
Κοκκινίδης, Μιχάλης
Τοκατλίδης, Κωνσταντίνος
Reviewer Πλαϊτάκης, Α.
Θερμού, Κ.
Καρδάσης, Δ.
Σπανάκη, Κ.
Abstract Background: Glutamate dehydrogenase (GDH) is an abundant mitochondrial enzyme which catalyzes the interconversion of glutamate to α-ketoglutarate. Humans possess two functional genes which encode for GDH, the ubiquitously expressed GLUD1 (coding for hGDH1) and the tissue specific GLUD2 (coding for hGDH2). The two isoenzymes, although sharing all but 15 out of their 505 amino acids in their mature form, differ significantly in their regulation properties. hGDH2 is mainly detected on brain, kidney and testis, where it is found to co-localize with hGDH1 in the mitochondrial matrix. Both iso-enzymes operate by forming homo-hexamers, but it is currently unknown if they form hetero-hexamers at the areas where they co-localize. Besides their physiological role in metabolism, genetic alterations in the GLUD1 and GLUD2 genes have been associated with pathophysiological conditions. Specifically, mutations of the GLUD1 gene lead to the hyperinsulinism / hyperammonemia syndrome, whereas a polymorphism in GLUD2 has been associated with accelerated Parkinson’s disease age of onset. Furthermore, there is evidence that hGDH plays a role in Alzheimer’s disease associated neurodegeneration (as its overexpression leads to age associated degenerative changes in the mouse hippocampus), even though proof for this in human patients suffering from dementia is lacking. Aims: Aim of this study is to further analyze hGDH1 and hGDH2 properties by employing functional, structural as well as genetic methods. Specifically, we investigated whether hGDH1 and hGDH2 are capable of forming hetero-hexamers when they are co-expressed, and what particular properties these complexes may have. Secondly, we aimed to obtain the so far unsolved crystal structure of hGDH2 which will enable us to perform a valid and accurate comparison with hGDH1 (the structure of which is already known). Lastly, we analyzed the genetic variations’ spectrum of GLUD1 and GLUD2 genes in the well characterized Cretan Aging Cohort of 201 subjects, comprising of patients suffering from dementia and mild cognitive impairment (MCI), as well as of cognitive normal controls. Methods: To accomplish the first part of our aims, we created hetero-hexameric hGDH complexes in Sf21 cells, using the Baculovirus expression system. We employed coimmunoprecipitation and affinity chromatography to discriminate between in vitro mixtures of separately expressed hGDHs and in vivo 1:1 co-expressed hGDHs. Then we performed a series of kinetic studies to characterize the possible hetero-hexamers’ properties. For the second part, we produced hGDH2 in large-scale Sf21 cultures and we purified it to homogeneity, before crystallization by repeated trials using different methods and reagents. For the genetic studies, we obtained Whole Exome Sequencing data from 201 Cretan elderly individuals, from which 100 suffered from dementia, 20 were classified as MCI and 81 were cognitive normal controls. After characterizing their dementia related genetic background, we focused on GLUD1 and GLUD2 genes in respect to their variations. Results: In the present study we show that hGDHs can form, apart from homohexamers, hetero-hexameric complexes, when they are co-expressed. These heterohexamers possess unique enzymatic properties, which under specific conditions (absence of ADP, L-leucine activation, heat stability) are intermediate between the properties of pure hGDH1 and hGDH2 (and thus comparable to the 1:1 in vitro mixture of the two homo-hexamers), whereas they tend to resemble hGDH2’s behavior in respect with GTP and DES inhibition. In the second part of the study, we managed for the first time to produce high-quality hGDH2 crystals of 3.2Å resolution for structure characterization. In the third part of the study (genetic analysis of GLUD1 and GLUD2 genes), we found the Ser498Ala GLUD2 variant to be more common in controls (16.05%, n=81) than in patients with dementia (3.0%, n=100; p=0.003), and these results were verified by including in our analyses an additional local cohort. Conclusion: Our study raises the possibility that hGDH1 and hGDH2 form heterohexamers in the cells where they are co-expressed. As the functional properties of these complexes are distinct from those of homo-hexamers, this association might provide versatility to human cells by equipping them with more than two iso-enzymes. Structural studies on hGDH2 are expected, not only to explore its particularities, but also to decipher the structural basis of its enzymatic differences from its highly homologous hGDH1 iso-enzyme. Finally, in the culturally and genetically homogeneous cohort of aged adults of the island of Crete, we found that the Ser498Ala GLUD2 variant was associated with lower risk for dementia in our population.
Language English
Subject Ctystallization
Hetero-hexamers
Whole exome sequencing
Αλληλούχιση εξώματος
Ετερο-εξαμερή
Κρυστάλλωση
Issue date 2016-12-13
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/e/1/1/metadata-dlib-1482481239-113953-11580.tkl Bookmark and Share
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