Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.

No abstract

Introduction: Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9 Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics. | 803 DIMOVASILI et AL.

Background: Apolipoprotein E gene (APOE) ɛ4 allele increases the risk for Alzheimer’s disease (AD). Furthermore, among patients with cognitive impairment, longer sleep duration is associated with worse cognitive performance. To date, literature examining the associations between APOE ɛ4 allele and objective sleep duration is limited. Objective: Our aim was to assess the association between APOE ɛ4 and objective sleep duration, among patients with mild cognitive impairment (MCI) and AD. A sub-sample of 89 patients with AD (n = 49) and MCI (n = 40) were recruited from a large, population-based cohort of 3,140 elders (>60 years) residing on Crete, Greece. Methods: All participants underwent medical history/physical examination, extensive neuropsychiatric and neuropsychological evaluation, 3-day 24 h actigraphy and APOE ɛ4 allele genotyping. Comparisons of sleep duration variables between APOE ɛ4 allele carriers and non-carriers were assessed using ANCOVA, controlling for confounders. Results: The sample included 18 APOE ɛ4 carriers and 71 non-carriers, aged 78.6±6.6 and 78.2±6.5 years, respectively. Comparisons between the APOE ɛ4 carriers and non-carriers revealed no significant differences in terms of demographic and clinical variables. In terms of objective sleep duration across the two groups, APOE ɛ4 carriers compared to non-carriers had significantly longer nighttime Total Sleep Time (nTST) (7.7±1.4 versus 7.2±1.3  h, respectively, p = 0.011), as well as 24 h TST (8.5±1.6 versus 7.8±1.5  h, respectively, p = 0.012). Conclusion: Among patients with MCI and AD, APOE ɛ4 carriers have longer objective nighttime and 24 h sleep duration compared to non-carriers. These findings further support that objective long sleep duration is a genetically-driven pre-clinical marker associated with worse prognosis in elderly with cognitive impairment.