Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import

Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17-35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary mechanisms that led to the large mitochondrial targeting signals of human GDHs.     

The human GLUD2 glutamate dehydrogenase and its regulation in health and disease

Whereas glutamate dehydrogenase in most mammals (hGDH1 in the human) is encoded by a single functional GLUD1 gene expressed widely, humans and other primates have acquired through retroposition an X-linked GLUD2 gene that encodes a highly homologous isoenzyme (hGDH2) expressed in testis and brain. Using an antibody specific for hGDH2, we showed that hGDH2 is expressed in testicular Sertoli cells and in cerebral cortical astrocytes. Although hGDH1 and hGDH2 have similar catalytic properties, they differ markedly in their regulatory profile. While hGDH1 is potently inhibited by GTP and may be controlled by the need of the cell for ATP, hGDH2 has dissociated its function from GTP and may metabolize glutamate even when the Krebs cycle generates GTP amounts sufficient to inactivate hGDH1. As astrocytes are known to provide neurons with lactate that largely derives from the Krebs cycle via conversion of glutamate to α-ketoglutarate, the selective expression of hGDH2 may facilitate metabolic recycling processes essential for glutamatergic transmission. As there is evidence for deregulation of glutamate metabolism in degenerative neurologic disorders, we sequenced GLUD1 and GLUD2 genes in neurologic patients and found that a rare T1492G variation in GLUD2 that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 interacted significantly with Parkinson's disease (PD) onset. Thus, in two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6-13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant shows enhanced catalytic activity that is resistant to modulation by GTP, but sensitive to inhibition by estrogens. These observations are thought to suggest that enhanced glutamate oxidation by the Ala445-hGDH2 variant accelerates nigral cell degeneration in hemizygous males and that inhibition of the overactive enzyme by estrogens protects heterozygous females. We then evaluated the interaction of estrogens and neuroleptic agents (haloperidol and perphenazine) with the wild-type hGDH1 and hGDH2 and found that both inhibited hGDH2 more potently than hGDH1 and that the evolutionary Arg443Ser substitution was largely responsible for this sensitivity. Hence, the properties acquired by hGDH2 during its evolution have made the enzyme a selective target for neuroactive steroids and drugs, providing new means for therapeutic interventions in disorders linked to deregulation of this enzyme.     

Mutations in human GLUD2 glutamate dehydrogenase affecting basal activity and regulation

Glutamate dehydrogenase (GDH) in human exists in GLUD1 and GLUD2 gene-encoded isoforms (hGDH1 and hGDH2, respectively), differing in their regulation and tissue expression pattern. Whereas hGDH1 is subject to GTP control, hGDH2 uses for its regulation, a novel molecular mechanism not requiring GTP. This is based on the ability of hGDH2 to maintain a baseline activity of <10% of its capacity subject to full activation by rising ADP/ l -leucine levels. Here we studied further the molecular mechanisms regulating hGDH2 function by creating and analyzing hGDH2 mutants harboring single amino acid substitutions in the regulatory domain (antenna, pivot helix) of the protein. Five hGDH2 mutants were obtained: two with an amino acid change (Gln441Arg, Ser445Leu) in the antenna, two (Lys450Glu, His454Tyr) in the pivot helix, and one (Ser448Pro) in the junction between the two structures. Functional analyses revealed that, while the antenna mutations increased basal enzyme activity without affecting its allosteric properties, the pivot helix mutations drastically reduced basal activity and impaired enzyme regulation. On the other hand, the Ser448Pro mutation reduced basal activity but did not alter allosteric regulation. Also, compared with wild-type hGDH2, the antenna mutants were relatively thermostable, whereas the pivot helix mutants were extremely heat labile. Hence, the present data further our understanding of the molecular mechanisms involved in the function and stability of hGDH2, an enzyme thought to be of importance for nerve tissue biology.     

"Prion-proof" for [PIN+]: infection with in vitro-made amyloid aggregates of Rnq1p-(132-405) induces [PIN+

Prions are self-propagating, infectious protein conformations. The mammalian prion, PrP(Sc), responsible for neurodegenerative diseases like bovine spongiform encephalopathy (BSE; "mad cow" disease) and Creutzfeldt-Jakob's disease, appears to be a beta-sheet-rich amyloid conformation of PrP(c) that converts PrP(c) into PrP(Sc). However, an unequivocal demonstration of "protein-only" infection by PrP(Sc) is still lacking. So far, protein only infection has been proven for three prions, [PSI(+)], [URE3] and [Het-s], all of fungal origin. Considerable evidence supports the hypothesis that another protein, the yeast Rnq1p, can form a prion, [PIN(+)]. While Rnq1p does not lose any known function upon prionization, [PIN(+)] has interesting positive phenotypes: facilitating the appearance and destabilization of other prions as well as the aggregation of polyglutamine extensions of the Huntingtin protein. Here, we polymerize a Gln/Asn-rich recombinant fragment of Rnq1p into beta-sheet-rich amyloid-like aggregates. While the method used for [PSI(+)] and [URE3] infectivity assays did not yield protein-only infection for the Rnq1p aggregates, we did successfully obtain protein-only infection by modifying the protocol. This work proves that [PIN(+)] is a prion mediated by amyloid-like aggregates of Rnq1p, and supports the hypothesis that heterologous prions affect each other's appearance and propagation through interaction of their amyloid-like regions.     

Activation of Cdc2 contributes to apoptosis in HPV E6 expressing human keratinocytes in response to therapeutic agents

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 oncogene induces apoptosis in cervical cancer precursor lesions but the mechanism is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in apoptosis, E6 also sensitizes cells to apoptosis under some experimental conditions. Here, we demonstrate that expression of E6 in human keratinocytes rendered sensitization to chemotherapeutic agents. The cell death was shown to be by apoptosis involving caspase activation and the mitochondria pathway. To explore mechanisms involved in sensitization of E6 expressing cells to apoptosis, we used a proteomic approach to identify proteins differentially expressed in E6 expressing and control keratinocytes. Among nearly a thousand proteins examined, Cdc2 was demonstrated to be the most dramatically up-regulated protein in E6 expressing cells. p53 degradation appears to be important for the up-regulation of Cdc2 by E6. Using genetic, pharmacologic, and siRNA strategies, a role for Cdc2 in E6 expression-conferred apoptosis was demonstrated. Thus, these results have important therapeutic implications in enhancing the efficacy of chemotherapy.     

Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange

Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.     

Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor,OdhI, involved in glutamate production in Corynebacterium glutamicum

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.     

Development of a novel beta-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures

Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med. 343 (2000) 230-238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407-415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259-266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A, 97 (2000) 7999-8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691-1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. In order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel beta-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3x. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3x construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and beta-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3x shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel beta-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting beta-like insulin-producing cells that have differentiated from stem cells or other progenitor cells.     

Orientation of Small Multidrug Resistance Transporter Subunits in the Membrane: Correlation with the Positive-Inside Rule

Small multidrug resistance (SMR) transport proteins provide a model for the evolution of larger two-domain transport proteins. The orientation in the membrane of 27 proteins from the SMR family was determined using the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology). Eighteen members were encoded in pairs on the chromosome and shown to insert in fixed orientations; the two proteins in each pair invariably had opposite orientations in the membrane. Interaction between the two proteins in pairs was demonstrated by copurification. The orientation in the membrane of either protein in the pair was affected only marginally by the presence of the other protein.For the proteins in pairs, the orientation in the membrane correlated well with the distribution of positively charges residues (R + K) over the cytoplasmic and extracellular loops (positive-inside rule). In contrast, dual-topology insertion of the singles was predicted less well by the positive-inside rule. Three singles were predicted to insert in a single orientation with the N-terminus and the C-terminus at the extracellular side of the membrane. Analysis of charge distributions suggests the requirement of a threshold number of charges in the cytoplasmic loops for the positive-inside rule to be of predictive value. It is concluded that a combined analysis of gene organization on the chromosome and phylogeny is sufficient to distinguish between fixed or dual topology of SMR members and, probably, similar types of membrane proteins. The positive-inside rule can be used to predict the orientation of members in pairs, but is not suitable as a sole predictor of dual topology.     

TAT-Hsp40 inhibits oxidative stress-mediated cytotoxicity via the inhibition of Hsp70 ubiquitination

Heat shock protein 40 (Hsp40) functions as a co-chaperone of mammalian Heat shock protein 70 (Hsp70) and facilitates the ATPase activity of Hsp70, and also promotes the cellular protein folding and renaturation of misfolded proteins. In an effort to assess the effects of Hsp40, we generated TAT-fused Hsp40 (TAT-Hsp40). The cells were transduced with TAT-Hsp40 and exposed to H(2)O(2). We demonstrated that the TAT-Hsp40-transduced cells were more resistant to cellular cytotoxicity and cell death. In particular, the degradation of Hsp70 was significantly reduced in TAT-Hsp40-containing cells as a consequence of reduced ubiquitin-proteasome activity after oxidative injury. These data support the notion that Hsp40 may confer resistance to oxidative stress via the prevention of proteasome activity.     

Enumeration of oligomerization states of membrane proteins in living cells by homo-FRET spectroscopy and microscopy: theory and application

Protein-protein interactions play a pivotal role in biological signaling networks. It is highly desirable to perform experiments that can directly assess the oligomerization state and degree of oligomerization of biological macromolecules in their native environment. Homo-FRET depends on the inverse sixth power of separation between interacting like fluorophores on the nanometer scale and is therefore sensitive to protein oligomerization. Homo-FRET is normally detected by steady-state or time-resolved fluorescence anisotropy measurements. Here we show by theory and simulation that an examination of the extent of homotransfer as measured by steady-state fluorescence anisotropy as a function of fluorophore labeling (or photodepletion) gives valuable information on the oligomerization state of self-associating proteins. We examine random distributions of monomers, dilute solutions of oligomers, and concentrated solutions of oligomers. The theory is applied to literature data on band 3 protein dimers in membranes, GPI-linked protein trimers in "rafts," and clustered GFP-tagged epidermal growth factor receptors in cell membranes to illustrate the general utility and applicability of our analytical approach.     

Functional implications of the conformational switch in AICD peptide upon binding to Grb2-SH2 domain

It has been hypothesized previously that synergistic effect of both amyloid precursor protein intracellular C-terminal domain (AICD) and Aβ aggregation could contribute to Alzheimer's disease pathogenesis. Structural studies of AICD have found no stable globular fold over a broad range of pH. Present work is based on the premises that a conformational switch involving the flipping of C-terminal helix of AICD would be essential for effective binding with the Src homology 2 (SH2) domain of growth factor receptor binding protein-2 (Grb2) and subsequent initiation of Grb2-mediated endo-lysosomal pathway. High-resolution crystal structures of Grb2-SH2 domain bound to AICD peptides reveal a unique mode of binding where the peptides assume a noncanonical conformation that is unlike other structures of AICD peptides bound to protein-tyrosine-binding domains or that of its free state; rather, a flipping of the C-terminal helix of AICD is evident. The involvement of different AICD residues in Grb2-SH2 interaction is further elucidated through fluorescence-based assays. Our results reveal the significance of a specific interaction of the two molecules to optimize the rapid transport of AICD inside endosomal vesicles presumably to reduce the cytotoxic load.     

Antiangiogenic,antimigratory and antiinflammatory effects of 2-methoxyestradiol in zebrafish larvae

2-Methoxyestradiol (2ME), an endogenous metabolite of 17β-estradiol, has been previously reported to possess antiangiogenic and antitumor properties. Herein, we demonstrate that the effects of this antiangiogenic steroid can be readily assayed in live zebrafish, introducing a convenient and robust new model system as a screening tool for both single cell and collective cell migration assays. Using the in vitro mammalian endothelial cell line EA.hy926, we first show that cell migration and angiogenesis, as estimated by wound assay and tube formation respectively, are antagonized by 2ME. In zebrafish (Danio rerio) larvae, dose-dependent exposure to 2ME diminishes (1) larval angiogenesis, (2) leukocyte recruitment to damaged lateral line neuromasts and (3) retards the lateral line primordium in its migration along the body. Our results indicate that 2ME has an effect on collective cell migration in vivo as well as previously reported anti-tumorigenic activity and suggests that the molecular mechanisms governing cell migration in a variety of contexts are conserved between fish and mammals. Moreover, we exemplify the versatility of the zebrafish larvae for testing diverse physiological processes and screening for antiangiogenic and antimigratory drugs in vivo.     

Involvement of membrane protein GDE2 in retinoic acid-induced neurite formation in Neuro2A cells

We show that a glycerophosphodiester phosphodiesterase homolog, GDE2, is widely expressed in brain tissues including primary neurons, and that the expression of GDE2 in neuroblastoma Neuro2A cells is significantly upregulated during neuronal differentiation by retinoic acid (RA) treatment. Stable expression of GDE2 resulted in neurite formation in the absence of RA, and GDE2 accumulated at the regions of perinuclear and growth cones in Neuro2A cells. Furthermore, a loss-of-function of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite formation. These results demonstrate that GDE2 expression during neuronal differentiation plays an important role for growing neurites.     

Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.     

Analysis of the human HP1 interactome reveals novel binding partners

Heterochromatin protein 1 (HP1) has first been described in Drosophila as an essential component of constitutive heterochromatin required for stable epigenetic gene silencing. Less is known about the three mammalian HP1 isotypes CBX1, CBX3 and CBX5. Here, we applied a tandem affinity purification approach coupled with tandem mass spectrometry methodologies in order to identify interacting partners of the mammalian HP1 isotypes. Our analysis identified with high confidence about 30–40 proteins co-eluted with CBX1 and CBX3, and around 10 with CBX5 including a number of novel HP1-binding partners. Our data also suggest that HP1 family members are mainly associated with a single partner or within small protein complexes composed of limited numbers of components. Finally, we showed that slight binding preferences might exist between HP1 family members.