Human brain glycogen content and metabolism: implications on its role in brain energy metabolism

The adult brain relies on glucose for its energy needs and stores it in the form of glycogen, primarily in astrocytes. Animal and culture studies indicate that brain glycogen may support neuronal function when the glucose supply from the blood is inadequate and/or during neuronal activation. However, the concentration of glycogen and rates of its metabolism in the human brain are unknown. We used in vivo localized 13C-NMR spectroscopy to measure glycogen content and turnover in the human brain. Nine healthy volunteers received intravenous infusions of [1-(13)C]glucose for durations ranging from 6 to 50 h, and brain glycogen labeling and washout were measured in the occipital lobe for up to 84 h. The labeling kinetics suggest that turnover is the main mechanism of label incorporation into brain glycogen. Upon fitting a model of glycogen metabolism to the time courses of newly synthesized glycogen, human brain glycogen content was estimated at approximately 3.5 micromol/g, i.e., three- to fourfold higher than free glucose at euglycemia. Turnover of bulk brain glycogen occurred at a rate of 0.16 micromol.g-1.h-1, implying that complete turnover requires 3-5 days. Twenty minutes of visual stimulation (n=5) did not result in detectable glycogen utilization in the visual cortex, as judged from similar [13C]glycogen levels before and after stimulation. We conclude that the brain stores a substantial amount of glycogen relative to free glucose and metabolizes this store very slowly under normal physiology.     

Gene expression profiling defines ATP as a key regulator of human dendritic cell functions

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPgammaS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.     

Functional expression of H4 histamine receptor in human natural killer cells, monocytes, and dendritic cells

We describe here the protein expression of H4 histamine receptor in cells of the innate immune system, which include NK cells, monocytes, and dendritic cells (DCs). Anti-H4R specifically stained permeabilized NK cells, THP-1 clone 15 monocytes, and DCs. This binding was inhibited by incubating anti-H4R Ab with its corresponding peptide. Histamine induced NK cells, THP-1 clone 15 cells, and DCs chemotaxis with high affinity. The ED(50) chemotactic effect was 5 nM, 6.8 nM, and 2.7 nM for NK cells, THP-1 clone 15 cells, and DCs, respectively. Thioperamide, an H3R/H4R antagonist, inhibited histamine-induced chemotaxis in all these cells. However, histamine failed to induce the mobilization of [Ca(2+)](i) in NK cells and THP-1 clone 15 cells, but it induced calcium fluxes in DCs. Using a new method of detecting NK cell-mediated cytolysis, it was observed that NK cells efficiently lysed K562 target cells and that histamine did not affect this NK cell activity. In summary, this is the first demonstration of the protein expression of H4 receptor in NK cells. Also, the results of the chemotactic effects of histamine on NK cells and THP-1 cells are novel. These results may shed some light on the colocalization of cells of innate immune arm at sites of inflammation. They are also important for developing drugs that target H4R for the treatment of various disorders, such as autoimmune and immunodeficient diseases.     

Extracellular Interaction between hCD98 and the PDZ Class II Domain of hCASK in Intestinal Epithelia

The extracellular domain of the glycoprotein-associated integrin hCD98 protrudes into the basolateral extracellular space of the intestine and contains a PDZ class II-binding domain (GLLLRFPYAA, amino acids 520-529). Protein-protein interaction studies in vitro as well as in human colonic sections and Caco2-BBE cells have revealed that hCD98 coimmunoprecipitated with the basolateral membrane-associated guanylate kinase hCASK and that this interaction occurred in a PDZ domain-dependent manner. These novel results, which provide the first evidence for a PDZ domain-dependent interaction between a membrane protein and an extracellular protein, open a new field of investigation related to extracellular signaling in cell biology.     

The heterodimeric primase from the euryarchaeon Pyrococcus abyssi: a multifunctional enzyme for initiation and repair?

We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase α–primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg²⁺ and Mn²⁺. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.     

The crystal structure of Trypanosoma cruzi glucokinase reveals features determining oligomerization and anomer specificity of hexose-phosphorylating enzymes

Glucose is an essential substrate for Trypanosoma cruzi, the protozoan organism responsible for Chagas' disease. The glucose is intracellularly phosphorylated to glucose 6-phosphate. Previously, a hexokinase responsible for this phosphorylation has been characterized. Recently, we identified an ATP-dependent glucokinase in T. cruzi exhibiting a tenfold lower substrate affinity compared to the hexokinase. Both enzymes, which belong to very different groups of the same family, are located inside glycosomes, the peroxisome-like organelles of Kinetoplastida that are known to contain the first seven glycolytic steps as well as enzymes of the oxidative branch of the pentose phosphate pathway. Here, we present the crystallographic structure of T. cruzi glucokinase, in complex with glucose and ADP. The structure suggests a loose tetrameric assembly formed by the association of two tight dimers. TcGlcK was previously reported to exist in a concentration-dependent equilibrium of monomeric and dimeric states. Here, we used mass spectrometry analysis to confirm the existence of TcGlcK monomeric and dimeric states. The analysis of subunit interactions and comparison with the bacterial glucokinases give insights into the forces promoting the stability of the different oligomeric states. Each T. cruzi glucokinase monomer contains one glucose and one ADP molecule. In contrast to hexokinases, which show a moderate preference for the alpha anomer of glucose, the electron density clearly shows the d-glucose bound in the beta configuration in the T.cruzi glucokinase. Kinetic assays with alpha and beta-d-glucose further confirm a moderate preference of the T. cruzi glucokinase for the beta anomer. Structural comparison of the glucokinase and hexokinases permits the identification of a possible mechanism for anomer selectivity in these hexose-phosphorylating enzymes. The preference for distinct anomers suggests that in T. cruzi hexokinase and glucokinase are not directly competing for the same substrate and are probably both present because they exert distinct physiological functions.     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

PrPc does not mediate internalization of PrPSc but is required at an early stage for de novo prion infection of Rov cells

We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface.