O(2)- and H(2)O(2)-dependent verdoheme degradation by heme oxygenase: reaction mechanisms and potential physiological roles of the dual pathway degradation

Heme oxygenase (HO) catalyzes the catabolism of heme to biliverdin, CO, and a free iron through three successive oxygenation steps. The third oxygenation, oxidative degradation of verdoheme to biliverdin, has been the least understood step despite its importance in regulating HO activity. We have examined in detail the degradation of a synthetic verdoheme IXalpha complexed with rat HO-1. Our findings include: 1) HO degrades verdoheme through a dual pathway using either O(2) or H(2)O(2); 2) the verdoheme reactivity with O(2) is the lowest among the three O(2) reactions in the HO catalysis, and the newly found H(2)O(2) pathway is approximately 40-fold faster than the O(2)-dependent verdoheme degradation; 3) both reactions are initiated by the binding of O(2) or H(2)O(2) to allow the first direct observation of degradation intermediates of verdoheme; and 4) Asp(140) in HO-1 is critical for the verdoheme degradation regardless of the oxygen source. On the basis of these findings, we propose that the HO enzyme activates O(2) and H(2)O(2) on the verdoheme iron with the aid of a nearby water molecule linked with Asp(140). These mechanisms are similar to the well established mechanism of the first oxygenation, meso-hydroxylation of heme, and thus, HO can utilize a common architecture to promote the first and third oxygenation steps of the heme catabolism. In addition, our results infer the possible involvement of the H(2)O(2)-dependent verdoheme degradation in vivo, and potential roles of the dual pathway reaction of HO against oxidative stress are proposed.     

Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle

The mechanical contribution of nucleus in adherent cells to bearing intracellular stresses remains unclear. In this paper, the effects of fluid shear stress on morphology and elastic properties of endothelial nuclei were investigated. The morphological observation suggested that the nuclei in the cytoplasm were being vertically compressed under static conditions, whereas they were elongated and more compressed with a fluid shear stress of 2 Pa (20 dyn/cm2) onto the cell. The elongated nuclei remained the shape even after they were isolated from the cells. The micropipette aspiration technique on the isolated nuclei revealed that the elastic modulus of elongated nuclei, 0.62+/-0.15 kPa (n=13, mean+/-SD), was significantly higher than that of control nuclei, 0.42+/-0.12 kPa (n=11), suggesting that the nuclei remodeled their structure due to the shear stress. Based of these results and a transmission electron microscopy, a possibility of the nucleus as an intracellular compression-bearing organelle was proposed, which will impact interpretation of stress distribution in adherent cells.     

Augmentation of T(H)-1 type response by immunoactive AT oligonucleotide from lactic acid bacteria via Toll-like receptor 9 signaling

Toll-like receptor 9, which is expressed on the surface of antigen presenting cells and which was recently identified in the cytoplasmic follicle, recognizes bacterial CpG oligodeoxynucleotides (ODNs), resulting in the induction of a potent immune response. However, in our previous study, we found that TLR9 potentially recognizes not only CpG ODN but also non-CpG ODN such as AT ODN. Therefore, in the present study, to investigate this possibility, we elucidated the effects of AT ODN on T(H)-1, T(H)-2 type cytokine induction via TLR9 by real-time quantitative PCR analysis and ELISA of the swine TLR9 transfectant. The results demonstrated that the T(H)-1 type cytokines such as interleukin (IL)-12p70 and interferon (IFN)-gamma were strongly induced by AT ODN compared to the unexposed controls, while T(H)-2 type cytokines were not induced. These results indicate that the AT ODN can augment the T(H)-1 immune response, which plays an important role in prevention of allergic responses. Moreover, the swine TLR9 transfectant demonstrated its usefulness for evaluation of immunostimulation by bacterial DNA through the detection of T(H)-1, T(H)-2 type cytokine induction via TLR9 signaling.     

Novel interactions of large P3 moiety and small P4 moiety in the binding of the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.     

Gain-of-function screen identifies a role of the Sec61alpha translocon in Drosophila postmitotic neurotoxicity

To elucidate the intrinsic mechanisms of neurotoxicity induction, including those underlying neural cell death and neurodegeneration, we developed a gain-of-function screen for gene products causing neural cell loss. To identify novel genes with a cell-death-related function in neurons, we screened 4,964 Drosophila GS lines, in which one or two genes from much of the Drosophila genome can be overexpressed. Approximately 0.68% of the GS lines produced phenotypes involving a loss of postmitotic neurons. Of these, we identified and characterized the endd2 gene, which encodes the Drosophila ortholog of Sec61alpha (DSec61alpha), an endoplasmic reticulum protein with protein translocation activity. Ectopic expression of DSec61alpha caused neural cell death accompanied by the accumulation of ubiquitinated proteins, which was mediated by DSec61alpha's translocon activity. This supported our previous observation that the DSec61alpha translocon contributes to expanded polyglutamine-mediated neuronal toxicity, which is also associated with ubiquitinated protein accumulation. These data suggest that the translocon may be a novel component of neural cell death and degeneration pathways. Our approach can be used to identify potential neurotoxic factors within the whole genome, which will increase our understanding of the molecular mechanisms of various types of cell death, including those associated with human neurodegenerative diseases.     

Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

Expression cloning of a human cDNA restoring sphingomyelin synthesis and cell growth in sphingomyelin synthase-defective lymphoid cells

Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-beta-cyclodextrin (MbetaCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MbetaCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile alpha motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MbetaCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.     

Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-gamma-inducible protein-10), and CXCL9 (monokine induced by IFN-gamma) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2-/-). A significant prolongation of islet allograft survival was noted in CCR2-/- recipients, with median survival time of 24 and 12 days for CCR2-/- and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62L(low)CD44(high)) in CCR2-/- compared with WT recipients. In addition, CCR2-/- recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2-/- recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.