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ABSTRACT: Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. The mechanism underlying this function is only partly understood. Here, we report that human Ngb exists in lipid rafts only during oxidative stress and that lipid rafts are crucial for neuroprotection by Ngb. The ferrous oxygen-bound form of Ngb, which exists under normoxia, is converted to the ferric bis-His conformation during oxidative stress, inducing large tertiary structural changes. We clarified that ferric bis-His Ngb, but not ferrous ligand-bound Ngb, specifically binds to flotillin-1, a lipid raft microdomain-associated protein, as well as to α-subunits of heterotrimeric G proteins (Gα(i/o)). Moreover, we found that human ferric bis-His Ngb acts as a guanine nucleotide dissociation inhibitor for Gα(i/o) that has been modified by oxidative stress. In addition, our data shows that Ngb inhibits the decrease in cAMP concentration that occurs under oxidative stress, leading to protection against cell death. Furthermore, by using a mutated Ngb protein that cannot form the bis-His conformation, we demonstrate that the oxidative stress-induced structural changes of human Ngb are essential for its neuroprotective activity.
Journal of Biological Chemistry 07/2012; 287(36):30128-38. · 4.77 Impact Factor
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ABSTRACT: Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Human Ngb is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. We previously demonstrated that, on the one hand, human ferric Ngb binds to the α-subunit of heterotrimeric G proteins (Gα(i)) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Gα(i). On the other hand, zebrafish Ngb does not exhibit GDI activity. By using wild-type and Ngb mutants, we demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. The crucial residues for both GDI and neuroprotective activity, corresponding to Glu53, Arg97, Glu118, and Glu151 of human Ngb, are conserved among boreotheria of mammalia. Recently, we found that zebrafish, but not human, Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. By performing site-directed mutagenesis, we showed that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for protein transduction activity. Because these residues are conserved among fishes, but not among mammals, birds, reptilians, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins. Moreover, we clarified that zebrafish Ngb interacts with negatively charged cell-surface glycosaminoglycan. Taken together, these results suggest that the function of Ngb proteins has been changing dynamically throughout the evolution of life.
Marine Genomics 09/2011; 4(3):137-42. · 1.55 Impact Factor
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ABSTRACT: CD25(+)CD4(+) regulatory T cells suppress T cell activation and regulate multiple immune reactions in in vitro and in vivo studies. To define the regulatory function of human CD25(+)CD4(+) T cells at various stages of maturity, we investigated in detail the functional differences of CD25(+)CD4(+) T cells from thymocytes, cord blood (CB), and adult peripheral blood (APB). CB CD25(+)CD4(+) T cells displayed low-FOXP3 protein expression level and had no suppressive activity. In contrast, CD25(+)CD4(+) T cells from thymocytes or APB expressed high expression level of FOXP3 protein associated with significant suppressive activity. Although CB CD25(+)CD4(+) T cells exhibited no suppressive activity, striking suppressive activity was observed following expansion in culture associated with increased FOXP3 expression and a shift from the CD45RA(+) to the CD45RA(-) phenotype. These functional differences in CD25(+)CD4(+) T cells from Thy, CB, and APB hence suggest a pathway of maturation for Treg in the peripheral immune system.
Clinical and Developmental Immunology 02/2008; 2008:305859. · 1.84 Impact Factor
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ABSTRACT: CD26 is a 110 kDa surface glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) activity that is expressed on numerous cell types and has a multitude of biological functions. CD26 role in immune regulation has been extensively characterized, with recent findings elucidating its linkage with signaling pathways and structures involved in T-lymphocyte activation as well as antigen presenting cell (APC)-T-cell interaction. In this paper, we will review emerging data on CD26-mediated T-cell costimulation, suggesting that CD26 may be an appropriate therapeutic target for the treatment of immune disorders. However, the identity of its putative natural ligand had not yet been clearly elucidated. Recently, using protein engineering and proteomic approach, we have recently characterized the putative costimulatory ligand for CD26 in T-cells and the proximal signaling events directly associated with the cytoplasmic region of CD26 in CD26-associated T-cell costimulation, processes that are independent of the CD28 costimulatory pathway. Our work therefore presents novel findings that contribute to the area of T-cell costimulation and signal transduction.
Frontiers in Bioscience 02/2008; 13:2299-310. · 3.52 Impact Factor
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Teruo Inamoto,
Taketo Yamada,
Kei Ohnuma,
Shinichiro Kina, Nozomu Takahashi,
Tadanori Yamochi,
Sakiko Inamoto,
Yoji Katsuoka,
Osamu Hosono,
Hirotoshi Tanaka,
Nam H Dang,
Chikao Morimoto
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ABSTRACT: CD26 is a 110-kDa cell surface antigen with a role in tumor development. In this report, we show that CD26 is highly expressed on the cell surface of malignant mesothelioma and that a newly developed humanized anti-CD26 monoclonal antibody (mAb) has an inhibitory effect on malignant mesothelioma cells in both in vitro and in vivo experiments.
Using immunohistochemistry, 12 patients' surgical specimens consisting of seven malignant mesothelioma, three reactive mesothelial cells, and two adenomatoid tumors were evaluated for expression of CD26. The effects of CD26 on malignant mesothelioma cells were assessed in the presence of transfection of CD26-expressing plasmid, humanized anti-CD26 mAb, or small interfering RNA against CD26. The in vivo growth inhibitory effect of humanized anti-CD26 mAb was assessed in human malignant mesothelioma cell mouse xenograft models.
In surgical specimens, CD26 is highly expressed in malignant mesothelioma but not in benign mesothelial tissues. Depletion of CD26 by small interfering RNA results in the loss of adhesive property, suggesting that CD26 is a binding protein to the extracellular matrix. Moreover, our in vitro data indicate that humanized anti-CD26 mAb induces cell lysis of malignant mesothelioma cells via antibody-dependent cell-mediated cytotoxicity in addition to its direct anti-tumor effect via p27(kip1) accumulation. In vivo experiments with mouse xenograft models involving human malignant mesothelioma cells show that humanized anti-CD26 mAb treatment drastically inhibits tumor growth in tumor-bearing mice, resulting in enhanced survival.
Our data strongly suggest that humanized anti-CD26 mAb treatment may have potential clinical use as a novel cancer therapeutic agent in CD26-positive malignant mesothelioma.
Clinical Cancer Research 08/2007; 13(14):4191-200. · 7.74 Impact Factor
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Kei Ohnuma,
Masahiko Uchiyama,
Tadanori Yamochi,
Kunika Nishibashi,
Osamu Hosono, Nozomu Takahashi,
Shinichiro Kina,
Hirotoshi Tanaka,
Xin Lin,
Nam H Dang,
Chikao Morimoto
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ABSTRACT: CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-kappaB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-kappaB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IkappaB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule.
Journal of Biological Chemistry 04/2007; 282(13):10117-31. · 4.77 Impact Factor
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ABSTRACT: We have synthesized and explored the feasibility of using a novel nuclear factor (NF) kappaB inhibitor, a dehydroxymethylepoxyquinomicin designated as DHMEQ, against prostate cancer. The activity of NFkappaB, evaluated by transient transfection of a luciferase reporter DNA containing a specific binding sequence for NFkappaB, was inhibited by DHMEQ in three human hormone-refractory prostate cancer cell lines, DU145, JCA-1, and PC-3. Statistically significant growth inhibition was achieved by 20 micro g/ml of DHMEQ, and marked levels of apoptosis were induced 48 h after DHMEQ administration in vitro. Electrophoretic mobility shift assay showed that DHMEQ completely inhibited NFkappaB DNA binding activity in JCA-1 cells. Furthermore, i.p. administrations of DHMEQ significantly inhibited pre-established JCA-1 s.c. tumor growth in nude mice without any side effects. Our result indicates the possibility of using a novel NFkappaB activation inhibitor, DHMEQ, as a new treatment strategy against hormone-refractory prostate cancer.
Cancer Research 02/2003; 63(1):107-10. · 7.86 Impact Factor
