LIGHT/TNFSF14研究进展

LIGHT／TNFSF14是近年来发现的肿瘤坏死因子超家族成员，具有共刺激T淋巴细胞、诱导iDC成熟、促进肿瘤细胞凋亡的生物学功能，在自身免疫病、移植物抗宿主病（GVHD）及抗肿瘤免疫中发挥重要的调节作用。     

Identification of a human brain-specific gene, calneuron 1, a new member of the calmodulin superfamily

The calmodulin superfamily includes the calmodulins, calcium-binding proteins, and related genes. Herein, we describe the cloning and characterization of human calneuron 1 (CALN1). CALN1 encodes a novel neuron-specific protein that maps to chromosome 7q11. CALN1 spans a large genomic region (>360 kb). Sequence comparison shows significant similarity with the calmodulin superfamily of genes, especially in the two conserved EF-hand motifs. The mouse orthologous gene (Caln1) shows little prenatal expression, with highest expression at Postnatal Day 21. In situ hybridization to adult mouse brain shows high expression in the cerebellum, hippocampus, and cortex. The high expression of this gene exclusively in brain, the developmental changes in expression levels, the high homology with calmodulin which indicates a potential role in signal transduction, and the cellular localization of the mRNA suggest that CALN1 has a significant role in the physiology of neurons and is potentially important in memory and learning.     

Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major

Pentamidine (PEN) is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance is not well understood. Here, we used a genetic strategy to search for loci able to mediate PEN resistance (PENr) when overexpressed in Leishmania major. A shuttle cosmid library containing genomic DNA inserts was transfected into wild-type promastigotes and screened for PEN-resistant transfectants. Two different cosmids identifying the same locus were found, which differed from other known Leishmania drug resistance genes. The PENr gene was mapped by deletion and transposon mutagenesis to an open reading frame (ORF) belonging to the P-glycoprotein (PGP)/MRP ATP-binding cassette (ABC) transporter superfamily that we named pentamidine resistance protein 1 (PRP1). The predicted PRP1 protein encodes 1,807 amino acids with the typical dimeric structure involving 10 transmembrane domains and two nucleotide-binding domains (NBDs). PRP1-mediated PENr could be reversed by verapamil and PRP1 overexpressors showed cross-resistance to trivalent antimony but not to pentavalent antimony (glucantime). Although the degree of PENr was modest (1.7- to 3.7-fold), this may be significant in clinical drug resistance given the marginal efficacy of PEN against Leishmania.     

Binding specificity and in vivo targets of the EH domain, a novel protein–protein interaction module

EH is a recently identified protein–protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine–proline–phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.     

The MRC OX-47 antigen is a member of the immunoglobulin superfamily with an unusual transmembrane sequence

The MRC OX-47 monoclonal antibody recognizes a membrane antigen present at low levels on many lymphocytes but whose expression is markedly increased on activation with mitogens. cDNA clones for the OX-47 antigen were isolated from an expression library and the protein sequence deduced. It contains a leader sequence giving a mature protein of 251 amino acids with a single putative transmembrane region, a cytoplasmic domain of 40 amino acids and an extracellular domain of 187 amino acids that contained two immunoglobulin-like domains. The putative transmembrane sequence includes a glutamic acid residue within the hydrophobic sequence. The presence of acidic residues within the hydrophobic sequence of transmembrane sequences usually indicates association with other polypeptides and this is predicted for the OX-47 antigen. A sequence of 37 amino acids that included all the transmembrane region was identical to that of the chicken HT7 antigen present on endothelium in brain and erythroblasts. The level of protein sequence identity in the Ig-like domains was lower but HT7 is almost certainly the chicken homologue of the rat OX-47 antigen. The ligand and function of the molecule are unknown. In addition to lymphoblasts the OX-47 antigen was localized on a variety of other cell types including various immature cells, endothelia and cells with excitable membranes.     

Molecular cloning and characterization of a novel member of the MAP kinase superfamily

BACKGROUND: Members of the MAP kinase superfamily play important roles in a wide variety of signal transduction pathways, and several members have been identified. However, the diversity and complexity of cellular responses in mammalian systems may imply existence of hitherto unidentified members of the MAP kinase superfamily. RESULTS: We report the molecular cloning and characterization of a novel member of the MAP kinase superfamily. We isolated full-length mouse and human cDNAs that encode complete open reading frames of a novel protein kinase, termed MOK. MOK consists of 419 (human) and 420 (mouse) amino acids, with a calculated molecular weight of 48kDa. MOK contains all of the protein serine/threonine kinase consensus motifs and shows a modest similarity to members of the MAP kinase superfamily and MAK and MAK-related kinase (MRK). In addition, MOK possesses a Thr-Glu-Tyr (TEY) motif in the activation loop domain, as do classical MAP kinases. MOK is widely expressed in normal tissues and organs and localizes to the cytoplasm. MOK is able to phosphorylate several known MAP kinase substrates and to undergo autophosphorylation. A mutation in the TEY motif to AEF abolished the kinase activity of MOK, and the treatment of cells with a phosphatase inhibitor, okadaic acid, enhanced the kinase activity of MOK, suggesting the existence of an upstream kinase. Phorbol ester TPA was found to stimulate the kinase activity of MOK, whereas serum stimulation, osmotic shock, or anisomycin treatment did not significantly activate MOK. CONCLUSION: These results indicate that MOK is distantly related to members of known subfamilies of the MAP kinase superfamily and can therefore be classified as a novel member.     

GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations

GITR (glucocorticoid-induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8+ and CD4+ peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti-CD3 stimulus. Furthermore, expression of the main activation-induced receptor (IL-2Ralpha) and production of IL-2 and IFN-gamma were increased more with a GITR costimulus than with anti-CD3 alone. GITR stimulation also enhanced anti-CD3-induced ERK phosphorylation, suggesting that GITR is involved in MAPK-pathway activation. Interestingly, CD4+CD25+ regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR(-/-) CD4+CD25+ and GITR(+/+) CD4+CD25+ cells were equally able to exert suppressor activity on CD4+CD25- responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.     

Man1, an inner nuclear membrane protein,regulates left–right axis formation by controlling nodal signaling in a node‐independent manner

Man1, an inner nuclear membrane protein, regulates transforming growth factor β signaling by interacting with receptor‐associated Smads. In Man1‐deficient (Man1^Δ/Δ) embryos, vascular remodeling is perturbed by misregulation of Smad activity. Here, we show that Man1^Δ/Δ embryos exhibit abnormal heart morphogenesis including the looping abnormality. We searched for the molecular basis underlying the heart abnormalities and found that the left side‐specific genes responsible for left–right (LR) asymmetry, Nodal, Lefty2, and Pitx2, were expressed bilaterally in the lateral plate mesoderm and that their expression was enhanced significantly in mutants. Notably, Lefty1, a marker for the midline barrier, was maintained in Man1^Δ/Δ mutants. Crossing Man1^Δ/+ with Nodal hypomorphs (Nodal^neo/+), in which Nodal signaling in the node is disrupted, to generate double homozygous embryos (Man1^Δ/Δ; Nodal^neo/neo) revealed that the bilateral Nodal was retained in Man1^Δ/Δ; Nodal^neo/neo embryos. These results suggest that Man1 regulates LR asymmetry by controlling Nodal signaling in a node‐independent manner. Developmental Dynamics 237:3565–3576, 2008. © 2008 Wiley‐Liss, Inc.     

Characterization of monoclonal antibody specific to the Z39Ig protein, a member of immunoglobulin superfamily

Z39Ig, a recently identified immunoglobulin (Ig) superfamily member, is localized in the pericentromeric region of human chromosome X and detectable in all human tissue, but it is predominantly expressed in fetal human tissues as well as in adult lungs and placenta. In the present study, we generated a monoclonal antibody against Z39Ig protein to investigate the immunological role of Z39Ig protein on various immune cells. The anti-Z39Ig mAb that we generated specifically bound to Z39Ig protein on human promonocytic THP-1 cells, monocytes isolated from human peripheral blood mononuclear cells (PBMC) and mature CD14(+) dendritic cells (DC) differentiated from umbilical-cord blood CD34(+) hematopoietic progenitor cells. In addition, a signal through the Z39Ig protein induced an obvious cell surface expression of HLA-DR on THP-1 cells mediated by MHC class II transactivator (CIITA). These data suggest that the Z39Ig protein might be a critical molecule to regulate an immune response mediated by phagocytosis and/or antigen presentation.     

Molecular and functional characterization of IRp60, a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human NK cells.

In this study we describe the functional and molecular characterization of IRp60 (inhibitory receptor protein 60), an inhibitory receptor expressed on all human NK cells. The IRp60 molecule has been identified by the generation of three novel monoclonal antibodies (mAb). Cross-linking of IRp60 by specific mAb strongly inhibits the spontaneous cytotoxicity of NK cells as well as the NK-mediated cytolytic activity induced via different non-HLA-specific or HLA-specific activating receptors. IRp60 is a 60-kDa glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates with the SH2-containing phosphatases SHP-1 and SHP-2. The IRp60 gene is located on human chromosome 17 and encodes a molecule belonging to the immunoglobulin (Ig) superfamily characterized by a single V-type Ig-like domain in the extracellular portion. The cytoplasmic tail contains three classical immunoreceptor tyrosine-based inhibitory motifs. Southern blot analysis revealed cross-hybridization with monkey and mouse genomic DNA, thus suggesting that IRp60 may be conserved among different species. Moreover, based on the use of different anti-IRp60 mAb, we could identify two IRp60 allelic variants. Since IRp60 is also expressed by other cell types, including T cell subsets, monocytes and granulocytes, it may play a more general role in the negative regulation of different leukocyte populations.     

LIGHT, a new TNF superfamily member, is essential for memory T helper cell-mediated activation of dendritic cells

LIGHT is a recently identified member of the TNF superfamily that is up-regulated on activated T cells and can cooperate with CD40L to condition DC for the priming of CTL. In this report, we show that an intracellular pool of LIGHT is found selectively in some CD45RA-CD27+CD4+ "central" memory T cells and to a greater extent in some CD45RA-CD27-CD4+ effector memory T cells. LIGHT, like CD40L, is rapidly up-regulated on memory CD4+ T cells upon activation. Unlike CD40L, LIGHT up-regulation on naive T cells is delayed. Stimulation of DC with each subset of memory T cells in the presence of superantigen revealed that CD40L and LIGHT are required for optimal secretion of IL-12. Moreover, effector memory T cells, which can interact with DC in peripheral tissues, contribute to CCR7 induction on DC. LIGHT and CD40L can also regulate IL-12 production by CCR7+ DC capable of migration to lymph nodes. These data suggest that both LIGHT and CD40L may be involved in the maintenance or reactivation of secondary Th1 responses.