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    Keywords: OPTIMIZATION ; PEPTIDE ; SPECTRA ; CELLS ; EXPRESSION ; INHIBITOR ; Germany ; PROTEIN ; PROTEINS ; MOLECULES ; MICE ; ACTIVATION ; COMPLEX ; COMPLEXES ; murine ; MEMBER ; MHC ; LYMPHOCYTES ; antigen presentation ; PEPTIDES ; STABILITY ; MHC class I ; DEGRADATION ; SUBUNITS ; TRANSLOCATION ; antigen processing ; CELL-LINE .220 ; HISTOCOMPATIBILITY COMPLEX ; LOADING COMPLEX ; MUTANT MICE ; NEWLY SYNTHESIZED PROTEINS
    Abstract: Tapasin is a member of the MHC class I loading complex where it bridges the TAP peptide transporter to class I molecules. The main role of tapasin is assumed to be the facilitation of peptide loading and optimization of the peptide cargo. Here, we describe another important function for tapasin. In tapasin- deficient (Tpn(-/-)) mice the absence of tapasin was found to have a dramatic effect on the stability of the TAP1/TAP2 heterodimeric peptide transporter. Steady-state expression of TAP protein was reduced more than 100-fold from about 3 x 10(4) TAP molecules per wild-type splenocyte to about 1 x 10(2) TAP per Tpn(-/-) splenocyte. Thus, a major function of murine tapasin appears to be the stabilization of TAR The low amount of TAP molecules in Tpn(-/-) lymphocytes is likely to contribute to the severe impairment of MHC class I expression. Surprisingly, activation of Tpn(-/-) lymphocytes yielded strongly enhanced class I expression comparable to wild-type levels, although TAP expression remained low and in the magnitude of several hundred molecules per cell. The high level of class I on activated Tpn(-/-) cells depended on peptides generated by the proteasome as indicated by blockade with the proteasome-specific inhibitor lactacystin. Lymphocyte activation induced an increase in ubiquitinated proteins that are cleaved into peptides by the proteasome. These findings suggest that in the presence of a large peptide pool in the cytosol, a small number of TAP transporters is sufficient to translocate enough peptides for high class I expression. However, these class I molecules were less stable than those of wild-type cells, indicating that tapasin is not only required for stabilization of TAP but also for optimization of the spectrum of bound peptides
    Type of Publication: Journal article published
    PubMed ID: 12594855
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    Keywords: PEPTIDE ; MODEL ; SITE ; MOLECULES ; MECHANISM ; ANTIGEN ; BINDING ; SEQUENCE ; SEQUENCES ; MOLECULE ; ACTIVE-SITE ; antigen presentation ; PEPTIDES ; LENGTH ; DEGRADATION ; EPITOPES ; antigen processing ; INTERFERON-GAMMA ; ENDOPLASMIC-RETICULUM ; RESIDUES ; TRANSPORTER ; LONG ; endoplasmic reticulum ; ARGININE AMINOPEPTIDASE ; CELL-PROTEINS ; LEUCINE AMINOPEPTIDASE ; LEUKOTRIENE-A(4) HYDROLASE ; PRESENTED PEPTIDES ; proteases ; THIMET OLIGOPEPTIDASE
    Abstract: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an IFN-gamma-induced aminopeptidase in the endoplasmic reticulum that trims longer precursors to the antigenic peptides presented on MHC class I molecules. We recently reported that purified ERAP1 trimmed N-extended precursors but spared peptides of 8 - 9 residues, the length required for binding to MHC class I molecules. Here, we show another remarkable property of ERAP1: that it strongly prefers substrates 9 - 16 residues long, the lengths of pepticles transported efficiently into the ER by the transporter associated with antigen processing (TAP) transporter. This aminopeptidase rapidly degraded a model 13-mer to a 9-mer and then stopped, even though the substrate and the product had identical N- and C-terminal sequences. No other aminopeptidase, including the closely related EIR-aminopeptidase ERAP2, showed a similar length preference. Unlike other aminopeptidases, the activity of ERAP1 depended on the C-terminal residue of the substrate. ERAP1, like most MHC class I molecules, prefers pepticles with hydrophobic C termini and shows low affinity for peptides with charged C termini. Thus, ERAP1 is specialized to process precursors transported by TAP to pepticles that can serve as MHC class I epitopes. Its "molecular ruler" mechanism involves binding the hydrophobic C terminus of the substrate 9 -16 residues away from the active site
    Type of Publication: Journal article published
    PubMed ID: 16286653
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    Keywords: CELLS ; ENDOTHELIAL-CELLS ; EXPRESSION ; Germany ; IN-VIVO ; PATHWAY ; SYSTEM ; SYSTEMS ; liver ; EFFICIENCY ; MICE ; ACTIVATION ; IFN-GAMMA ; INDUCTION ; ANTIGEN ; ANTIGENS ; DENDRITIC CELLS ; T-CELL ; T-CELLS ; TOLERANCE ; BONE-MARROW ; MATURATION ; knockout ; MOUSE ; LINE ; DEGRADATION ; IMMUNITY ; NAIVE ; CYTOTOXICITY ; CROSS-PRESENTATION ; endothelial cells ; PH ; development ; KNOCKOUT MICE ; CYTOKINE PRODUCTION ; CELL TOLERANCE ; dendritic cell ; INDUCE ; T cell tolerance ; KUPFFER CELLS ; ADOPTIVE TRANSFER ; CD8 T cell tolerance ; ENDOTOXIN ; oral tolerance ; scavenger endothelial cells
    Abstract: After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver simisoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2k(b) by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2k(b)-restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2(-/-) knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-gamma in CD8 T cells. Using a new transgenic mouse line expressing H-2k(b) only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K(b)-restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens
    Type of Publication: Journal article published
    PubMed ID: 16163670
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    Keywords: CELLS ; Germany ; human ; INHIBITION ; SITE ; COMPLEX ; COMPLEXES ; DOMAIN ; ANTIGEN ; T-CELLS ; BINDING ; GLYCOPROTEIN ; TRANSPORT ; CELL-LINE ; antigen presentation ; DOMAINS ; ENDOPLASMIC-RETICULUM ; MAJOR HISTOCOMPATIBILITY COMPLEX ; CLASS-I MOLECULES ; BINDING-SITE ; function ; BLOCKADE ; TAP ; cytomegalovirus ; INHIBITS PEPTIDE TRANSLOCATION ; PROTEIN ICP47 ; VACCINIA VIRUS
    Abstract: The endoplasmic reticulum-resident human cytomegalovirus glycoprotein US6 (gpUS6) inhibits peptide translocation by the transporter associated with antigen processing ( TAP) to prevent loading of major histocompatibility complex class I molecules and antigen presentation to CD8+ T cells. TAP is formed by two subunits, TAP1 and TAP2, each containing one multispanning transmembrane domain (TMD) and a cytosolic nucleotide binding domain. Here we reported that the blockade of TAP by gpUS6 is species-restricted, i.e. gpUS6 inhibits human TAP but not rat TAP. Co-expression of human and rat subunits of TAP demonstrates independent binding of gpUS6 to human TAP1 and TAP2, whereas gpUS6 does not bind to rat TAP subunits. gpUS6 associates with preformed TAP1/2 heterodimers but not with unassembled TAP subunits. To locate domains of TAP required for gpUS6 binding and function, we took advantage of reciprocal human/rat intrachain TAP chimeras. Each TAP subunit forms two contact sites within its TMD interacting with gpUS6. The dominant gpUS6-binding site on TAP2 maps to an N-terminal loop, whereas inhibition of peptide transport is mediated by a C-terminal loop of the TMD. For TAP1, two gpUS6 binding domains are formed by loops of the C-terminal TMD. The domain required for TAP inactivation is built by a distal loop of the C-terminal TMD, indicating a topology of TAP1 comprising 10 endoplasmic reticulum transmembrane segments. By forming multimeric complexes, gpUS6 reaches the distant target domains to arrest peptide transport. The data revealed a nonanalogous multipolar bridging of the TAP TMDs by gpUS6
    Type of Publication: Journal article published
    PubMed ID: 16356928
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    Keywords: RECEPTOR ; CELLS ; tumor ; TUMOR-CELLS ; CELL ; human ; TOOL ; MONOCLONAL-ANTIBODY ; ACTIVATION ; LIGAND ; BINDING ; BIOLOGY ; MOLECULAR-BIOLOGY ; RECOGNITION ; antibodies ; TARGET ; FUSION PROTEINS ; CLASS-I ; glycosylation ; PROTEIN INTERACTIONS ; PERFORMANCE LIQUID-CHROMATOGRAPHY ; molecular biology ; molecular ; RECOMBINANT ; secretion ; LEVEL ; analysis ; USA ; glycosaminoglycan ; heparan sulfate ; KILLER-CELLS ; natural killer ; NATURAL CYTOTOXICITY RECEPTOR ; NCR ; NK CELL ACTIVATION ; NKp30 ; NKP46
    Abstract: NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFN gamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells
    Type of Publication: Journal article published
    PubMed ID: 18006589
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    Keywords: DOMAIN, DOMAINS, ER, MECHANISM, PLASMA, PROTEIN, PROTEINS, YEAST
    Abstract: Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS(Ist2)) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS(Ist2) targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P(2) at the PM created ER-PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca(2+) entry (SOCE). The CSS(Ist2) and the C-terminus of the ER-located Ca(2+) sensor STIM2 were sufficient to bind PI(4,5)P(2) and PI(3,4,5)P(3) at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P(2), suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.
    Type of Publication: Journal article published
    PubMed ID: 19845919
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    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; INHIBITOR ; tumor ; TUMOR-CELLS ; carcinoma ; CELL ; Germany ; human ; DISEASE ; PROTEIN ; PROTEINS ; TUMOR-NECROSIS-FACTOR ; ACTIVATION ; LIGAND ; RESPONSES ; INFECTION ; MECHANISM ; mechanisms ; BINDING ; RECOGNITION ; ACID ; antibodies ; antibody ; PARTICLES ; TARGET ; virus ; NECROSIS-FACTOR-ALPHA ; MELANOMA ; LIGANDS ; NATURAL-KILLER-CELLS ; NK cells ; NKG2D ; SIALIC-ACID ; INTERFERON ; melanoma cells ; RECEPTORS ; CYTOTOXICITY ; APOPTOSIS-INDUCING LIGAND ; GAMMA ; MELANOMA-CELLS ; HEPARAN-SULFATE ; Newcastle disease virus ; USA ; macrophage ; ANTITUMOR VACCINATION ; NECROSIS ; paramyxovirus ; virology ; MODIFIED TUMOR-CELLS ; CYTOTOXICITY RECEPTORS ; NATURAL-KILLER-CELL ; NKG2D ligands ; PARTICLE ; CYTOMEGALOVIRUS UL16 GLYCOPROTEIN ; INFECTED CELLS ; INTRACELLULAR RETENTION ; KILLER-CELL ; natural killer cell
    Abstract: The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3 zeta lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV
    Type of Publication: Journal article published
    PubMed ID: 19515783
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