Biomedical subjects

R Tampé

Publications and source records attributed to R Tampé.

At least 19 recordsLinked to original sources

PubMed2006

The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation.

A challenging task for the adaptive immune system of vertebrates is to identify and eliminate intracellular antigens. Therefore a highly specialized antigen presentation machinery has evolved to display fragments of newly synthesized proteins to effector cells of the immune system at the cell surface. After proteasomal degradation of unwanted proteins or defective ribosome products, resulting peptides are translocated into the endoplasmic reticulum by the transporter associated with antigen processing and loaded onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are transported via the secretory pathway to the cell surface where they are then inspected by cytotoxic T lymphocytes, which can trigger an immune response. This review summarizes the current view of the intracellular machinery of antigen processing and of viral immune escape mechanisms to circumvent destruction by the host.

ATP-Binding Cassette Transporters↗

PubMed2006

Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus.

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

Adenosine Triphosphatases↗

PubMed2002

Herpes viral proteins blocking the transporter associated with antigen processing TAP--from genes to function and structure.

In adaptation to the immune system, viruses have developed manifold mechanisms to evade the immune response, causing lifelong persistence in the host. Several members of the herpesvirus family are known to interfere with antigen presentation via MHC class I molecules. Here we compare the mechanistic and structural aspects of two unrelated herpesviral proteins, both of which have selected the transporter associated with antigen processing (TAP) as target for immune evasion. However, ICP47 (IE12) encoded by the herpes simplex virus and US6 from human cytomegalovirus utilize entirely different strategies to block TAP function. Detailed knowledge of the function and structure of these viral factors will help to understand TAP function and to design novel immune suppressors or vectors for gene transfer.

Amino Acid Sequence↗

PubMed2001

Immune escape of melanoma: first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway.

Sequence analyses of the transporter associated with antigen processing (TAP) in tumor cell lines with deficient MHC class I surface expression identified a bp deletion at position 1489 near the ATP-binding domain of Tap1, causing a frameshift in one melanoma cell line. The impaired TAP1 protein expression was associated with deficient TAP2 protein expression, peptide binding, translocation, and MHC class I surface expression. Stable TAP1 gene transfer reconstitutes the described defects, whereas lysis by HLA-A2-restricted CTLs was still abrogated. This was attributable to a 2-bp insertion at position 890 in the HLA-A2 gene and was corrected after HLA-A2 cotransfection. This study describes for the first time mutations in two distinct components of the MHC class I antigen processing pathway, suggesting an immune selection against CTLs recognizing both TAP-dependent and -independent T-cell epitopes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Molecular mechanism and structural aspects of transporter associated with antigen processing inhibition by the cytomegalovirus protein US6.

The human cytomegalovirus (HCMV) has evolved a set of elegant strategies to evade host immunity. The HCMV-encoded type I glycoprotein US6 inhibits peptide trafficking from the cytosol into the endoplasmic reticulum and subsequent peptide loading of major histocompatibility complex I molecules by blocking the transporter associated with antigen processing (TAP). We studied the molecular mechanism of TAP inhibition by US6 in vitro. By using purified US6 and human TAP co-reconstituted in proteoliposomes, we demonstrate that the isolated endoplasmic reticulum (ER)-luminal domain of US6 is essential and sufficient to block TAP-dependent peptide transport. Neither the overall amount of bound peptides nor the peptide affinity of TAP is affected by US6. Interestingly, US6 causes a specific arrest of the peptide-stimulated ATPase activity of TAP by preventing binding of ATP but not ADP. The affinity of the US6-TAP interaction was determined to 1 microm. The ER-luminal domain of US6 is monomeric in solution and consists of 19% alpha-helices, 25% beta-sheets, and 27% beta-turns. All eight cysteine residues are involved in forming a stabilizing network of four intramolecular disulfide bridges. Glycosylation of US6 is not required for function. These findings point to fascinating mechanistic and structural properties, by which specific binding of US6 at the ER-luminal loops of TAP signals across the membrane to the nucleotide-binding domains to prevent ATP hydrolysis of TAP.

ATP-Binding Cassette Transporters↗

PubMed2001

Enhanced expression of human ABC-transporter tap is associated with cellular resistance to mitoxantrone.

Multidrug resistance (MDR) phenotypes have been associated with the overexpression of various members of the superfamily of ATP binding cassette (ABC) transporters. Here we demonstrate that a member of the ABC-transporter family, the heterodimer 'transporter associated with antigen processing' (TAP), physiologically involved in major histocompatibility complex class I-restricted antigen presentation, is significantly overexpressed in the human gastric carcinoma cell line EPG85-257RNOV exhibiting a mitoxantrone-resistant phenotype. This tumor cell line shows an atypical MDR phenotype in the absence of 'P-glycoprotein' or 'MDR-associated protein' overexpression but with an enforced 'breast cancer resistance protein' expression level. Transfection of both TAP subunits encoding cDNA molecules, TAP1 and TAP2, into the drug-sensitive parental gastric carcinoma cell line EPG85-257P conferred a 3.3-fold resistance to mitoxantrone but not to alternative anti-neoplastic agents. Furthermore, cell clones transfected with both, but not singularly expressed TAP1 or TAP2, reduced cellular mitoxantrone accumulation. Taken together, the data suggest that the heterodimeric TAP complex possesses characteristics of a xenobiotic transporter and that the TAP dimer contributes to the atypical MDR phenotype of human cancer cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP.

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide-MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis-Menten kinetics with a maximal velocity V(max) of 2 micromol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.

ATP-Binding Cassette Transporters↗

PubMed2001

Single molecule research on surfaces: from analytics to construction and back.

The study of single molecules opens a new dimension in understanding nature down to its finest ramifications. While much progress was achieved in the last decade concerning the detection techniques, suitable techniques for manipulating and handling the biomolecules still bear a challenge. Primarily, the task is keeping an individual, active molecule of a certain lifespan in the spot. Here, we will focus on techniques for the functional immobilization of (single) molecules on surfaces to enable their observation at one position over a time period. Presenting the main methods of reversible immobilization we will accentuate the chelator lipid concept as combining all features prerequisite for functional, reversible and well-defined immobilization. This will also show that single molecule research in principle is the synthesis of an insight into the function of nature and nano-biotechnology (manipulation): thus of analytics, construction, and back.

Biotechnology↗

PubMed2001

Downregulation of the constitutive tapasin expression in human tumor cells of distinct origin and its transcriptional upregulation by cytokines.

Human tumor cells frequently exhibit abnormalities in the major histocompatibility complex (MHC) class I surface expression which can be due to structural alterations and/or dysregulation of various components of the MHC class I antigen processing machinery, such as HLA class I heavy and light chains, the peptide transporter and the proteasome subunits. Although several cofactors critical for proper MHC class I assembly have been identified, their contribution to the immune escape phenotype of tumor cells has not been analyzed. In order to determine whether tapasin deficits are an integral part of immune escape mechanisms of human tumors, we studied the constitutive and cytokine-regulated expression pattern of tapasin in malignant cells of distinct histology. Heterogeneous and reduced expression levels of tapasin were found in small-cell lung carcinoma, pancreatic carcinoma, colon carcinoma, head an neck squamous cell carcinoma and renal cell carcinoma cell lines. Tapasin downregulation was also prominent in surgically removed tumor lesions when compared to normal controls. The impaired tapasin expression is often associated with low MHC class I cell surface expression. In addition, various cytokines, including interferon (IFN)-alpha, IFN-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-4, but not granulocyte-macrophage colony stimulating factor (GM-CSF), transcriptionally upregulate to a distinct extent and in a time-dependent manner tapasin expression in tumor cells. Thus, deficient tapasin expression appears to be a frequent event in human tumor cells. Its restoration by cytokines further suggests that impaired tapasin expression in tumors is rather due to dysregulation than to structural alterations.

Antiporters↗

PubMed2000

Affinity, specificity, diversity: a challenge for the ABC transporter TAP in cellular immunity.

The immune system is a perfect piece of machinery that serves only one purpose: to protect and defend our organism. The fact that we are still alive, although surrounded by a hostile environment, demonstrates impressively how well the immune system performs this task. Over the past decades our knowledge about the functions and components of the immune system has increased exponentially. Especially for the humoral immune system and its key players, the antibodies, a detailed knowledge about structure and function as well as regulation and communication between the individual components exists. In case of the cellular immune system that knowledge is not as profound as for the humoral counterpart. In this article we do not intend to give a complete overview about both branches of the immune system. Rather, we want to focus on an essential protein of the cellular immune system, the transporter associated with antigen processing (TAP). This transmembrane protein complex displays unique properties with respect to recognition and translocation of a vast spectrum of antigenic peptides. Its important role within the major histocompatibility complex (MHC) class I mediated immune response has been utilized by some viruses which target TAP to hide and escape from a deadly attack by the immune system. Here we summarize the existing data and analyze structural and mechanistic aspects of recognition and transport by which this transporter performs its task.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Pairing of the nucleotide binding domains of the transporter associated with antigen processing.

The transporter associated with antigen processing (TAP) comprises two structurally related subunits, TAP1 and TAP2, that form stable complexes in endoplasmic reticulum (ER) membranes. TAP complexes function in the translocation of peptides from the cytosol into the ER lumen for presentation by major histocompatibility complex class I molecules. Each TAP subunit contains an N-terminal membrane-spanning region with multiple membrane-spanning segments, and a C-terminal, cytosolic nucleotide binding region. To study the nature of the interactions occurring on the cytosolic face of TAP1/TAP2 complexes, we investigated quaternary associations mediated by two C-terminal fragments of human TAP1 (T1c, residues 452-748 and T1ctr, residues 472-748) and two C-terminal fragments of human TAP2 (T2c, residues 399-686 and T2ctr, residues 433-686). Each of these constructs contains the core nucleotide binding region as well as a long or short N-terminal extension. We show stable complex formation between T1c and T2c but not between T1ctr and T2ctr. The mechanistic implications of these results are discussed. We also show that each of the constructs except T1ctr interacts with wild type TAP1 and TAP2, indicating possibilities for homodimerization of TAP1 and TAP2, or of oligomerization of TAP1/TAP2 heterodimers on membranes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Design of supported membranes tethered via metal-affinity ligand-receptor pairs.

Model lipid layers are very promising in investigating the complex network of recognition, transport and signaling processes at membranes. We have developed a novel and generic approach to create supported lipid membranes tethered by metal-affinity binding. By self-assembly we have generated various interfaces that display histidine sequences (6xHis) via polymer spacers. These histidine-functionalized interfaces are designed to allow specific docking and fusion of vesicles containing metal-chelating lipids. By means of surface plasmon resonance and atomic force microscopy we analyzed the formation and subsequently the structure of these solid-supported membranes. Although the affinity constant of single ligand-receptor pairs is only in the micromolar range, very stable immobilization of these membranes was observed. This behavior can be explained by multivalent interactions resembling many features of cell adhesion. The process is highly specific, because vesicle docking and bilayer formation are strictly dependent on the presence of metal-affinity ligand-receptor pairs. The surface accessibility and geometry of these tethered membranes were probed by binding of histidine-tagged polypeptides. The supported membranes show adsorption kinetics and values similar to planar supported monolayers. Using various combinations of metal-chelating and histidine-tagged lipids or thiols these metal-affinity-tethered membranes should make a great impact on probing and eventually understanding the dynamic dialog of reconstituted membrane proteins.

Affinity Labels↗

PubMed2000

A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy.

In recent years, the atomic force microscope (AFM) has contributed much to our understanding of the molecular forces involved in various high-affinity receptor-ligand systems. However, a universal anchor system for such measurements is still required. This would open up new possibilities for the study of biological recognition processes and for the establishment of high-throughput screening applications. One such candidate is the N-nitrilo-triacetic acid (NTA)/His-tag system, which is widely used in molecular biology to isolate and purify histidine-tagged fusion proteins. Here the histidine tag acts as a high-affinity recognition site for the NTA chelator. Accordingly, we have investigated the possibility of using this approach in single-molecule force measurements. Using a histidine-peptide as a model system, we have determined the binding force for various metal ions. At a loading rate of 0.5 microm/s, the determined forces varied from 22 +/- 4 to 58 +/- 5 pN. Most importantly, no interaction was detected for Ca(2+) and Mg(2+) up to concentrations of 10 mM. Furthermore, EDTA and a metal ion reloading step demonstrated the reversibility of the approach. Here the molecular interactions were turned off (EDTA) and on (metal reloading) in a switch-like fashion. Our results show that the NTA/His-tag system will expand the "molecular toolboxes" with which receptor-ligand systems can be investigated at the single-molecule level.

Chelating Agents↗

PubMed2000

Gene transfer of human interferon gamma complementary DNA into a renal cell carcinoma line enhances MHC-restricted cytotoxic T lymphocyte recognition but suppresses non-MHC-restricted effector cell activity.

Even though renal cell carcinomas (RCC) are thought to be immunogenic, many tumors express variations in surface molecules and intracellular proteins that hinder induction of optimal antitumor responses. Interferon gamma (IFNgamma) stimulation can correct some of these deficiencies. Therefore, we introduced the complementary DNA (cDNA) encoding human IFNgamma into a well-characterized RCC line that has been selected for development of an allogeneic tumor cell vaccine for treatment of patients with metastatic disease. Studies were performed to determine how endogenous IFNgamma expression influences tumor cell immunogenicity. IFNgamma transductants showed minimal increases in surface expression of MHC class I and adhesion molecules but expression of class II molecules was induced. Proteins of the transporter associated with antigen processing (TAP) and low molecular weight polypeptide (LMP) were constitutively expressed at high levels. The transductants stimulated allospecific cytotoxic T lymphocytes (CTL); however, they were not better than unmodified tumor cells in this capacity. Endogenous IFNgamma expression enhanced tumor cell recognition by MHC-restricted, tumor antigen-specific CTL but suppressed recognition by non-MHC-restricted cytotoxic cells. Thus, the functional consequences of IFNgamma expression varied with respect to the type of effector cell and were not always beneficial for tumor cell recognition.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

The transporter associated with antigen processing TAP: structure and function.

The transport of antigenic peptides from the cytosol to the lumen of the endoplasmic reticulum (ER) is an essential process for presentation to cytotoxic T-lymphocytes. The transporter associated with antigen processing (TAP) is responsible for the intracellular translocation of peptides across the membrane of the ER. Efficient assembly of MHC-peptide complex requires the formation of a macromolecular transport and chaperone complex composed of TAP, tapasin and MHC class I molecules. Therefore, structure and function of TAP is important for the understanding of the immune surveillance.

Antigens↗

PubMed1999

Kinetic analysis of peptide binding to the TAP transport complex: evidence for structural rearrangements induced by substrate binding.

The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the proteasome complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the fluorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this peptide-induced structural reorganization may reflect the crosstalk of structural information between the peptide binding site and both nucleotide-binding domains within the TAP complex.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Function of the transport complex TAP in cellular immune recognition.

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility complex (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. The MHC-encoded ABC transporter works in concert with the proteasome and MHC class I molecules for the antigen presentation on the cell surface for T cell recognition. TAP forms a heterodimer where each subunit consists of a hydrophilic nucleotide binding domain and a hydrophobic transmembrane domain. The transport mechanism is a multistep process composed of an ATP-independent peptide association step which induces a structural reorganization of the transport complex that may trigger the ATP-driven transport of the peptide into the endoplasmic reticulum lumen. By using combinatorial peptide libraries, the substrate selectivity and the recognition principle of TAP have been elucidated. TAP maximizes the degree of substrate diversity in combination with high substrate affinity. This ABC transporter is also unique as it is closely associated with chaperone-like proteins involved in bonding of the substrate onto MHC molecules. Most interestingly, virus-infected and malignant cells have developed strategies to escape immune surveillance by affecting TAP expression or function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Structure of the active domain of the herpes simplex virus protein ICP47 in water/sodium dodecyl sulfate solution determined by nuclear magnetic resonance spectroscopy.

ICP47 encoded by herpes simplex virus (HSV) is a key factor in the evasion of cellular immune response against HSV-infected cells. By specific inhibition of the transporter associated with antigen processing (TAP), ICP47 prevents peptide transport into the endoplasmic reticulum and subsequent loading of major histocompatibility complex (MHC) class I molecules. Amino acid residues 3-34 have been identified as the active domain. This domain appeared to be unstructured in aqueous solution, whereas after binding to membranes an alpha-helical conformation was observed. Here, we have analyzed the structure of ICP47(2-34) in a lipidlike environment by nuclear magnetic resonance (NMR) spectroscopy. In micellar solution of deuterated sodium dodecyl sulfate, the viral TAP inhibitor adopts an ordered structure. There are two helical regions extending from residues 4 to 15 and from residues 22 to 32. Arg-16 is found on the C-terminus of the first helix, and Gly-33 serves as a terminator of the second helix. A loop between residues 17 and 21 is also evident in the structure. The relative orientation of the helices toward each other, however, could not be determined due to the paucity of NOEs from residues 18-21.

ATP Binding Cassette Transporter, Subfamily B, Mem↗