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R Tampé

Publications and source records attributed to R Tampé.

At least 37 records · Page 2Linked to original sources
PubMed1999

High-resolution AFM-imaging and mechanistic analysis of the 20 S proteasome.

As macromolecular protease complex, the 20 S proteasome is responsible for the degradation of cellular proteins and the generation of peptide epitopes for antigen presentation. Here, structural and functional aspects of the 20 S proteasome from Thermoplasma acidophilum have been investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR). Due to engineered histidine tags introduced at defined positions, the proteasome complex was pre-oriented at ultra-flat chelator lipid membranes allowing for high-resolution imaging by AFM. Within these two-dimensional protein arrays, the overall structure of the proteasome and the organization of individual subunits was resolved under native conditions without fixation or crosslinking. In addition, the substrate-proteasome interaction was monitored in real-time by SPR using a novel approach. Instead of following enzyme activity by product formation, the association and dissociation kinetics of the substrate-proteasome complex were analyzed during proteolysis of the polypeptide chain. By blocking the active sites with a specific inhibitor, the substrate binding step could be dissected from the degradation step thus resolving mechanistic details of substrate recognition and cleavage by the 20 S proteasome.

Adsorption↗
PubMed1999

Specificity of the proteasome and the TAP transporter.

The generation of antigenic peptides and their transport across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules are essential steps in antigen presentation to cytotoxic T lymphocytes. Recent studies have characterized the substrate specificities of the proteasome and the transporter associated with antigen processing. It is interesting to compare the specificity of this transporter to the wide spectrum of peptides generated by the proteasome, to the binding motifs of MHC class I molecules and in particular to the principles of T cell recognition.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts.

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and LMP-7, and whether it can be restored by gene transfer. In comparison to parental NIH3T3 cells, the ras oncogenic transformants revealed reduced TAP and LMP mRNA expression and impaired function of these genes, leading to deficient peptide transport and peptide loading of MHC class I molecules resulting in instable expression of the MHC class I complex on the cell surface. Enhanced H-2 surface expression due to stabilization of the MHC class I complex could be achieved by culturing ras transformants at low, unphysiological temperature (26 degrees C) or by loading these cells with either exogenous human beta2-microglobulin or MHC class I-binding peptide alone or in combination. Furthermore, interferon-gamma treatment was capable to enhance the expression of TAP, LMP and MHC class I molecules in both parental as well as ras-transformed fibroblasts. Stable transfection of the human TAP-1 cDNA into ras transformants caused a partial reconstitution of the peptide transport and an enhancement of the MHC class I surface expression, whereas the level of MHC class I biosynthesis was not affected by TAP-1 overexpression in parental cells. Together these results point to the existence of an association between oncogenic transformation and deficiencies in the MHC class I antigen-restricted immunosurveillance, suggesting intervention strategies involving specific MHC class I-binding peptides or transfection of the LMP and/or TAP genes to overcome the expression of the immune escape phenotype.

3T3 Cells↗
PubMed1997

Peptide binding and photo-crosslinking to detergent solubilized and to reconstituted transporter associated with antigen processing (TAP).

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally active, heterologously expressed human TAP from microsomal membranes. The efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions. The activity of the solubilized TAP complex was analyzed over time, using a non-radioactive crosslinking assay with a photo-activateable peptide, in the presence or absence of external lipid. The detergent CHAPS was found optimally to retain activity and thus allowed us to reconstitute detergent-solubilized, active TAP into proteoliposomes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

The active domain of the herpes simplex virus protein ICP47: a potent inhibitor of the transporter associated with antigen processing.

The herpes simplex virus type 1 (HSV-1) protein ICP47 binds specifically to the transporter associated with antigen processing (TAP), thereby blocking peptide-binding and translocation by TAP and subsequent loading of peptides onto MHC class I molecules in the endoplasmic reticulum. In consequence, HSV-infected cells are masked for immune recognition by cytotoxic T-lymphocytes. To investigate the molecular details of this, so far, unique transporter-inhibitor interaction, the active domain and critical amino acid residues were identified by using short overlapping fragments and systematic deletions of the viral inhibitor. A fragment of 32 amino acid residues, ICP47(3-34), was found to be the minimal region harboring an activity to inhibit peptide-binding to TAP comparable to the action of the full-length protein and therefore representing the active domain. Further N or C-terminal truncations cause an abrupt loss in activity. Within the identified active domain, various mutants and chimeras of ICP47 derived from HSV-1 and HSV-2 helped to identify amino acid residues critical for TAP inhibition. On the basis of these results, therapeutic drugs could be designed that are applicable in treatment of allograft rejection or in novel vaccination strategies against HSV, restoring the ability of the immune system to recognize HSV-infected cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10.

Virally infected cells degrade intracellular viral proteins proteolytically and present the resulting peptides in association with major histocompatibility complex (MHC) class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). These cells are normally prone to CTL-mediated elimination. However, several viruses have evolved strategies to avoid detection by the immune system that interfere with the pathway of antigen presentation. Epstein-Barr virus (EBV) expresses a predominantly late protein, the BCRF1 gene product vIL-10, that is similar in sequence to the human interleukin-10 (hIL-10). We show here that vIL-10 affects the expression of one of the two transporter proteins (TAPs) associated with antigen presentation. Similarly, hIL-10 showed the same activity. Expression of the LMP2 and TAP1 genes but not expression of TAP2 or LMP7 is efficiently downregulated, indicating a specific IL-10 effect on the two divergently transcribed TAP1 and LMP2 genes. Downregulation of TAP1 by IL-10 hampers the transport of peptide antigens into the endoplasmatic reticulum, as shown in the TAP-specific peptide transporter assay, their loading onto empty MHC I molecules, and the subsequent translocation to the cell surface. As a consequence, IL-10 causes a general reduction of surface MHC I molecules on B lymphocytes that might also affect the recognition of EBV-infected cells by cytotoxic T cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes.

Newly assembled major histocompatibility complex (MHC) class I molecules, together with the endoplasmic reticulum chaperone calreticulin, interact with the transporter associated with antigen processing (TAP) through a molecule called tapasin. The molecular cloning of tapasin revealed it to be a transmembrane glycoprotein encoded by an MHC-linked gene. It is a member of the immunoglobulin superfamily with a probable cytoplasmic endoplasmic reticulum retention signal. Up to four MHC class I-tapasin complexes were found to bind to each TAP molecule. Expression of tapasin in a negative mutant human cell line (220) restored class I-TAP association and normal class I cell surface expression. Tapasin expression also corrected the defective recognition of virus-infected 220 cells by class I-restricted cytotoxic T cells, establishing a critical functional role for tapasin in MHC class I-restricted antigen processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries.

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing D amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

ATP-Binding Cassette Transporters↗
PubMed1997

Structure of the viral TAP-inhibitor ICP47 induced by membrane association.

Herpes simplex virus type I protein ICP47 (IE12) turns off antigen presentation by specifically binding to and blocking the major histocompatibility complex- (MHC-) encoded transporter associated with antigen processing (TAP). Due to the lack of translocated peptides inside the endoplasmic reticulum, MHC class I molecules fail to assemble and therefore MHC-peptide complexes do not reach the cell surface for immune recognition by cytotoxic T-lymphocytes. Here we investigated the structure of ICP47 representing the first natural inhibitor of an ATP-binding-cassette (ABC) transporter identified so far. First, we demonstrate that the N-terminal half of ICP47 is as active in inhibition of human TAP as the full-length protein and therefore serves as an ideal model to investigate structural and functional aspects of the inhibitor. Second, from circular dichroism analysis, the viral inhibitor of TAP appears to be mainly unstructured in aqueous solution. However, in the presence of membrane mimetics or lipid membranes an alpha-helical structure is induced. Third, circular dichroism and fluorescence spectroscopy reveal that membrane adsorption and conformational change of ICP47 are directly dependent on the surface charge density of the lipid membrane. Therefore we conclude that docking to membranes induces a conformational change in ICP47 that may be prerequisite to blocking TAP function.

ATP-Binding Cassette Transporters↗
PubMed1997

Intracellular location, complex formation, and function of the transporter associated with antigen processing in yeast.

Peptide transport across the membrane of the endoplasmic reticulum (ER) gains increasing importance in view of its potential function in selective protein degradation and antigen processing. An example for peptide transport in the ER is the transporter associated with antigen processing (TAP), which supplies peptides for the formation of major-histocompatibility-complex class-I complexes. Here, we have expressed human TAP1 and TAP2 in the yeast Saccharomyces cerevisiae. Expression of both genes resulted in the formation of a stable TAP heterodimer that was localized mainly in the ER. Although a minor fraction of TAP is found in the plasma membrane, TAP is unable to restore a-factor secretion in a mutant cell line that lacks the yeast mating-factor transporter Ste6. Nevertheless, in vitro studies with microsomal vesicles demonstrated that the TAP complex is fully functional in the ER membrane in terms of selective peptide binding, ATP-dependent transport, and specific inhibition by the viral protein of herpes simplex virus ICP47. This offers opportunities for topological, structural and mechanistic studies as well as genetic screenings for TAP functionality.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Diffusion measurement of fluorescence-labeled amphiphilic molecules with a standard fluorescence microscope.

The lateral diffusion of fluorescence-labeled amphiphilic tracer molecules dissolved within a two-dimensional matrix of lipids was measured by continuous illumination of an elongated rectangular region. The resulting spatial concentration profile of unbleached tracer molecules was observed with a standard epifluorescence microscope and analyzed with digital image-processing techniques. These concentration profiles are governed by the mobility of the tracers, their rate of photolysis, and the geometry of the illuminated area. For the case of a long and narrow illuminated stripe, a mathematical analysis of the process is given. After prolonged exposure, the concentration profile can be approximated by a simple analytical function. This fact was used to measure the quotient of the rate of photolysis, and the diffusion constant of the fluorescent probe. With an additional measurement of the rate of photolysis, the mobility of the tracer was determined. As prototype experiments we studied the temperature dependence of the lateral diffusion of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidyl++ + ethanolamine in glass-supported bilayers of L-alpha-dimyristoylphosphatidylcholine. Because of its simple experimental setup, this technique represents a very useful method of determining the lateral diffusion of fluorescence-labeled membrane molecules.

Chemical Phenomena↗
PubMed1997

Proteasome subunits, low-molecular-mass polypeptides 2 and 7 are hyperexpressed by target cells in autoimmune thyroid disease but not in insulin-dependent diabetes mellitus: implications for autoimmunity.

Autoimmune thyroid diseases (AITD) and insulin-dependent diabetes mellitus (IDDM) are two autoimmune syndromes of unknown etiology with common immune features. One is that the target cells, thyrocytes and pancreatic islet beta cells respectively, hyperexpress several proteins encoded in the HLA region: HLA class I, HLA class II and transporter associated with antigen processing (TAP-1): the clinical course and many aspects of the immunopathology are, however, quite different. Low-molecular-mass polypeptides 2 and 7 (LMP2 and LMP7) are proteasome subunits that increase the efficiency of endogenous antigen processing and are encoded in close vicinity to the TAP genes. We investigated whether LMP2 and LMP7 are hyperexpressed in thyrocytes and islet cells in AITD and IDDM. Thyroid tissue from Graves' disease patients (GD, n = 8) and Hashimoto thyroiditis (HT, n = 1) and pancreatic tissue from IDDM patients (n = 4) as well as control tissues were examined by the two-color indirect immunofluorescence technique. The results demonstrate that, in normal glands, thyrocytes and pancreatic islet cells express comparable moderate to low levels of LMP2 and LMP7. In AITD and IDDM, expression of LMP2/7 in the endocrine cells was disparate: while in AITD glands there was hyperexpression of LMP2 and 7 parallel to that of HLA class I and TAP-1, in the islet cells of recent onset diabetic pancreases (n = 2) the level of LMP2 and 7 expression was totally normal, including islets that were infiltrated by lymphocytes and hyperexpressed HLA class I and TAP-1. These observations suggest different mechanisms of endogenous peptides generation at the target cells in AITD from IDDM. Since this is a key step for the maintenance of peripheral tolerance, it may help to understand some of the different clinical features of the two autoimmune diseases.

Adolescent↗
PubMed1997

Expression and function of the peptide transporters in escape variants of human renal cell carcinomas.

The transporter associated with antigen processing (TAP) complex is a heterodimeric transmembrane pump consisting of the TAP-1 and TAP-2 subunits; these subunits translocate peptides from the cytoplasm into the lumen of the endoplasmic reticulum, where they bind nascent major histocompatibility complex (MHC) class I molecules. Loss or reduced expression of the TAP genes results in the synthesis of unstable peptide free MHC class I molecules that are only weakly expressed on the cell surface. In a number of human tumor cell lines, downregulation of MHC class I expression has been found to be associated with reduced or absent TAP expression. To investigate whether alterations in MHC class I expression occur during transformation and subsequent progression and whether MHC class I suppression is caused by impaired TAP function, we analyzed the protein expression of MHC class I heavy and light chain and TAP-1 in three renal cell carcinoma (RCC) cell lines and short-term cultures from corresponding normal kidney tissue. In one case a cell line established from a metastatic lesion was also available. Compared with normal epithelial cells, suppression of TAP-1 and MHC class I molecules was detected in all three primary RCC cells and was even more pronounced in the metastatic cell line. In contrast to normal epithelial cells, MHC class I membrane expression of two RCC lines was enhanced by culture in the presence of MHC class I binding peptides or at low temperature (26 degrees C) instead of 37 degrees C. Unstable MHC class I surface expression is caused by dissociation of the MHC class I heavy and light chain molecules as a result of functional defects in the antigen processing machinery, e.g., impaired peptide transport. Attempts to counteract the reduced immunogenicity by transferring the TAP genes into these cells demonstrated that TAP-1-modified RCC cells expressed higher levels of MHC class I molecules. These data indicate that downregulation and instability of MHC class I surface expression in RCC cells is at least partially caused by deficient loading with endogenous peptides and can be restored by TAP gene transfer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.

Adenosine Triphosphate↗
PubMed1996

Functional immobilization of a DNA-binding protein at a membrane interface via histidine tag and synthetic chelator lipids.

The coupling of a DNA-binding protein to self-organized lipid monolayers is examined at the air-water interface by means of film balance techniques and epifluorescence microscopy. We used two recombinant species of the heat shock factor HSF24 which differ only in a carboxy-terminal histidine tag that interacts specifically with the nickel-chelating head group of a synthetic chelator lipid. As key function, HSF24 binds to DNA that contains heat-shock responsible promoter elements. In solution, DNA-protein complex formation is demonstrated for the wild type and fusion protein. Substantial questions of these studies are whether protein function is affected after adsorption to lipid layers and whether a specific docking via histidine tag to the chelator lipid leads to functional immobilization. Using lipid mixtures that allow a lateral organization of chelator lipids within the lipid film, specific binding and unspecific adsorption can be distinguished by pattern formation of DNA-protein complexes. At the lipid interface, functional DNA-protein complexes are only detected, when the histidine-tagged protein was immobilized specifically to a chelator lipid containing monolayer. These results demonstrate that the immobilization of histidine-tagged biomolecules to membranes via chelator lipids is a promising approach to achieve a highly defined deposition of these molecules at an interface maintaining their function.

Adsorption↗
PubMed1996

Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides.

The proteasome is responsible for the non-lysosomal degradation of misfolded, transient, or ubiquitin-tagged proteins. This fact and the identification of two major-histocompatibility-complex-(MHC)-encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20S proteasomes from a wild-type and a LMP2/7-deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time-dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse-phase chromatography, pool sequencing, or mass spectrometry. The 20S proteasome can cleave insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20S proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone.

Amino Acid Sequence↗
PubMed1996

A functionally defective allele of TAP1 results in loss of MHC class I antigen presentation in a human lung cancer.

Tumours express a variety of novel epitopes which represent potential immune targets, and thus clinically evident tumours are thought to have effectively avoided immune recognition and elimination. Transporters associated with antigen presentation (TAP) are thought to be responsible for conveying intracellular peptides into the endoplasmic reticulum for complex formation with class I MHC and subsequent recognition by cytotoxic T lymphocytes. In this study, we evaluated 79 human solid tumours and cell lines for genetic abnormalities in TAP1 that might have led to an acquired loss of antigen presenting ability. A novel sequence (R659Q) was discovered near the ATP binding site in a human small cell lung cancer (SCLC) cell line, H1436. This cell line is heterozygous for this allele, but only the R659Q allele is transcribed into RNA. Even though the R659Q protein is expressed, these cells act as if they were TAP deficient by peptide binding and antigen presentation studies, which are restored after transfection of a functional TAP1 allele. This is the first evidence for a naturally occuring protein structural defect resulting in defective peptide transport in a human solid tumour.

ATP Binding Cassette Transporter, Subfamily B, Mem↗