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Biomedical subjects

K Früh

Publications and source records attributed to K Früh.

At least 37 records · Page 2Linked to original sources
PubMed1996

In vivo characterization of the proteasome regulator PA28.

A proteasome regulator, termed PA28, has been shown to modulate peptidase activities of the proteasomes in vitro. Two different but homologous PA28 molecules, designated as PA28alpha and PA28beta, have been cloned. Both alpha and beta polypeptides of PA28 are found in PA28 complexes isolated from cells, indicating that both are constituents of functional PA28 complexes. Using antisera specific to PA28alpha, PA28beta, and epitope-tagged PA28 molecules, we show that expression of PA28alpha and PA28beta is coordinately induced by various cytokines in different cell lines and that PA28 subunits and proteasomes have almost identical half-lives. In addition, we show that PA28 complexes are associated with 20 S but not 26 S proteasomes in vivo. Moreover, we demonstrate that PA28 complex is a heterohexamer composed of both alpha and beta subunits with a stoichiometry of alpha3beta3 in an alternating order.

Amino Acid Sequence↗
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

A point mutation in the human transporter associated with antigen processing (TAP2) alters the peptide transport specificity.

The heterodimeric transporter associated with antigen processing (TAP1/TAP2) translocates peptides from the cytosol into the endoplasmic reticulum where loading of major histocompatibility complex class I molecules takes place. TAP transporters from different species are known to exhibit distinct transport specificities with regard to the C-terminal amino acid (aa) of peptides. Thus, human TAP (hTAP), and rat TAP (rTAP) containing the rTAP2a allele are rather promiscuous, whereas mouse TAP (mTAP), and rTAP containing the rTAP2a allele are restrictive and select against peptides with C-terminal small polar/hydrophobic or positively charged aa. The structural basis for this selectivity is not clear. To assess the relative contribution of the TAP1 and TAP2 subunits to transport specificity, we have constructed and analyzed interspecies TAP hybrids and point mutants of hTAP2 expressed in Sf9 insect cells and in TAP-deficient T2 cells. Transport assays with 20 C-terminal variants of the peptide RYWANATRSX showed that: first, transport specificity with regard to C-terminal aa is mainly influenced by TAP2, but TAP1 can also contribute. Second, the selective transport of peptides with C-terminal positively charged aa is critically controlled by the amino-terminal region (1-361) on the TAP2 chain, while transport of peptides with C-terminal small polar/hydrophobic aa is determined by residues located within as well as outside the region 1-361. Third, a single point mutation in hTAP2 (374A-->D) resulted in a drastic alteration of the transport pattern. These results indicate that both TAP1 and TAP2 contribute to efficient peptide transport and that single point mutations in hTAP2 are able to alter the peptide transport specificity. This opens the possibility that naturally occurring mutations in one of the hTAP subunits may alter epitope selection in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

In vivo assembly of the proteasomal complexes, implications for antigen processing.

The multicatalytic and multisubunit proteasomal complexes have been implicated in the processing of antigens to peptides presented by class I major histocompatibility complex molecules. Two structural complexes of this proteinase, 20 S and 26 S proteasomes, have been isolated from cells. By analyzing in vivo assembly of the proteasomal complexes we show that the 20 S proteasomal complexes are irreversibly assembled via 15 S assembly intermediates containing unprocessed beta-type subunits. The 20 S proteasomes further associate reversibly with proteasome activators PA28 or pre-existing ATPase complexes to form 26 S proteasomal complexes. Our findings that not all of the 20 S proteasomal complexes are assembled into 26 S proteasomal complexes within cells and that all of PA28 and ATPase complexes are associated with 20 S proteasomes strongly suggest that all proteasomal complexes coexist within cells. We further demonstrate that 26 S proteasomal complexes are predominantly present in the cytoplasm and a significant portion of the 20 S proteasomal complexes is associated with the endoplasmic reticulum membrane. Taken together, our findings suggest that depending upon their associated regulatory components, 26 S and 20 S-PA28 proteasomal complexes serve different housekeeping functions within the cells, while they degrade antigens in a cooperative manner in antigen processing.

Animals↗
PubMed1995

A viral inhibitor of peptide transporters for antigen presentation.

Cytotoxic T lymphocytes lyse target cells after T-cell-receptor-mediated recognition of class I major histocompatibility complex molecules presenting peptides. Antigenic peptides are generated in the cytoplasm by proteasomes and translocated into the lumen of the endoplasmic reticulum (ER) by peptide transporters (TAP). Herpes simplex virus (HSV) expresses a cytoplasmic protein, ICP47, which seems to interfere with such immune surveillance by mediating retention of 'empty' class I molecules in the ER. By expressing ICP47 in HeLa cells under an inducible promoter, we show that ICP47 efficiently inhibits peptide transport across the ER membrane such that nascent class I molecules fail to acquire antigenic peptides. This inhibition was overcome by transfecting murine TAP. Further, we demonstrate that ICP47 colocalizes and physically associates with TAP within the cell. Inhibition of peptide translocation by a viral protein indicates a previously undocumented potential mechanism for viral immune evasion.

ATP-Binding Cassette Transporters↗
PubMed1994

Nucleotide binding of the C-terminal domains of the major histocompatibility complex-encoded transporter expressed in Drosophila melanogaster cells.

The C-terminal domains of the mouse transporter associated with antigen processing (TAP) were expressed as soluble proteins in Drosophila melanogaster cells and labeled by [alpha-32P]8-azido-ATP after UV-irradiation. The relative potencies of the nucleotides in preventing azido-ATP labeling were in the order of ATP > GTP > CTP > ITP > UTP for both the TAP1 and TAP2 C-terminal domains, suggesting ATP to be the natural substrate of the transporter. Our data provide the first evidence that the individual C-terminal domain of either TAP1 or TAP2 can be expressed as a functional ATP-binding protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.

Amino Acid Sequence↗
PubMed1993

A prospective study of the association between the human humoral immune response to Plasmodium falciparum blood stage antigen gp190 and control of malarial infections.

The human humoral immune response to the Plasmodium falciparum merozoite surface antigen gp190 was analyzed to determine the rate of reinfection by the parasite and the ability to control parasite density. The prospective study was carried out in a West African village where malaria is hyperendemic. No correlation between the antibody titers and protection against infection was observed within the group of children. Positive and negative associations of antibody specificities with protection against and/or control of parasitemia were, however, found for adolescents and adults, respectively. Thus, in adolescents, the presence of antibodies to gp190 fragment M6 correlates with a 50% reduced risk of P. falciparum infection and an increased ability to control parasitemia, whereas in adults, the humoral response to some of the polymorphic regions of gp190 associates with an increased risk of infection.

Adult↗
PubMed1993

Analysis of the human antibody response to thrombospondin-related anonymous protein of Plasmodium falciparum.

Thrombospondin-related anonymous protein (TRAP) of the malaria parasite Plasmodium falciparum shares two sequence motifs with other proteins which possess adhesive properties. Recently, findings indicate that TRAP is an antigen which contributes to antisporozoite immunity. We have cloned and expressed the TRAP coding sequences in Escherichia coli to investigate the human humoral immune response against this protein in a region of malaria endemicity of West Africa characterized by a seasonal transmission. Our results show that antibodies against TRAP are present in infected individuals. The anti-TRAP antibodies were analyzed in both a longitudinal and a prospective study. The longitudinal analysis shows seasonal fluctuations of the levels of specific antibodies as well as age-dependent quantitative differences. The immune response is long-lived in most of the adults and some of the older children but short-lived in young children. More importantly, the prospective analysis suggests that the presence of anti-TRAP antibodies in older children before the beginning of malaria transmission correlates with the subsequent control of parasite densities.

Adolescent↗
PubMed1992

Alternative exon usage and processing of the major histocompatibility complex-encoded proteasome subunits.

The finding that two subunits of the proteasome, LMP2 and LMP7, are encoded in the major histocompatibility complex (MHC) has linked the proteasome which represents a major extralysosomal proteolytic system to the processing of intracellular antigens. Here we describe a second form of the human LMP7 cDNA, LMP7-E2, which has been identified during the characterization of novel genes in the MHC. The analysis of the genome organization of LMP7 revealed that LMP7-E1 and LMP7-E2 arise by alternative exon usage. Using specific antibodies against LMP2 and LMP7, we show that they are co-expressed with class I MHC molecules as well as a putative peptide transporter. The polypeptides encoded by LMP7 and LMP2 undergo proteolytic processing when incorporated into proteasomes, and the LMP7 precursor is derived mainly from LMP7-E2. Furthermore, our data suggest that LMP7 and LMP2 are mutually dependent for their incorporation into the proteasomal complex.

Amino Acid Sequence↗
PubMed1992

Major histocompatibility complex (MHC)-encoded HAM2 is necessary for antigenic peptide loading onto class I MHC molecules.

The mutant murine lymphoma cell line RMA-S is unable to present endogenous antigens due to its inability to efficiently assemble class I major histocompatibility complex molecules and antigenic peptides. Therefore, it has been suggested that RMA-S cells are defective either in peptide generation or in peptide transport into the endoplasmic reticulum, where class I major histocompatibility complex molecule assembly is believed to occur. As proteasomes and the putative peptide transporters HAM1 and HAM2 have been implicated in class I antigen processing, we have investigated their expression in RMA-S and its wild-type counterpart RMA. Both proteasomes and HAM1 proteins are expressed at similar levels and show identical subcellular distributions in the two cell lines. However, only one copy of the HAM2 gene is present in RMA-S cells, and it contains a point mutation that leads to a premature stop codon. Thus, the HAM2 protein is absent from RMA-S cells. These data demonstrate that HAM2 is essential for peptide loading onto class I molecules.

Amino Acid Sequence↗
PubMed1992

Proteasomes are regulated by interferon gamma: implications for antigen processing.

Class I major histocompatibility complex (MHC) molecules present antigenic peptides of cytoplasmic origin to T cells. As the lengths of these peptides seem restricted to eight or nine amino acids, an unusual proteolytic system must play a role in antigen processing. Proteasomes, a major extralysosomal proteolytic system, are responsible for the degradation of cytoplasmic proteins. We demonstrate that several proteasomal subunits, including MHC-encoded subunits, are regulated by interferon gamma. These data and the finding that MHC-encoded and other interferon gamma-regulated proteasomal subunits are uniquely associated with proteasomes strongly suggest that the immune system has recruited proteasomes for antigen processing.

Animals↗
PubMed1991

Epitopes of the human malaria parasite P. falciparum carried on the surface of HBsAg particles elicit an immune response against the parasite.

The development of recombinant subunit vaccines against pathogenic organisms requires not only the identification of epitopes eliciting a protective immune response but also suitable carriers with adjuvant function. B- and T-cell epitopes of the malaria vaccine candidate gp190 were selected on the basis of a systematic search along the gp190 molecule and by computer prediction based on the amino acid sequence. Using some of the epitopes identified, we have redesigned the surface of the hepatitis B surface antigen lipoprotein particles by replacing the major antigenic determinants with malaria-specific sequences of up to 61 amino acids in length. Upon expression via vaccinia virus the hybrid particles elicit an anti-gp190 immune response in animals. Monoclonal antibodies derived from such infections recognize the native parasite.

Amino Acid Sequence↗
PubMed1991

Human antibody response to the major merozoite surface antigen of Plasmodium falciparum is strain specific and short-lived.

The precursor of the major merozoite antigen of Plasmodium falciparum, gp190, is considered a candidate for inclusion in a malaria vaccine. This protein, which consists of conserved, dimorphic, and polymorphic sequences, is very immunogenic in humans. In a longitudinal study carried out with 94 inhabitants of a rural community in Mali, West Africa, we show that in this endemic area naturally acquired gp190-specific antibodies are predominantly directed against the dimorphic parts of one of the main alleles of gp190. The presence of antibodies against these dimorphic regions correlates with the prevalence of the corresponding antigen in the infecting parasite population. Moreover, qualitative as well as quantitative differences were found in the time course of the humoral immune response to the dimorphic regions in adults and children, who differ in their susceptibility to malaria infection.

Adolescent↗
PubMed1990

The T cell reactivity against the major merozoite protein of Plasmodium falciparum.

We have undertaken a systematic search for T cell epitopes within the sequence of the major merozoite surface antigen (GP190) of Plasmodium falciparum. Recombinant polypeptides expressed in E. coli were used to evaluate the reactivity of peripheral blood mononuclear cells (PBMC) from both inhabitants of a rural community of West Africa exposed to P. falciparum transmission and from German patients with diagnosis of acute malaria. Although the proliferative response of the PBMC was in most cases very low, several T cell clones could be established. Deletion analysis of each gp190-derived polypeptide allowed the identification of six different T cell epitopes. Epitopes could be mapped within the dimorphic region of gp190, which also contains the sequences most frequently recognized by sera from adult individuals living in endemic areas.

Adult↗
PubMed1989

A new tool for the serodiagnosis of acute Plasmodium falciparum malaria in individuals with primary infection.

We have developed an ELISA which detects, with high specificity, antibodies against a major surface protein of P. falciparum merozoites which is a processing product of the precursor glycoprotein gp190. This assay can be used in the diagnosis of acute malaria in individuals with primary infection. Two partial sequences of gp190 were expressed in E. coli as beta-galactosidase (beta-Gal) fusion proteins. The same sequences fused to chloramphenicol acetyltransferase (CAT) or mouse dihydrofolate reductase (DHFR) react with high frequency when sera of acute malaria patients are analyzed in immunoblots. Antibodies from such sera crosslink, via their antigen binding sites, the beta-Gal fusions to the corresponding CAT or DHFR fusions adsorbed to a solid phase as demonstrated by the captured beta-Gal activity. The assay is highly specific, shows extremely low cut off values and should therefore be widely applicable.

Animals↗