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M Groettrup

Publications and source records attributed to M Groettrup.

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

Selective proteasome inhibitors: modulators of antigen presentation?

The proteasome is the main nonlysosomal endoprotease in the cytoplasm and nucleus of all eukaryotic cells. It is responsible for the generation of most antigenic peptides as ligands for major histocompatibility complex (MHC) class I proteins. The proteasome hence qualifies as a target for modifying or silencing antigen processing and presentation to cytotoxic T cells, which are important players in transplant rejection and autoimmune disease. The authors summarize recent progress in the understanding of antigen processing by the proteasome and discuss the potential of novel and selective proteasome inhibitors as drugs for suppressing or modifying the cytotoxic immune response.

Journal Article↗
PubMed1998

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses.

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Animals↗
PubMed1998

Inactivation of a defined active site in the mouse 20S proteasome complex enhances major histocompatibility complex class I antigen presentation of a murine cytomegalovirus protein.

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the delta/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and H-2Dd molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the delta/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.

Animals↗
PubMed1998

The proteasome inhibitor lactacystin prevents the generation of an endoplasmic reticulum leader-derived T cell epitope.

The presentation of viral antigens on MHC class I molecules requires their intracellular fragmentation into peptides of appropriate length and anchor residue positions. Evidence has accumulated that the proteasome is the endoprotease in charge of the generation of MHC class I ligands in the cytoplasm. The generation of T cell epitopes derived from the leader peptides of endoplasmic reticulum (ER) targeted proteins, however. has been reported to be independent of the proteasome. Here we show that the H-2Db restricted antigen presentation of the immunodominant T cell epitope derived from the ER leader of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) is completely abolished by administration of the proteasome inhibitor lactacystin. Thus our data support the role of the proteasome in class I restricted antigen processing and extend it to an ER leader derived epitope from a viral glycoprotein.

Acetylcysteine↗
PubMed1997

The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome.

Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon gamma (IFN-gamma)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-gamma but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-gamma-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation.

Animals↗
PubMed1997

Expression and subcellular localization of mouse 20S proteasome activator complex PA28.

We have cloned the mouse PA28 proteasome activator cDNAs. Northern blot demonstrates high PA28 mRNA levels in liver, kidney and lung. mRNA levels are low in thymus, spleen and brain. In contrast, PA28 protein levels vary little between these tissues. Immunocytological analysis and cell fractionation experiments demonstrate that both subunits are almost equally distributed between the cytoplasm and the nucleus. Interestingly, PA28alpha spares nucleoli, while PA28beta is strongly enhanced in the nucleolus. This indicates for the first time that the PA28alpha and PA28beta subunits may serve nuclear functions which may be different from and independent of each other.

Amino Acid Sequence↗
PubMed1997

Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-gamma-modulated proteasome subunits.

The primary structures of the interferon-gamma-inducible mouse 20S proteasome subunit MECL-1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL-1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in non-lymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL-1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and delta as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells.

Amino Acid Sequence↗
PubMed1996

Coordinated dual cleavages induced by the proteasome regulator PA28 lead to dominant MHC ligands.

The eukaryotic 20S proteasome is known to associate with the IFN gamma-inducible regulator PA28. We analyzed the kinetics of product generation by 20S proteasomes with and without PA28. In the absence of PA28, the 20S proteasome rapidly generates peptides that have been cleaved only once, while internal fragments accumulate only slowly. In the presence of PA28, products generated by two flanking cleavages appear immediately as main products while the generation of single-cleavage products is strongly reduced. Kinetic data support a PA28-induced, coordinated double-cleavage mechanism. In particular, degradation of peptides derived from mouse cytomegalovirus pp89 and JAK1 kinase in the presence of PA28 leads to strongly enhanced production of the respective major histocompatibility complex ligands and potential precursors. These results show that PA28 profoundly alters the cleavage mechanism of the proteasome and appears to optimize the generation of dominant T-cell epitopes.

Amino Acid Sequence↗
PubMed1996

A role for the proteasome regulator PA28alpha in antigen presentation.

Cytotoxic T cells recognize viral proteins as peptide fragments which are produced in the cytosol and transported on major histocompatibility complex (MHC) class I proteins to the cell surface. Viral peptides that meet the stringent binding characteristics of class I proteins are generated by the 20S proteasome. The interferon (IFN)-gamma-inducible activator of the 20S proteasome, PA28, strongly influences the proteasomal cleavage pattern in vitro. This led us to investigate whether changes in cellular levels of PA28 affect the efficiency of viral antigen processing. A mouse fibroblast line expressing the murine cytomegalovirus pp89 protein was transfected with either the human or murine gene encoding the PA28alpha subunit, which is sufficient to activate the peptide-hydrolysing activity of the 20S proteasome in vitro. Here we report that enhanced expression of PA28alpha at a level similar to that obtained after IFN-gamma induction resulted in a marked enhancement of recognition by pp89-specific cytotoxic T cells; the presentation of influenza nucleoprotein was also significantly improved. These results demonstrate a fundamental in vivo function for PA28alpha in antigen processing.

Amino Acid Sequence↗
PubMed1996

LMP-associated proteolytic activities and TAP-dependent peptide transport for class 1 MHC molecules are suppressed in cell lines transformed by the highly oncogenic adenovirus 12.

Expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the in vivo oncogenicity of this virus. In primary embryonal fibroblasts (H-2b) that express transgenic swine class I antigen (PD1), Ad12-mediated transformation results in inhibition in transport of newly synthesized class I molecules, as well as significant reduction in transporter associated with antigen presentation (TAP) gene expression. In this report we show that reexpression of TAP molecules either by stable transfection of mouse TAP genes or by infection with recombinant vaccinia viruses expressing human TAP genes, only partially reconstitutes the expression and transport of the class I molecules. Further analysis of Ad12-transformed cells revealed that the expression of both LMP2 and LMP7, but not of other proteasome complex components, was downregulated, resulting in altered proteolytic activities of the 20S proteasomes. Reconstitution of both TAP and LMP expression resulted in complete restoration of PD1 cell surface expression and enhanced expression of the endogenous H-2D(b) molecules encoded by recombinant vaccinia viruses, in reconstituted Ad12-transformed cells, efficient transport of H-2 class I molecules could only be achieved by treatment of the cells with gamma-interferon. These data suggest that an additional factor(s) that is interferon-regulated plays a role in the biosynthetic pathway of the class I complex, and that its function is deficient in this cell system. Thus, Ad12 viral transformation appears to suppress the expression of multiple genes that are important for antigen processing and presentation, which allows such transformed cells to escape immune surveillance. This coordinate downregulation of immune response genes must likely occur through their use of common regulatory elements.

ATP-Binding Cassette Transporters↗
PubMed1996

A third interferon-gamma-induced subunit exchange in the 20S proteasome.

The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, delta and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-gamma (IFN-gamma)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-gamma-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-gamma and replaces a sofar barely characterized beta subunit designated 'MC14'. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation.

Amino Acid Sequence↗
PubMed1996

A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation.

CTL epitope (KSPWFTTL) encoded by AKV/MCF type of murine leukemia virus (MuLV) differs from the sequence in Friend/Moloney/Rauscher (FMR) type in one residue (RSPWFTTL). CTL experiments indicated defective processing of the FMR peptide in tumor cells. Proteasome-mediated digestion of AKV/MCF-type 26-mer peptides resulted in the early generation and higher levels of epitope-containing fragments than digestion of FMR-type peptides, explained by prominent cleavage next to R in the FMR sequence. The fragments were identified as 10- and 11-mer peptides and were efficiently translocated by TAP. The naturally presented AKV/MCF peptide is the 8-mer, indicating ER peptide trimming. In conclusion, a single residue exchange can cause CTL epitope destruction by specific proteasomal cleavage.

ATP-Binding Cassette Transporters↗
PubMed1995

The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro.

Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-gamma, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.

Amino Acid Sequence↗
PubMed1995

Incorporation of major histocompatibility complex--encoded subunits LMP2 and LMP7 changes the quality of the 20S proteasome polypeptide processing products independent of interferon-gamma.

The 20S proteasome is the enzyme complex responsible for the processing of antigens bound by major histocompatibility complex class I molecules. The role of the interferon-gamma (IFN-gamma)-inducible proteasome subunits LMP2 and LMP7 in this process is, however, still controversial. We have studied the effects of IFN-gamma-independent LMP incorporation on the quality of peptides processed from the murine cytomegalovirus IE pp89 25-mer polypeptide substrate through dual cleavages by 20S proteasomes. The incorporation of a single LMP subunit or both LMP2 and LMP7 induces changes in 20S proteasome subunit stoichiometry, alters its cleavage site preference and in consequence, the quality of the generated peptides. When the several hydrolytic activities are tested with short fluorogenic peptide substrates, the Vmax, S0.5 (Km), or both values of 20S proteasomes are altered, depending on the combination of LMP. There exists, however, no obvious correlation between the observed changes in hydrolytic activities against short fluorogenic peptides and the changes in dual cleavage site usage within the 25-mer polypeptide substrate. As judged from the calculated Hill coefficients, the presence of both LMP subunits induces a drastic increase in positive cooperativity between the proteasome subunits.

Amino Acid Sequence↗
PubMed1994

Analysis and expression of a cloned pre-T cell receptor gene.

The T cell antigen receptor (TCR) beta chain regulates early T cell development in the absence of the TCR alpha chain. The developmentally controlled gene described here encodes the pre-TCR alpha (pT alpha) chain, which covalently associates with TCR beta and with the CD3 proteins forms a pre-TCR complex that transduces signals in immature thymocytes. Unlike the lambda 5 pre-B cell receptor protein, the pT alpha chain is a type I transmembrane protein whose cytoplasmic tail contains two potential phosphorylation sites and a Src homology 3 (SH3)-domain binding sequence. Pre-TCR alpha transfection experiments indicated that surface expression of the pre-TCR is controlled by additional developmentally regulated proteins. Identification of the pT alpha gene represents an essential step in the structure-function analysis of the pre-TCR complex.

Amino Acid Sequence↗
PubMed1993

A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein.

We describe a novel signal-transducing protein complex, which consists of the T cell receptor (TCR) beta chain that is disulfide linked to a 33 kd glycoprotein and noncovalently associated with proteins of the CD3 complex on the surface of the pre-T cell line SCB.29. This 33 kd glycoprotein, provisionally designated gp33, represents neither of the known TCR chains and has escaped previous detection because it labels poorly by surface iodination. This glycoprotein is absent from the surface of mature T cell lines. A TCR beta complex with identical molecular masses before and after reduction can be immunoprecipitated from surface-iodinated large thymocytes of TCR alpha-deficient mice. The novel gp33-TCR beta complex may be entirely or partly responsible for control of early T cell development exerted by the TCR beta protein.

Animals↗
PubMed1993

T cell receptor beta chain dimers on immature thymocytes from normal mice.

During T cell development the T cell receptor (TCR) beta chain is expressed before the TCR alpha chain. Experiments in TCR beta transgenic severe combined immune deficiency (SCID) mice have shown that the TCR beta protein can be expressed on the cell surface of immature thymocytes in the absence of the TCR alpha chain and that the TCR beta protein controls T cell development with regard to cell number, CD4/CD8 expression and allelic exclusion of the TCR beta chain. Subsequent experiments have shown that on the surface of thymocytes from TCR beta transgenic SCID mice the TCR beta protein can be expressed in a monomeric and dimeric form whereas only the dimeric form was found on the surface of a TCR beta-transfected, immature T cell line. The results presented here show that normal thymocytes from 16-day-old fetuses likewise express only the dimeric form and that the monomeric form on the surface of thymocytes from transgenic mice results from glycosyl phosphatidylinositol linkage. Our results show for the first time that under physiological conditions a TCR beta dimer can be expressed on the cell surface without the TCR alpha chain.

Animals↗