Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the ß5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

The role of the proteasome in the generation of MHC class I ligands and immune responses.

The ubiquitin-proteasome system (UPS) degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. While the UPS is functional in all mammalian cells, its subunit composition differs depending on cell type and stimuli received. Thus, cells of the hematopoietic lineage and cells exposed to (pro)inflammatory cytokines express three proteasome immunosubunits, which form the catalytic centers of immunoproteasomes, and the proteasome activator PA28. Cortical thymic epithelial cells express a thymus-specific proteasome subunit that induces the assembly of thymoproteasomes. We here review new developments regarding the role of these different proteasome components in MHC class I antigen processing, T cell repertoire selection and CD8 T cell responses. We further discuss recently discovered functions of proteasomes in peptide splicing, lymphocyte survival and the regulation of cytokine production and inflammatory responses.

Immunoproteasome subunit LMP2 expression is deregulated in Sjogren's syndrome but not in other autoimmune disorders.

BACKGROUND: The proteasome system has a pivotal role in the control of the immune response, which suggests that it might be involved in the pathogenesis of autoimmune disorders. OBJECTIVE: To investigate the expression profile of selected proteasomal genes in human peripheral blood mononuclear cells in patients with a variety of autoimmune diseases compared with healthy subjects. METHODS: Real time quantitative RT-PCR was used to analyse the mRNA expression pattern of the proteasome activator subunits PA28alpha and PA28beta and of constitutive proteasome and interferon-gamma-inducible immunoproteasome subunits in peripheral blood mononuclear cells. Simultaneously, protein expression of selected proteasome subunits was quantified by immunoblotting. RESULTS: Under systemic inflammatory conditions the proteasome subunits LMP2 (beta1i), LMP7 (beta5i), MECL1 (beta2i), and PA28alpha were expressed abundantly at the protein level in the vast majority of systemic autoimmune disorders. However, simultaneous mRNA and protein quantification showed a characteristic proteasome expression signature in primary Sjögren's syndrome. At the transcript level, the interferon-gamma-responsive subunits LMP2 (beta1i), MECL1 (beta2i), and the proteasome activator subunit PA28alpha were markedly up regulated. In contrast, LMP2 (beta1i) deficiency was evident at the protein level, indicating deregulation of proteasome expression in Sjögren's syndrome. CONCLUSIONS: These data provide evidence for a regulatory defect in the proteasome system in human autoimmune disorders, pointing to a unique role for LMP2 (beta1i) in the pathogenesis of primary Sjögren's syndrome.

Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding.

Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).

The components of the proteasome system and their role in MHC class I antigen processing.

By generating peptides from intracellular antigens which are then presented to T cells, the ubiquitin/26S proteasome system plays a central role in the cellular immune response. The proteolytic properties of the proteasome are adapted to the requirements of the immune system by proteasome components whose synthesis is under the control of interferon-gamma. Among these are three subunits with catalytic sites that are incorporated into the enzyme complex during its de novo synthesis. Thus, the proteasome assembly pathway and the formation of immunoproteasomes play a critical regulatory role in the regulation of the proteasome's catalytic properties. In addition, interferon-gamma also induces the synthesis of the proteasome activator PA28 which, as part of the so-called hybrid proteasome, exerts a more selective function in antigen presentation. Consequently, the combination of a number of regulatory events tunes the proteasome system to gain maximal efficiency in the generation of peptides with regard to their quality and quantity.

Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases.

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.

The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide.

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.

Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine.

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.

20S proteasome biogenesis.

26S proteasomes are multi-subunit protease complexes responsible for the turnover of short-lived proteins. Proteasomal degradation starts with the autocatalytic maturation of the 20S core particle. Here, we summarize different models of proteasome assembly. 20S proteasomes are assembled as precursor complexes containing alpha and unprocessed beta subunits. The propeptides of the beta subunits are thought to prevent premature conversion of the precursor complexes into matured particles and are needed for efficient beta subunit incorporation. The complex biogenesis is tightly regulated which requires additional components such as the maturation factor Ump1/POMP, an ubiquitous protein in eukaryotic cells. Ump1/POMP is associated with precursor intermediates and degraded upon final maturation. Mammalian proteasomes are localized all over the cell, while yeast proteasomes mainly localize to the nuclear envelope/endoplasmic reticulum (ER) membrane network. The major localization of yeast proteasomes may point to the subcellular place of proteasome biogenesis.

Subtypes of 20S proteasomes from skeletal muscle.

20S proteasomes from tissues and cells are a mixture of several subtypes. From rat skeletal muscle we have tentatively separated six different subtypes of 20S proteasomes purified from rat skeletal muscle by high-resolution anion exchange chromatography. Immunoblot analysis using antibodies to the beta-subunits LMP2, LMP7 and their constitutive counterparts delta and MB1 revealed that two of the three major subtypes (subtypes I and II) are constitutive proteasomes, whereas two of the three minor subtypes belong to the subpopulation of immuno-proteasomes. Subtype III and IV are intermediate-type proteasomes. Enzymological characterisation of the six subtypes revealed clearly different V(max) values for hydrolysis of fluorogenic peptide substrates as well as significantly different activities measured with a 25-mer polypeptide of the murine cytomegalovirus IE pp89 protein as substrate. Our data show that the properties of 20S proteasomes isolated from a given tissue or cells are always the average of the properties of the whole set of proteasome subtypes.

Antigen processing by the proteasome.

The proteasome is an essential part of our immune surveillance mechanisms: by generating peptides from intracellular antigens it provides peptides that are then 'presented' to T cells. But proteasomes--the waste-disposal units of the cell--typically do not generate peptides for antigen presentation with high efficiency. How, then, does the proteasome adapt to serve the immune system well?

The role of the ubiquitin-proteasome pathway in MHC class I antigen processing: implications for vaccine design.

Proteasomes are multisubunit enzyme complexes that reside in the cytoplasm and nucleus of eukaryotic cells. By selective protein degradation, proteasomes regulate many cellular processes including MHC class I antigen processing. Three constitutively expressed catalytic subunits are responsible for proteasome mediated proteolysis. These subunits are exchanged for three homologous subunits, the immunosubunits, in IFNgamma-exposed cells and in cells with specialized antigen presenting function. Both constitutive and immunoproteasomes degrade endogenous proteins into small peptide fragments that can bind to MHC class I molecules for presentation on the cell surface to cytotoxic T lymphocytes. However, immunoproteasomes seem to fulfill this function more efficiently. IFNgamma further induces the expression of a proteasome activator, PA28, which can also enhance antigenic peptide production by proteasomes. In this review, we will introduce the ubiquitin-proteasome system and summarize recent findings regarding the role of the IFNgamma-inducible proteasome subunits and proteasome regulators in antigen processing. We review the different ways by which tumors and viruses have been found to target the proteasome system to avoid MHC class I presentation of their antigens, and discuss recent progressions in the development of computer assisted approaches to predict CTL epitopes within larger protein sequences, based on proteasome cleavage specificity. The availability of such programs as well as a general insight into the proteasome mediated steps in MHC class I antigen processing provides us with a rational basis for the design of new antiviral and anticancer T cell vaccines.

The effect of heat shock on 20S/26S proteasomes.

We have studied the consequences of heat shock on 20S/26S proteasome activity and activation, the proteasomal subunit composition, proteasome assembly, subunit mRNA stability as well as on the intracellular distribution of proteasomes. Our data show that heat shock locks 20S proteasomes in their latent inactive state and impairs further activation of the 26S proteasome by ATP. Proteasome mRNA levels are decreased after heat shock and the assembly of the proteasome complex is inhibited. Heat shock also induces a rapid reorganisation of the cellular distribution of the proteasome which appears to be connected with proteasome activity and the change of the cellular architecture after heat shock.

Different proteasome subtypes in a single tissue exhibit different enzymatic properties.

It is concluded from many experiments that mammalian tissues and cells must contain a heterogeneous population of 20 S proteasome complexes. We describe the purification and separation by chromatographic procedures of constitutive 20 S proteasomes, 20 S immuno-proteasomes and intermediate-type 20 S proteasomes from a given tissue. Our data demonstrate that each of these three groups comprises more than one subtype and that the relative ratios of the subtypes differ between different rat tissues. Thus, six subtypes could be identified in rat muscle tissue. Subtypes I and II are constitutive proteasomes, while subtypes V and VI comprise immuno-proteasomes. Subtypes III and IV belong to a group of intermediate-type proteasomes. The subtypes differ with regard to their enzymatic characteristics. Subtypes I-III exhibit high chymotrypsin-like activity and high peptidylglutamylpeptide hydrolysing activity, while these activities are depressed in subtypes IV-VI. In contrast, trypsin-like activity of subtypes IV-VI is enhanced in comparison to subtypes I-III. Importantly, the subtypes also differ in their preferential cleavage site usage when tested by digestion of a synthetic 25mer polypeptide substrate. Therefore, the characteristics of proteasomes purified from tissues or cells represent the average of the different subtype activities which in turn may have different functions in vivo.

A kinetic model of vertebrate 20S proteasome accounting for the generation of major proteolytic fragments from oligomeric peptide substrates.

There is now convincing evidence that the proteasome contributes to the generation of most of the peptides presented by major histocompatibility complex class I molecules. Here we present a model-based kinetic analysis of fragment patterns generated by the 20S proteasome from 20 to 40 residues long oligomeric substrates. The model consists of ordinary first-order differential equations describing the time evolution of the average probabilities with which fragments can be generated from a given initial substrate. First-order rate laws are used to describe the cleavage of peptide bonds and the release of peptides from the interior of the proteasome to the external space. Numerical estimates for the 27 unknown model parameters are determined across a set of five different proteins with known cleavage patterns. Testing the validity of the model by a jack knife procedure, about 80% of the observed fragments can be correctly identified, whereas the abundance of false-positive classifications is below 10%. From our theoretical approach, it is inferred that double-cleavage fragments of length 7-13 are predominantly cut out in "C-N-order" in that the C-terminus is generated first. This is due to striking differences in the further processing of the two fragments generated by the first cleavage. The upstream fragment exhibits a pronounced tendency to escape from second cleavage as indicated by a large release rate and a monotone exponential decline of peptide bond accessibility with increasing distance from the first scissile bond. In contrast, the release rate of the downstream fragment is about four orders of magnitude lower and the accessibility of peptide bonds shows a sharp peak in a distance of about nine residues from the first scissile bond. This finding strongly supports the idea that generation of fragments with well-defined lengths is favored in that temporary immobilization of the downstream fragment after the first cleavage renders it susceptible for a second cleavage.

Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28.

The activation kinetics of constitutive and IFNgamma-stimulated 20S proteasomes obtained with homomeric (recPA28alpha, recPA28beta) and heteromeric (recPA28alphabeta) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28alpha/RecP28beta/RecP28alpha + RecP28beta) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28alphabeta complex is about two orders of magnitude higher than towards the homomeric PA28alpha and PA28beta complexes. Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.

Differential influence on cytotoxic T lymphocyte epitope presentation by controlled expression of either proteasome immunosubunits or PA28.

The proteasome is the principal provider of major histocompatibility complex (MHC) class I-presented peptides. Interferon (IFN)-gamma induces expression of three catalytically active proteasome subunits (LMP2, LMP7, and MECL-1) and the proteasome-associated activator PA28. These molecules are thought to optimize the generation of MHC class I-presented peptides. However, known information on their contribution in vivo is very limited. Here, we examined the antigen processing of two murine leukemia virus-encoded cytotoxic T lymphocyte (CTL) epitopes in murine cell lines equipped with a tetracycline-controlled, IFN-gamma-independent expression system. We thus were able to segregate the role of the immunosubunits from the role of PA28. The presence of either immunosubunits or PA28 did not alter the presentation of a subdominant murine leukemia virus (MuLV)-derived CTL epitope. However, the presentation of the immunodominant MuLV-derived epitope was markedly enhanced upon induction of each of these two sets of genes. Thus, the IFN-gamma-inducible proteasome subunits and PA28 can independently enhance antigen presentation of some CTL epitopes. Our data show that tetracycline-regulated expression of PA28 increases CTL epitope generation without affecting the 20S proteasome composition or half-life. The differential effect of these IFN-gamma-inducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response.

Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes.

Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits. A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein). We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome. Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma. To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied. In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent. Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes. As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells.

Overexpression of the proteasome subunits LMP2, LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus T cell epitope.

The proteasome is a large protease complex that generates most of the peptide ligands of MHC class I molecules either in their final form or in the form of N-terminally extended precursors. Upon the stimulation of cells with IFN-gamma, three constitutively expressed subunits of the 20S proteasome are replaced by the inducible subunits LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1 (multicatalytic endopeptidase complex-like-1) to form so-called immunoproteasomes. We show in this study that overexpression of these three subunits in triple transfectants led to a marked enhancement in the H-2Ld-restricted presentation of the immunodominant nonameric epitope NP118, which is derived from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. Overexpression of the alpha and beta subunits of the IFN-gamma-inducible proteasome regulator PA28, in contrast, did not have a comparable effect. In vitro, immunoproteasomes as compared with constitutive proteasomes generated higher amounts of 11- and 12-mer fragments containing the NP118 epitope. These are likely to be cytosolic precursors of NP118, as a proline anchor residue in the second position of NP118 may interfere with TAP-mediated transport of the nonameric epitope itself. In conclusion, we provide evidence that up-regulation of the three inducible subunits, LMP2, LMP7, and MECL-1, can result in a marked improvement of Ag presentation and that, depending on the epitope, PA28 and immunoproteasomes may differentially affect Ag processing.